Download PDF: Microbehunter (January 2012) (297)

Paper version: Order printed version

Welcome to the 10th issue of MicrobeHunter magazine. You can download the PDF and order a printed version.

At this point I would also like to encourage all readers to participate by writing articles (short and long). Read the submissions page for more information.

If you encounter any errors or mistakes, please inform me, I will correct these and upload a new version.

Contents

Victorian “Live Box” Microscope Capability in 40mm
These small microscopes are not only attractive collectibles, but also practical excursion companions.
R. Jordan Kreindler

High Dynamic Range (HDR) Imaging of Micrographs
Sometimes it is impossible to show both bright and dark areas of a micrograph correctly exposed. HDR imaging combines micrographs of different exposures to obtain a single, correctly exposed picture.
Oliver Kim

Closterium costatum CORDA ex RALF
Starting this issue, Mike Guwak presents a different Desmid every month.
Mike Guwak

Making Stereoscopic Micrographs
The free program Picolay can be used to compute stereoscopic images from a focus stack.
Oliver Kim

Gallery of Micrographs
Images by Jeff Clinedienst, Gino I. Bianchi, James D. Crosby, Anthony Thomas and Luca Monzo.

Amateur Microscopy – Good for the Soul
Thinking about eye-diving into a microscope? Or are you already a devotee? A life-long fan ruminates on our fondness for looking at little things.
David B. Borg

Mosquito wing as a lens test subject
How sharp is your objective?
Anthony Thomas

Some basics first

There are several options for connecting a camera to a microscope. The camera system can either be connected via a dedicated photo tube on a trinocular head, or can be connected to one of the microscope’s eyepieces. Depending on the set-up, there can be either intermediate optics between the camera and the microscope’s objective, or not.

• The image produced by the microscope objective can be directly picked up by the sensor of a camera, without an eyepiece or other intermediate optics. Here objective of a microscope produces a real image directly on the camera’s sensor. The objective produces a relatively large image, compared to the small sensor of many cameras. Unless the sensor is large, there may be quite much empty magnification and the brightness of the image is low.
• The image produced by the microscope objective can also be passed through a reduction lens before reaching the camera sensor. This way the image produced by the microscope objective is reduced in size to better match the small sensor size of the digital camera. The reduction lens produces a real image on the camera sensor. Without the reduction lens the image would be magnified too much. The reduction lens also results in a brighter image. This is an improvement to the first point from above. Eyepiece cameras for microscopes use this system. The reduction lens is not a compensating photo eyepiece and therefore does not correct lens errors produced by the objectives.
• The image produced by the microscope objective is first passed through a regular eyepiece. A virtual image is produced this way, which can not be used to directly make a picture. A camera (with its own objective) then picks up the virtual image and projects it on the sensor. The camera works like the eye, which converts a virtual image to a real image. This system is used in afocal photography, in which a regular compact camera (with its own objective and all) is attached in front of the eyepiece.
• The image produced by the microscope objective is passed through a photo projection ocular (photo eyepiece), which then projects a real image on the sensor of an SLR camera. There are no camera objectives involved. The projection eyepiece corrects optical errors which are produced by the microscope objective. These photo projection eyepieces are compensating optical elements. This means that they are designed to correct various lens errors that the objectives produce, including field curvature and chromatic aberration. These projection oculars are therefore manufacturer dependent and must correspond to the objectives of the manufacturer. Besides image quality, another advantage is, that parfocality is maintained between the camera and the eyepieces (i.e. both images are in focus at the same, and there is no focus deviation).

Let’s now have a look at a few real life applications:

Connecting a webcam (home-made solution)

This was one of my earlier attempts of connecting a camera to a microscope. I completely dismounted a webcam and removed all of the optics. Leaving the webcam optics in place (used in afocal photography) would result in a too small image because most webcams have wide-angle optics. I then placed the electronics with the attached sensor into a separate plastic box and attached a short metal tube to the box for easy placement on the trinocular head. There were no intermediate optics involved and the image was directly projected from the microscope’s objective. Make sure that the blue filter is still in pace in front of the sensor (they are quite red sensitive), otherwise you have to use a blue “daylight” filter (for photography) on top of the halogen lamp of the microscope.

DIY webcam mounted without intermediate optics to the microscope.

The webcam sensor.

The electronics are held in place with some foam material.

The sensor is mounted directly to the main board.

Advantages:

• This was a low-cost solution and worked reasonably well. The plastic case was not expensive at all and the short metal tube I obtained from an old discarded telescope. I was lucky that the diameter was just right.
• The webcam produces a live image on the computer screen, which can be easily focused.
• The webcam was released through the computer and therefore there was no camera shake, resulting a steadier image.

Disadvantages:

• The resolution of the camera was quite low (640×480 pixels), but this can be resolved by using a different webcam.
• The far bigger disadvantage was, that the objective generated a relatively large image and the sensor of the camera was quite small. For this reason there was much empty magnification. Intermediate optics (reduction lenses) would have reduced the size of the image and would therefore also have made a brighter and sharper picture. These optics were not available to me, however.
• Some lack of parfocality can also be a problem. The focus of the camera and of the eyepieces are not the same. The objectives produced an image 10mm down in the phototube. This place was of course not accessible by the webcam. If the deviation is too large then this can become a problem for the objective to slide distance during focusing.
• Webcams often have a blue filter covering the sensor to reduce the effect of infra red light, which gives the whole image a reddish hue. If you remove this filter with the optics, then you need to add a blue filter over the microscope illumination or into the condenser

Connecting an analog video camera (home-made solution)

The principle here was the same as in the webcam solution. I ordered a color surveillance camera module, without case, only the electronics, and mounted this into an aluminum case, which a friend of mine made for me using the appropriate tools. I connected the camera both to a VCR recorder and a TV. Advantages and disadvantages were quite similar to the webcam solution.

A surveilance camera in a case made of aluminum. No intermediate optics.

The camera body is made of one piece.

The camera sensor can be seen.

The main board (with sensor) is held in place by teflon rings (white ring inside).

Advantages

• Fast video display on a TV is possible. This makes it suitable for use in classrooms that have TV screens. There is no need to convey the signal over a computer.
• The speed is in real time and faster than when using slower webcams.

Disadvantages

• Unlike webcams, a separate power supply is necessary.
• Analog video is now somewhat outdated and the resolution is lower than with modern webcams.
• The surveillance camera must be compatible with the TV system (NTSC, PAL or SECAM).
• Capturing still images on a computer (or simply viewing the images on a computer) requires an analog video input, which not every computer has.

Connecting an SLR

“Big name” microscope manufactuerers all have dedicated imaging solutions for their microscopy systems. This one was the most expensive solution, but also the one giving the best results. The image from the microscope’s objective is further processed by a special photo-projection ocular, which then projects it directly on the sensor of an SLR (single-lens-reflex) camera. The projection eyepiece corrects all remaining lens errors from the objectives. I have worked a lot with this system, and the resolution of the pictures is very high. I now have an 18 megapixel SLR camera and this resolution is much higher than the resolution produced by the microscope and specimen.

The photo eyepiece adapter is mounted on the phototube of the trinocular head and holds the photo ocular.

The Canon EOS 600D has a swing-out LCD screen. This makes it possible to focus while sitting.

Advantages:

• The best image quality is produced. The projection ocular corrects chromatic aberration and also generates a flat field of view (so that the sides of the image are not out of focus).
• SLRs have a high dynamic range (both bright and dark areas can be captured at the same time, without much information loss).
• Some SLRs allow for the capturing of RAW images. These are able to capture an even higher color depth for a high dynamic range.
• SLRs have a high resolution, often much higher than dedicated microscope cameras (for the same cost).
• The sensor of SLRs is quite large, therefore the signal to noise ratio is high. This makes it suitable for low-light photography or high ISO photography and very short exposure times.
• It is possible to couple the SLR with a flash system for the microscope, for microflashing. I never tried this, though.
• The SLR can also be controlled from the computer for automatic photography (called tethered shooing).
• Some modern digital SLRs also allow for video recording. This is a major advantage, if needed.

Disadvantages:

• Generally a high cost of both SLR camera and associated adaptor tubes, projection oculars, etc. The projection eyepiece and phototube are manufacturer specific and can be relatively expensive, especially when the components are not manufactured anymore. I read somewhere, that there are also non-manufacturer specific adapter tubes available for SLR cameras, but one has to do some research for this. For best results, the projection ocular must match the objectives and sensor size of the camera.
• A dedicated trinocular head with phototube is necessary. It is not possible to connect the SLR and phototube in front of an eyepiece tube (which extends 45 degrees horizontally), due to its weight.
• Camera shake during the release can be much higher than with other cameras and can blur the image a bit, but there are solutions for this (long exposure time, using mirror lock up).
• Live-view on the computer screen is not always possible, this depends on the SLR.
• The photo projection ocular should be from the same manufacturer as the microscope objectives, otherwise lens errors are not properly corrected.
• My system uses a photo projection ocular which was designed for analog 36mm film cameras. My current digital camera has a smaller sensor size and therefore there is extra magnification and the field of view is not as wide. Stitching images together with panorama software can be necessary. The field of view is much wider than in the webcam and analog video solution (from above), however. Different projection oculars for smaller sensors do exist, they are not manufactured anymore, and are thus relatively expensive.
• SLRs can not be used for focal photography, the lens diameter is too large.
• Not every microscope manufacturer may offer SLR adapter tubes and corresponding compensating photo eyepieces and it may be necessary to use manufacturer independent products. These may not deliver the highest image quality, however.

Connecting compact camera (afocal photography)

Fortunately it is possible to make good quality pictures by using regular digital compact cameras as well. This solution is more cost effective. The front lens of the compact camera must be sufficiency small, which is usually the case. The camera is mounted on a tripod and the picture is taken through the eyepiece of the microscope. It is necessary to zoom in and it is also necessary to adjust the camera-eyepiece distance properly. There are some adapters available, which allow one to clamp a compact camera to the base of the eyepiece, without any modifications to the camera. These clamps use the tripod connector of the camera.

The triplod holding the camera can either be placed on the table or on the floor.

Proper centering and correct camera to eyepiece distance is important.

Advantages

• No trinocular head needed and one can use a camera that one already has.
• It is also possible to take pictures freehand, without tripod, if there is sufficient light for short exposure times (but this is a bit difficult sometimes).
• Due to the high resolution of compact cameras, much image information can be captured.
• It is also possible to zoom in and out.
• Many modern compact cameras also allow for the recording of (HD) video clips!

Disadvantages

• There is the possibility of vignetting if the system is not properly set up. For proper photography one also needs to use a tripod, which takes space.
• The theoretically best image quality can not be achieved (compared to connecting an SLR with projection eyepiece), but often the quality loss is irrelevant. The reason for this is because there are simply more lens elements in the light path (camera objective) and because the regular eyepieces are not designed for photography.
• The suitability of the compact camera for microscopy has to be tested first, not all compact cameras may work equally well (depends on diameter of camera objective). If the camera objective is larger than the exit pupil of the eyepiece, then there is the possibility of vignetting (solution: zoom in more).
• Field curvature may possibly be a problem. In this case the side of the image is out of focus, while the center is in focus. This effect can be reduced by zooming in.
• The set up with tripod is not very convenient. It is possible to make an adapter tube which is connected directly to the camera. This tube then can be inserted into the microscope like an eyepeice. It is possible to use compact cameras that have a filter thread and connect the adapter tube to this thread.

Using a mobile phone camera

Yes, this too works! The objective lens of the mobile phone is smaller than the image produced by the eyepiece, so this works. The camera focuses to infinity.

Shaky, a bit improvised, but possible. The correct eyepiece to camera distance is critical.

Advantages

• Fast, simple, no extra cost and no microscope adaptations are necessary.
• There is a microscopy iPhone app which calculates magnification, it is worth checking out.
• The pictures can be directly sent from the phone, if this is needed.

Disadvantages:

• Very steady hand needed to hold the camera at the correct distance from the eyepiece. A wrong camera to eyepiece distance will result in vignetting.
• The camera is usually wide-angle and covers the complete field of view. The image is in a circle. For this reason pixels are wasted.
• The solution is somewhat improvised (having to hold the mobile phone by hand) and I have not used it on a regular basis.

Connecting a dedicated microscope camera

This is my favorite solution, because of its high convenience. It is possible to do microscopic observations without looking through the eyepiece, only the computer monitor. This can be more relaxing at times. These cameras are connected directly to the computer and are controlled entirely over the computer. Full manual control is possible with the camera that I have. This is absolutely essential (especially when one wants to make panoramic images). There are reduction lenses in front of the camera, so the brightness of the image and field of view are increased (compared the the DIY webcam solution from before). And for those of you who do not have a trinocular head: these cameras can be mounted instead of an eyepiece!

The camera has a reduction lens and can be mounted on the phototube.

The camera can also be attached in place of an eyepiece.

The camera can also be attached in place of an eyepiece - another view.

Advantages

• Most convenient, relatively small (compared to connecting an SLR).
• The image sensor (in combination with the reduction lens) also covers a wide field of view but does not make the image appear in a circle (like when using mobile phone cameras).
• The camera can stay on the microscope and I do not have to detach it like I had to for my SLR.
• There is no shutter release shake and all of the pictures are stored on the computer’s hard disk. It is therefore possible to take many images for time-lapse photography, without having to worry about the memory card becoming full.
• No trinocular head needed! These cameras are light enough to be used instead of an eyepiece.
• These cameras often have a C-mount or CS-mount to which the reduction lens is connected. It is therefore possible to exchange the reduction lens to obtain a different magnification. Unfortunately, these reduction lenses are difficult to obtain alone, without camera.

Disadvantages

• Compared to the camera resolution, these systems are quite expensive (but not unaffordable). I paid over $260 (EUR 200) for a 3.1 megapixel microscope camera with reduction lens. For my uses, a higher resolution is not needed, however.
• It is also necessary to have a computer around and running for taking pictures, as the camera is not able to store images.
• Another disadvantage is that the USB cable connection is too slow to allow for high-quality video recording. Movements are not recorded smoothly. Using a lower resolution may resolve the problem.
• The reduction lens can not be compared to a compensating photo projection eyepiece, which corrects lens errors (and is manufacturer specific).

Other possibilities, which I did not try

• Using a camera system which was specially designed for the microscope. Bigger microscope companies offer these solutions. The camera has a mount which fits only to the microscopes of a particular manufacturer. Often the optics of the camera are also match the optics of the remaining microscope (compensating lens errors etc.). Sometimes these cameras also have other features that are not commonly found on digital cameras, such as stand-alone operation (no computer connection needed), LAN connectivity, or even a separate camera control panel. I would guess that these cameras are interesting for research institutions, who want to have a solution, which works out of the box.
• Removing the objective of a compact camera and attaching a custom-made adapter (with or without reduction lens) to the body of the camera. These adapters may need to be designed specifically for the camera. You may be interested in this link: Making Digital Camera Microscope Adapters
• Attaching a custom adapter to a compact camera for easy afocal photography. The camera’s objective stays on the camera. The adapter goes over the camera’s objective and can be connected to the eyepiece without the use of a tripod.
• Using a clamp to hold the camera for afocal photography. I have found one German shop specializing in amateur astronomy (!!) selling these holders for microscopes and telescopes. The holder is clamped to the tube of the microscope and also holds a compact camera. Check the following links to see what I mean (I am not affiliated with this shop):
  Link 1 | Link 2 | Link 3 | Amazon also has them

Do you have any further suggestions on how to connect a camera? USe the comments section below to share your thoughts!

Download PDF: Microbehunter (December 2011) (670)

Paper version: Order printed version

Welcome to the 10th issue of MicrobeHunter magazine. You can download the PDF and order a printed version.

At this point I would also like to encourage all readers to participate by writing articles (short and long). Read the submissions page for more information.

If you encounter any errors or mistakes, please inform me, I will correct these and upload a new version.

Contents

My Perfect Microscope
My perfect microscope is assembled from 3 junked and discarded units.
Wilhelm Resch

Gallery
Images by Jeff Clinedinst, Rodney Brightwell, Anthony Thomas, Manfred Rath and Brian Loxton

Microscope Slides: Styles, Features and Preparation Techniques
Everything you ever wanted to know about glass slides!
Hayley Anderson

Improving the performance of the optical microscope
Some simple adaptations to the microscope can dramatically improve its performance. In this article the author explores the possibilities of darkfield and polarization.
César Guazzaroni

Observing Freshwater Planarians
These little flatworms are ideal specimens for low-power microscopy.
Charles Guevara

A Stage Platform for the Shop Inspection Microscope
The “shop inspection microscope” is a small monocular microscope using top-illumination. In this article the author describes the construction of a stage platform, which makes transmitted-light observation possible.
Glenn Shipley

Make your Photomicrographs look like DIC
Differential Interference Contrast (DIC) optics produces beautiful images that appear 3D. These optics are also very expensive. Luckily it is possible to achieve a DIC-like effect with PhotoShop’s Emboss tool.
Suphot Punnachaiya

Index of Contributors for Volume 1 (January – December 2011)

How much can I expect to pay for a microscope for children? The minimum price for a useable microscope is around EUR 150/USD 200 and up. You would probably like to spend a bit more, but cheaper devices also exist which provide a useful picture, but may be less convenient and stable to use. EUR 300/USD 400 should give you already a very decent device. This is the price that many educational microscopes in schools have. Anything much less than EUR 150/USD 200 is likely not to be of sufficient quality, but simply because a microscope is expensive does not mean that it is automatically suitable. Specialized optics may quickly drive the price up, but may not be suitable or required. If you are buying blindly then you may spend money on unnecessary things, or waste money on a quite useless device. As a matter of fact, some individual microscope objectives can be more expensive than the whole microscope (microscopes are modular).

How are cheaper microscopes different from more expensive ones? Modularity of the microscope, use of more specialized objectives and optics (plan objectives, apochromatic objectives… not needed for children) and quality of machining as well as brand name drive the price up. Devices containing many metal parts are more stable but also more expensive to manufacture.

What can I expect to see under the microscope? This depends to a large extent on three factors: The type of microscope (stereo or compound), the quality (resolution) of the optics and (of course) the specimens that you look at. The type of microscope determines to a certain extent also the specimens that you can look at. With stereo microscopes you can observe opaque objects, such as rocks, whole plant parts or insects. With compound microscopes you can observe the much smaller cells.

Which type of microscope, stereo or compound, should I buy? The choice of the right microscope type (stereo vs compound) is a fundamental issue. After all, you do want to keep the child interested. Buy stereo microscopes if the child is very young (up to 10 years) or if you want to do uncomplicated natural observation without much specimen preparation. Buy stereo microscopes also if you want to extend an already existing hobby or interest such as stamp collecting, collecting coins, minerals, rocks, insects and butterflies or plants. These objects can all be directly viewed under the stereo microscope, without destroying them. Buy compound microscopes if you or your child is also interested in specimen preparation or if you are interested in seeing smaller objects. Older children may be more interested in compound microscopes, as this is the kind that gives more “interactivity” and more possibilities for preparing specimens. Older children may also be interested in making a slide collection. The children can use different magnifications and have to learn to operate both the coarse and fine focus knobs. If you want to to observe water samples and cells, then compound microscopes are the way to go. Be aware that some children may not consider stereo microscopes as “real” microscopes and that they may be disappointed if they are not able to observe paramecia and other small water life that they read about in books. Also be aware that compound microscopes need more guidance and practice, especially if specimens are to be prepared. Many of the following FAQ will deal with compound microscopes. For more information about these two types of microscopes, read: Types of Light Microscopes.

Where can I buy microscopes? Microscopes can be bought from specialized microscope dealers. These often also supply schools and universities with microscopes. Do not buy second hand devices unless you really know what you are doing. There are simply too many things that can go wrong, even if the quality of the second hand microscope is otherwise quite good. It’s well possible that second hand microscopes are equipped with specialized objectives that are not suitable (or simply too expensive) for children. Unlike consumer products, which come out of the box, microscopes are commonly assembled according to the research needs and second hand microscopes may have a research or medical background. It is probably best to personally get advice from a microscope shop.

Is there one single criterion that I should look out for when buying a compound microscope? Look for two things in microscopes. The microscope should be made of metal (and be heavy) and the objectives should be DIN standard. Look at the objective of the microscope and check if it has the number 160 written on it. This refers to a 160 mm tube length. Microscopes that are able to accept these optics often (not always) have a minimum quality. Most educational and routine microscopes use these, plastic toy microscopes do not. These objectives are interchangeable with each other. Microscopes that use infinity corrected objectives have an infinity sign printed on them and are expensive and can be found more on research microscopes. I just mention this for the sake of completion. The material of which the body of the microscope is made is also relevant. Devices made mostly of plastic can be considered toys, and these do not provide the stability and optical quality to keep children interested over a longer time period.

Do my children need support? A microscope is a scientific instrument and it use requires appropriate education and support. After all, inappropriate handling may damage the device (crashing the objective into the slide, for example). Sooner or later the child will have observed all the provided slides and samples and will want to observe new things. Guidance is then needed to prepare more samples (unless you buy ready-made slides). Safety issues must also be considered: How can you protect the microscope and how can you protect the child? Some chemicals used for preparing samples are toxic, do not use them and do not blindly trust them. There are also many non-toxic alternatives around, however, and the parent may need to do a bit of research. There is also the danger of cutting oneself, when preparing samples. You may also need to do some research on the different types of specimens that can be observed – yes a microscope does require some guidance.

Should I buy a second hand microscope? Unless you have worked with microscopes yourself and unless you know what you are doing, I would not buy them second hand. Maybe you know a trustworthy second hand dealer, in this case I would also take second hand microscopes into consideration. Be aware that a quality second hand microscope (such as the “Zeiss Standard”) can be obtained for a fairly low price, but that this microscope provides much greater value than new no-name devices, which may be more expensive. Without advice you run the risk of buying a microscope with objectives that are not appropriate for education, or microscopes that are not operating reliably. There is no way to see from a picture if the objectives are intact, if there is no stage drift and if the gear operate smoothly. Hospitals and research institutions sometimes sell useful used microscopes, but these may be equipped with specialized optics. Also do not buy microscopes from people who do not know much about them. Non experts are not able to assess the quality of a microscope. There are so many things that you have to look out for, that it is not possible for me to summarize this in a few lines. I may write a second FAQ about them.

Is there anything that I should not buy? Do not buy second hand microscopes unless you also buy them from a shop, which is able to give warranty and service. Do not buy specialized microscopes such as inverted microscopes, metallurgical or polarizing microscopes. Again, if you search Ebay, you may not always know the difference. Do not buy scopes that have only a mirror instead of a lamp. Kids may point them to the sun and destroy their eye sight. Mirrors also do not provide enough light intensity. Do not buy historical microscopes. They should go into the museum and also may not have the optical quality (fungal growth on the optical surfaces is a problem, etc.).

What’s the problem with plastic (“toy”) microscopes? These are microscopes that are sold in a colorful cardboard and Styrofoam box together with a wide range of different accessories. There is a general agreement among enthusiast microscopists and teachers that these microscopes should not be bought. First, they are not as cheap as one may think and for a little more money one can already obtain a microscope with substantially better optics. Toy microscopes are often difficult to focus often lacking a coarse and fine-focus knob. Do not forget, that the tolerances of the mechanics has to be extremely narrow. Plastic gears simply can’t keep up with metal gears. They do not have standardized objectives and the resolution of the picture is low. Often the magnification is also advertised as unrealistically high (1000x). The low light intensity (battery operated or mirror) makes it difficult to see the specimens properly. If money is indeed an issue, then it’s better to get a simple but solid stereo microscope. They are more fun to use. In my opinion, children need stable and solid devices that produce a sharp, contrasty and bright image. Kids are demanding these days. The images that the microscope produces has to compete with the strong visual impressions from television, the Internet and magazines. A low-contrast, washed-out, dark picture produced by a toy microscope will not captivate the children for an extended time. My 2 cents. Download Microbehunter Magazine (November 2011) for a comparison between toy microscopes and more suitable microscopes.

I already bought a toy microscope! What should I do? Keep it and buy a “real” one and compare the image quality. Then write an article about it for this magazine.

Where can I save money? You do not need: Köhler illumination (for photography through the microscope), 100x oil immersion objective (more expensive and difficult to use for children). Actually I really dis-advise getting a 100x oil immersion objective. This requires the use of immersion oil, which is messy to use and has more specific applications. You also do not need phase contrast and DIC, these are expensive anyway. Plan objectives are more expensive and useful for photography. I mention this, because second-hand devices may come with these. A bright-field condenser with a filter holder beneath the stage is highly recommended, however. This allows for simple dark-field microscopy (bright specimen on dark background), if you insert a dark-field patch-stop into the filter holder. A device with a mechanical stage (and not only stage clips) is very recommended. It makes operating the microscope easier. A mechanical stage allows you to move the specimen slide horizontally and vertically by turning two knobs.

Which objectives and eyepieces should I buy? Buy achromatic DIN objectives with the magnifications 4x, 10x, 40x and a 10x eyepiece. A 100x oil immersion is not needed and may even be counter-productive. This objective requires advanced techniques and is more expensive. Better to get a 60x objective instead (more rare), but this is optional. This is a standard combination and microscope dealers supplying for schools will already offer these combinations.

Should I buy a microscope with a “name” or a no-name device? This is a long question to answer and the opinions diverge on this issue. From a quality perspective, all “big four” microscope manufacturers (Olympus, Nikon, Leica, Zeiss) produce quality microscopes and also have cheaper introductory microscopes for schools in their program. Still the cost of these microscopes is often higher, but also their resale value. Many no-name devices do carry the name of the importer, and the quality can cover a wide range. I personally have a rather pragmatic view on the issue. If one wants is not able or willing to spend much money on a microscope, then a “no-name” device is probably the only way to go (unless one buys second hand). Some people think that it is better to buy a used “big four” microscope than a new “no-name” microscope of the same price, also because of the higher resale value. I would dare to say that for beginners it may be very difficult to judge the quality of a used device. Of course one can also buy used (and well serviced) microscopes from a dealer, and this is indeed a possibility.

During my beginning days of microscopy, I once talked to a “big four” microscope manufacturer. I was quite surprised that he gave me a surprisingly balanced advice on which microscope combination to buy. He could easily have sold me a microscope which would have been much more expensive (and also not suitable for my needs). I was a beginner. The salesman was quite honest and told me that they have no interest in selling me a microscope which is too expensive and not suitable, for the sake of earning quick money. I summarize his words: “We have a long-term view. The beginning microscopy users of today are the researchers of tomorrow. We want to keep beginners and children interested in microscopy. If the microscopy enthusiasts have a good view about our company (and do not feel ripped-off), then they will also purchase our microscopes when they are in a position to decide if they should equip a whole laboratory with microscopes.” Interesting point.

What about “computer microscopes” Back in 1999, Intel introduced the QX3 Play microscope, which needed to be connected to a computer. The QX3 was later replaced by the QX5. You can read an extensive QX5 USB microscope review here. The image quality of this device seems to be good, and I already have read several positive reviews about this device. It has the advantage that this microscope is able to cover both worlds, the world of compound and of stereo microscopes. Still, these microscopes are sold as “toys” and (according to a review I read) are not able to provide the same image quality as dedicated student and educational microscopes (of comparable price). A disadvantage is, that it is necessary to connect the device to a computer in order to see something (it has no eyepiece). Microscopes like these are not standard and if you want to teach children proper microscopy use (operating the fine and coarse focus, operating the diaphragm, changing objectives, proper microscope cleaning, etc.) then I would get a standard device. You can also take pictures through a regular microscope with a compact camera using afocal photography.

What accessories are needed You also need: an introductory book about microscopy (to keep the children motivated), slides and cover glasses, and tweezers. These things are not expensive. I also highly recommend that you get a slide box with ready-made samples from a wide range. Do not get slides made for medical students, which show a wide range of different anatomical sections (boring). Get slide boxes that contain both plants, insects, animal tissue, water samples, sand, radiolaria, etc, etc. To keep the children interested. This way the children have something to look at right away, without the need to prepare slides on the day they receive the microscope.

This all may sound complicated. What is the easiest approach? Find a dedicated shop selling microscopes and contact them. Often educational supplies companies will have several microscopes in their product range. Other companies are specifically specialized for microscopes. Study the catalog, do some research (your kids will need support preparing the samples anyway). Write them an email and be honest about the needs. Tell them that you need a scope for your kids and also tell them if it should be a compound or stereo microscope. A serious dealer will know the requirements and will not sell you an inappropriate device. They are interested in long term customer relationships and not in quick money.

Comments and opinions are appreciated!

Download PDF: Microbehunter (November 2011) (794)

Paper version: Order printed version

Welcome to the 10th issue of MicrobeHunter magazine. You can download the PDF and order a printed version.

At this point I would also like to encourage all readers to participate by writing articles (short and long). Read the submissions page for more information.

If you encounter any errors or mistakes, please inform me, I will correct these and upload a new version.

Contents

A Review of Essential Oils as clearing agents for natural history specimens
Certain essential oils have previously been used in microscopy as clearing agents. While they certainly smell better than xylene do they actually perform?
Andrew Chick

Bad Pictures
Why report back only the successful microscopy attempts? This one resulted in quite disappointing images.
Oliver Kim

New Techniques in Microscopy
Smartphones as microscopes, scanners that scan slides instead of traditional microscopes…. Where will the future take us?
Oliver Kim

Image Gallery
Pictures by Anthony Thomas and Manfred Rath.

Field Notes on Science and Nature
Keep a scientific notebook which documents your observations! A book review.
Wayne Wilson

Polarizing Microscope and Applications
What are the areas of applications of polarizing microscopes?
Monty Apollo

A Wonderful Gift for Christmas?
How well do toy microscopes perform? Wilhelm Resch bought one to give it a test.
Wilhelm Resch

Observing Yogurt Bacteria
Yogurt bacteria can be observed in a wet mount or after heat-fixing and staining them.
Oliver Kim

Large phototrophic purple sulfur bacteria on a leaf
A magenta-colored bloom can be an indication for the presence of purple phototrophic bacteria. Here, aggregates of these bacteria were found on a decaying leaf.
Charles Guevara

Staining Epithelial Cells with Ink
Regular fountain-pen will stain epithelial cells.
Oliver Kim

• Microscopy related articles
• B. J. Ford: The Royal Society and the microscope
• J. Friesen: Essay review. Robert Hooke under the microscope

A possible health hazard: Biofilm on the underside of a bathtub stopper.

Much less has been written about the precautions that one should take when dealing with the organisms themselves. It is now my objective to address some precautionary measures when dealing with organisms that are to be microscoped.

Amateur microscopy certainly can not be considered a high-risk hobby, especially when one looks at ready-made permanent slides. Here the organisms in question safely killed and embedded in mounting medium. The issue starts to look a little different when one engages in collecting, concentrating and possibly even growing microorganisms for microscopic observation. Safety issues like this are not only relevant to amateur microscopists, but also for teachers who want to conduct basic microbiological and microscopic work in a school laboratory. In this case the organisms are alive and depending on the type of organism, they may pose a possible health hazard.

It it not, and can not be the intention of this article to give a detailed overview of the aseptic procedures used in a microbiology laboratory. I am not going to address the growing of bacterial colonies on agar petri dishes or the the making of a nutrient broth for the enrichment of bacteria. I am also not going to address the proper use of an inoculation loop and a Bunsen burner for sterile colony transfer. These laboratory methods are, in my opinion, too specific for the majority of amateur microscopists and require a properly equipped lab and appropriate training. Such issues can also not be covered in the little space available. The growth of (unknown) bacteria on agar plates or liquid culture medium also poses a potential health hazard, as the bacterial densities can be extremely high, and I generally would be cautious when working with nutrient media. There are also legal issues associated with these methods, as the legislation of some countries only permit the enrichment and growth of bacteria for certified laboratories. As a matter of fact, the growth of unknown bacteria isolated form the environment even requires the application of aspetic methods of an elevated biohazard level. Readers who are interested in these methods should consult introductory microbiology books, which cover these aspects in detail.

Rather, I would like to place a focus on the methods that are relevant for microscopists. In particular, I would like to address the making of a hay infusion, the observation of pond water as well as the observation of molds and other fungi.

Aseptic technique

The term aseptic technique refers to a medical or laboratory procedure that is performed under sterile conditions. The aseptic technique fulfils several objectives. First, the technique should protect the sample under investigation from contamination. This is of particular importance when culturing microorganisms, as fast-growing contaminants may possibly grow faster than the microorganism that one is interested in. The sample may thus quickly become overgrown by unwanted microorganisms.

While still working in a microbiology lab, I was told that a student working towards his diploma thesis accidentally sub-cultured a contaminant for several months. All of the experimental tests were performed on this contaminant and at the end of the thesis work the obtained data of several months was considered worthless. A quick check of the microorganism under the microscope would have quickly revealed the mix-up. For those of you who were wondering: Luckily I was not the unfortunate student.

Second, the aseptic technique should protect oneself from infection by potentially pathogenic microorganisms. The procedure therefore includes measures that prevent the inhalation of microorganism containing aerosols as well as the prevention of skin contact and ingestion.
Last, the environment and other people should be protected as well. Proper disposal of petri dishes and microorganism-containing sample materials is therefore necessary and often also required by law.

Risk Assessment

The dangers of contacting an infection depend on several aspects:

• Immune status of the person: The weaker the immune system of the person, the higher the chance of contacting an infection. For this reason, only handle unknown bacteria if you are healthy and have no immune system problems.
• Infectivity of the organism: Some pathogens can be infective at a low concentration, others require a higher concentration. Keep the concentration of the microorganisms low.
• Density of the organism: The higher the density, the higher the chance that a critical level of the microorganism is reached to cause infection. Just as above, keep the density of the microorganisms low and only grow them if it is not possible to observe them from natural samples.
• Mode of transmission: Different pathogens prefer a different method of transmission. Certain pathogens, for example, are transmitted over the air, others over water and still others over food. Others require insect vectors for transmission.

Most microorganisms are harmless, but one never knows what substances they are producing when grown at a higher concentration. Certain Cyanobacteria, for example, are known to cause eye irritations or allergies.

Hay infusion issues

A hay infusion is a culture medium which is commonly used to grow protists, such as the well-known Paramecium, for microscopic observation. Hay infusions have been popular since the beginning days of microscopy and are still a popular way of obtaining protozoa for educational uses in schools and universities.
There are two ways in which a hay infusion can be made. A handful of hay is boiled with water to extract nutrients, which serve as a food source for the microorganisms. The obtained culture medium must then be inoculated with the microorganisms that one wants to enrich. Pond water containing ciliates, for example, can be used. Generally this procedure is not recommended, as heat-resistant spored of potentially pathogenic bacteria can survive the boiling process. Alternatively one can try to enrich the microorganisms that can be naturally found on the hay. In this case the hay-water mixture is not boiled, but simply left standing for 24-48 hours. A thin iridescent bio-film will start to form on the water surface. This film is teeming with bacteria. In the presence of ciliates, the number of bacteria may decrease over time, and a progression of different organisms can be observed. Be aware that some countries have laws that regulate the use of hay infusions (and growth ob bacteria in general) for educational purposes.

One should be aware that unknown (and therefore potentially pathogenic) microorganisms may also start to grow in the hay infusion. The boiling process does not necessarily kill all of the microorganisms present on the hay. It is not uncommon to find heat-resistant spores of Bacillus and Clostridium on the hay. After the cooling of the infusion, these spores may start to germinate giving rise to live, possibly pathogenic, bacteria. The fact is, that you simply do not know what you are growing and appropriate safety precautions should be taken.

It is not possible to determine the pathogenicity of bacteria by microscopic observation. A range of biochemical and genetic tests are necessary. The enthusiast microscopist should therefore treat such a hay infusion with utmost care. Do not ingest the hay infusion, avoid skin contact (especially if there are open wounds), do not inhale the aerosols and prevent spills. Generally avoid contact of the liquid with mucous membranes, including the eye. Also make sure that the hay is clean and has not been in contact with excrements of animals. You may otherwise enrich bacteria from the animal’s digestive system. If a spillage or skin contact has occurred, then use 70% ethanol for disinfection (mix 7 parts of alcohol with 3 parts of water). A higher concentration of alcohol may actually have a lower disinfection efficiency.

Do not simply flush the hay infusion down the toilet. This may cause aerosol formation. Add chlorine bleach to the infusion and allow the substance to work for a few hours. Some people may be concerned that the bleach will then also find its way into the waste water, which is not very environmentally friendly. I would agree, but have no solution to this issue. Be aware that the addition of 70% ethanol to the infusion will dramatically lower the concentration of the alcohol. You can add concentrated alcohol to the infusion but this is a cost issue (and the glass jar may not be large enough).

Pond water safety

Even pond water may be the source of some unexpected surprises. I recently introduced mosquito larvae into my household this way. The mosquitoes caused me quite some irritation at night. Be aware that keeping a jar of standing water may even be illegal in countries with Malaria, which can be spread by certain mosquitos.
Other issues relate to the water quality of the pond water, may or may not be very high. Decomposing animals close to the sampling site can give rise to microorganisms that one does not want to have in the household.

Ponds which are clean enough for swimming should not be problematic, there are rare cases, where people did get infected by certain protozoa, however. Water samples from ponds which are rich in (potentially irritating) Cyanobacteria or eutrophicated should be handled with more care. Some ponds may be close to agricultural areas and there is the possibility for manure to run into the ponds.

Molds

Molds can be easily grown by treating an appropriate substrate (such as bread) with a soil-water suspension. Fungi will start to grow and release spores into the air. These spores may not only contaminate other types of food in the household, but may also be responsible for allergic reactions when inhaled. Many types of mold also produce potent toxins, which are capable of causing severe health problems.

Prevent the spreading of spores by keeping the container with the mold closed and avoid air currents which may distribute the spores.
If you want to investigate molds for educational purposes, then I would suggest that you try to first use edible molds, as can be found on foods, such as cheeses.

Biofilms

Biofilms are composed of microorganisms that stick together and to a surface. They can often be found on objects that are moist. The slimy covering of rocks in a pond are an example. Biofilms that harbor bacteria from human sources (e.g. bathroom stoppers) may pose a possible health hazard, also because the bacterial density can be quite high. Harmful microorganisms can also be found on other places in the household, such as dishwashers. What does this have to do with microscopy? Microscopy enthusiasts should establish clear procedures when taking samples from these sources to prevent contact.

General Advice

Here is some general advice when handling samples that contain microorganisms.

• Open wounds: Do not handle microorganism-containing media if you have open wounds or cuts in your skin. Intact skin can be considered as a very effective physical barrier against infection and open wounds can be problematic.
• Disinfection: Disinfect hands and surfaces with 70% ethanol. More concentrated ethanol may actually work less efficiently in killing microorganisms.
• Disposal: Autoclave the used culture medium at 120°C for 30 minutes. This should also be able to kill spores. If you do not have an autoclave available, then cover the petri-dishes or culture medium with chlorine bleach. Allow sufficient time for these substances to work. When you add bleach, be aware that this is a corrosive substance when concentrated. Eye and skin contact must really be avoided. Also be aware that liquid bleach becomes more diluted when you add it to liquid culture medium, losing its efficiency.
• Avoid aerosolization: Some microorganisms spread over air. Avoid spillage of the culture medium and carefully add the disinfectant to the medium before disposal, avoiding splattering of the liquid.
• Keep bacterial counts low: Make sure that the sample (such as a hay infusion) contains many ciliates that consume the bacteria. Keep the level of nutrients low to avoid too many bacteria from forming and ensure that the medium has sufficient oxygen supply for the ciliates to grow.
• Do not use polluted water: Dirty and polluted water can contain contains many bacteria and a lower count of the more interesting ciliates. If the water sample was isolated from a stream that came in contact with household waste water, then it may be possible that pathogenic enterobacteria are present.
• Do not decompose food: Some teachers like to decompose food to demonstrate the spoiling process to children. Be aware that Clostridium perfringens may be found on spoiled meat or poultry. This bacterium can cause food-borne illnesses. Personally, I would not use microorganisms from spoiled food for educational microscopy. I would resort to much safer and easily available bacteria and fungi. These can be isolated from fresh cheese, or example.
  Do not culture bacteria obtained from humans: In particular, do not inoculate growth medium with bacteria from the skin. You may be growing Staphylococcus, otherwise.
• Keep petri dishes closed and sealed: This minimizes the risk of accidentally touching the agar surface, which may be covered by bacterial colonies. Generally speaking, I do not recommend the growth of unknown bacteria in petri dishes by people who do not have basic microbiological training in aseptic technique. The bacterial concentrations are simply too high to be safe.

What is the take-home message? A good portion of common sense and basic hygienics will greatly reduce the possibility of you catching an infection and will hopefully keep you healthy.

Download PDF: Microbehunter (October 2011) (928)

Paper version: Order printed version

Welcome to the 10th issue of MicrobeHunter magazine. You can download the PDF and order a printed version.

At this point I would also like to encourage all readers to participate by writing articles (short and long). Read the submissions page for more information.

If you encounter any errors or mistakes, please inform me, I will correct these and upload a new version.
 
 

Contents

How to find Tardigrades
Tardigrades, or water bears, are cute little creatures that can easily be observed with a stereo microscope.
Mike Shaw

A Beautiful Microscope Slide
Victorian microscope slides are not only nice to look at from outside. Some of the specimens that they carry are at least as interesting to observe.
Leonardo Balbi

Introductory Microscopy Projects for Children and Students
Are you searching for simple microscopy projects for classrooms or children? Here is a list of ideas.
Oliver Kim

Safety issues for Microscopists
Safety first! Do you know which microorganisms can be found in your sample?
Oliver Kim

Some Lichen Terminology
An understanding of basic mycological terminology is necessary to fully understand the anatomy of fungi and lichens.
Oliver Kim

Microscopic observations of Lichens
Lichens are fungi that live symbiotically with a photosynthetic partner. Who would have guessed that lichens can can be parasitically infected by other fungi as well?
Mike Guwak

Image Gallery

Diatom Cities
If your freshwater field microscopy agenda “turns sour” – then it’s time to make some lemonade of the microscopy hike!
Charles E. Guevara

Download PDF: Microbehunter (September 2011) (878)

Paper version: Order printed version

Welcome to the 9th issue of MicrobeHunter magazine. You can download the PDF and order a printed version.

At this point I would also like to encourage all readers to participate by writing articles (short and long). Read the submissions page for more information.

If you encounter any errors or mistakes, please inform me, I will correct these and upload a new version.

Contents

The Novitiate’s Odyssey Part 7
Episode Seven: Bumbling and Stumbling into Phase Contrast, a Tour of the Laboval 4 and Other Assorted Ramblings
G. Joseph Wilhelm

Of Life in the Water Fountain
You do not need a pond in order to observe pond microorganisms. Sometimes a forgotten water fountain will do as well.
Oliver Kim

The World of Sponge Spicules
Sponge spicules make up the skeleton of sponges. They are useful for sponge classification.
Oliver Kim

Female detectors
Moth antenna in close-up
Anthony W. Thomas

Seed Shrimps (Crustacea: Ostracoda)
Aquatic ‘seeds’ that swim.
Anthony W. Thomas

A Microscope Projection Screen
Projection screens allow for a more relaxed viewing, but they also have some disadvantages.
Oliver Kim

Download PDF: Microbehunter (August 2011) (962)

Paper version: Order printed version

Welcome to the 8th issue of MicrobeHunter magazine. You can download the PDF and order a printed version.

At this point I would also like to encourage all readers to participate by writing articles (short and long). Read the submissions page for more information.

If you encounter any errors or mistakes, please inform me, I will correct these and upload a new version.

Contents

How not to see double: Collimating the older Spencer Model 20 Series stereomicroscopes
In this episode, the author describes the repairing of a stereo microscope.
G. Joseph Wilhelm

Image Gallery
Pictures by David B Richman, Alan Partridge,and Rodney Brightwell

A Huge Protozoan: Monocystis – an Earthworm Parasite
Stole it from a bird! Examining Monocystis parasites from a worm.
Anthony Thomas

Utilizing Offset Rheinberg Illumination
Using Rheinberg Filters achieve a pseudo-DIC effect
Ken Wrench

Making Patch Stop Filters for Oblique Illumination
Patch stop filters made of cardboard
Oliver Kim

Of Furs and Fibers
A short photo journey through the world of fibers that can be found in a household.
Oliver Kim

Giant Chromosomes in Drosophila spp.
In this article, the author explains how to obtain and prepare giant chromosomes from a fruit fly
César Guazzaroni

Making a Microscopy Bench for Summer Stream Microscopy
Sometimes it is more convenient to take the microscope out into nature, than to take nature into the lab.
Charles E. Guevara

1. Air bubbles that are too large may not be recognized as air bubbles.
2. Air bubbles attract ciliates and other organisms, as they are a source of oxygen. These organisms can sometimes be seen to aggregate around the air bubbles. If algae or other photosynthetic organisms are present (which produce oxygen), then the ciliates may aggregate around them instead.
3. Air bubbles can limit the movement of ciliates. This can be seen as an advantage or disadvantage.

Bubbles can be more problematic if the specimen is thin and flat (such as larger microtome sections or onion skin). In this case it is possible that bubbles are caught below the specimen and it can be more difficult to remove these bubbles. Hydrophobic, furry specimens (insect hair etc.) also catch a lot of air and water may not be able to reach all parts of the specimen. In this case it may be easier to use a hydrophobic mounting medium (Euparal etc.) or to add a small amount of detergent to the water to break the surface tension. The detergent may also remove some oil from the surface of the insect hair, which makes the surface repel water.

This post was written in response to a reader’s question.

Ethyl alcohol has several effects on the cells:

• It removes water, it dehydrates the cells. This is important when mounting the cells in non-aqueous mounting medium.
• It denatures proteins. This way the metabolism of the cell is stopped and the cell dies. The metabolism is dependent on enzymes, which are proteins.
• It dissolves and removes lipids. The cell membrane(s) of the bacteria is harmed by the alcohol.

Bacteria are, however, generally fixed in a different way and not by using alcohol. The bacteria are streaked on the slide, dried and then heat-fixed. Heat-fixing sticks the bacterial cells to the glass slide so that they can not be washed away during the subsequent staining process.

Read the following for further information on heat-fixing: Heat-fixing and staining human cheek cells

This post is in response to a reader’s question.

A wet mount is probably one of the most universal ways of preparing a slide. The organism should still meet these criteria:

• The organism must be sufficiently thin. Otherwise it is not possible to place it between slide and cover glass. Water fleas and other similar creatures can be viewed by placing a spacer beneath the cover glass.
• The organism should have a refractive index which is different from that of the mounting medium (i.e. water). Bacteria have a similar refractive index and are therefore difficult to see, unless one uses DIC or phase contrast methods.
• The organism should have a color but should not be opaque. It should allow light to pass through. Otherwise one is only able to see a dark shadow.
• The organism’s natural habitat should be compatible with the mounting medium. For example, if you attempt to view salt water organisms, then it may not be a good idea to use fresh water for making the wet mount. This may damage the organism.

This post is in response to a reader’s question.

]]> http://www.microbehunter.com/2011/07/24/what-organisms-can-be-viewed-using-a-wet-mount/feed/ 0 http://www.microbehunter.com/2011/07/17/how-to-prepare-squash-specimen-samples-for-microscopic-observation/ http://www.microbehunter.com/2011/07/17/how-to-prepare-squash-specimen-samples-for-microscopic-observation/#comments Sun, 17 Jul 2011 07:51:52 +0000 Oliver http://www.microbehunter.com/?p=3402 Specimens have to be sufficiently thin and transparent to be viewed under the microscope. One can use a microtome to thinly section the material. These samples have to be sufficiently solid to be easily cut. Soft samples can not be easily cut, and must be dehydrated first in alcohol, which hardens them. There is also another possibility to prepare specimens. It is also possible to squash specimens between the coverslip and the slide.
1. Place a drop of water on the slide and then a small piece of the specimen into the water.
2. Carefully position the specimen in the center and place the cover glass on top, as if making a regular wet mount.
3. Using a soft round object, such as an eraser, carefully press down on the coverslip without horizontal movement, which would introduce shearing forces. This is the testing stage, to check if the specimen is sufficiently soft. The cover glass may break otherwise. This is, why you should use an eraser, to protect yourself.
4. If the sample is sufficiently soft, you can press down with more force. The sample should form a thin, almost transparent film between coverslip and slide. Again, do not introduce a horizontal movement.
5. Excess water should be soaked up with tissue paper. If some of the specimen starts to appear from beneath the cover glass, then you used too much specimen.
6. You can use any objective to observe under the microscope.

Samples that are too solid need to be softened first. Some plant material can be made softer by boiling, but this may not be enough to soften the cellulose of the cell walls. The cellulose of the cell walls can be made softer by heating with an acid, such as diluted HCl or acetate (careful, dangerous). Rinse the specimen after acid treatment with water and compress it.

Staining the samples should take place before squashing the specimen, as it is otherwise difficult for the stain to reach the cells.

Suitable specimens include soft fruits and fungi. Squashing may introduce artifacts. The cells are separated from each other and it is not possible to see the original place of the cells. If you want to see the arrangement of cells, as they occur naturally, then you need to resort to microtoming. The advantage of squashing is, that it is a fast and easy method to obtain very thin specimen samples.

]]> http://www.microbehunter.com/2011/07/17/how-to-prepare-squash-specimen-samples-for-microscopic-observation/feed/ 0 http://www.microbehunter.com/2011/07/11/microbehunter-magazine-july-2011/ http://www.microbehunter.com/2011/07/11/microbehunter-magazine-july-2011/#comments Mon, 11 Jul 2011 20:21:19 +0000 Oliver http://www.microbehunter.com/?p=3383  
 
 
]]>
Volume 1, Number 7, July 2011

Download PDF: Microbehunter (July 2011) (855)

Paper version: Order printed version

Welcome to the seventh issue of MicrobeHunter magazine. You can download the PDF and order a printed version.

At this point I would also like to encourage all readers to participate by writing articles (short and long). Read the submissions page for more information.

If you encounter any errors or mistakes, please inform me, I will correct these and upload a new version.

 

 

Contents

What’s in your bath?
Shallow concrete bowls which are used by birds for drinking and bathing initially start out clear, then go green, and then red.
Anthony W. Thomas

A depth-adjustable wet mount
Please do not squash me! How to prevent the coverslip from crushing the specimen.
Anthony W. Thomas

Mental forays into education, microscopy related safety and a challenge
Joseph Wilhelm continues his musings and thoughts about microscopy. In this episode he focuses on education and safety.
G. Joseph Wilhelm

Gallery of Micrographs
Floie Barrows, Oliver Kim

DIY Fluorescence Illumination System
Several blue LEDs and a highlighting marker – Enough for some simple fluorescence work.
César Guazzaroni

Observing Bacteria
Observing unstained bacteria requires a phase contrast microscope. What can you expect to see?
Oliver Kim

Using GIMP to Measure Distances
It is possible to use the free program GIMP to do measurements on micrographs.
Oliver Kim

Rediscovering the Art of Slide Wrapping
Many antique microscope slides were wrapped in decorative paper. Here is an attempt to rediscover this art.
Oliver Kim

]]> http://www.microbehunter.com/2011/07/11/microbehunter-magazine-july-2011/feed/ 4 http://www.microbehunter.com/2011/06/25/dandelion-pollen-in-dark-field/ http://www.microbehunter.com/2011/06/25/dandelion-pollen-in-dark-field/#comments Sat, 25 Jun 2011 06:24:39 +0000 Oliver http://www.microbehunter.com/?p=3191

Stacked image of dandelion pollen in dark field

The pollen grains from a dandelion (Taraxacum sp.) were collected and air-mounted (no liquid mounting medium used). Eleven separate images were stacked together to increase the depth of field and to produce the final image. The color contrast was then adjusted. Dark-field patch stop was used.

]]> http://www.microbehunter.com/2011/06/25/dandelion-pollen-in-dark-field/feed/ 5 http://www.microbehunter.com/2011/06/08/microscopic-observation-of-ehec/ http://www.microbehunter.com/2011/06/08/microscopic-observation-of-ehec/#comments Wed, 08 Jun 2011 15:08:34 +0000 Oliver http://www.microbehunter.com/?p=3341

Electron microscopic image of E. coli bacteria (10000x). Image credit: Eric Erbe, digital colorization by Christopher Pooley, both of USDA, ARS, EMU.

I do not know to what extent the news is also dominating in other parts of the world, but here in Europe the last few weeks saw one of the worst bacterial outbreaks since decades. The media, naturally, was (and still is) full with reports on this epidemic. The EHEC (enterohemorrhagic Escherichia coli) bacterium is a new E. coli strain which is not only highly infective, but can also cause a range of serious, life threatening conditions, including bloody diarrhea, kidney failure and neurological disorders. The bacterium is contracted over food, at least this is the suspected mode of transmission. Most infections occurred in Germany.

The general public scare resulted in a dramatic decrease in the purchase and consumption of fresh vegetables, even in those parts of Europe that were not affected by the outbreak. Consumers were (and are) simply afraid and this had a dramatic impact on the sales statistics of vegetables.

Microscopic observation of EHEC?

The public scare, emerging conspiracy theories (is the epidemic a biological warfare experiment?), as well as sometimes contradictory information (first Spanish cucumbers were the source of infection, later this was taken back), may motivate some people to take things into their own hands. Is it possible to test vegetables and other food, as well as one owns stool for the presence of EHEC, using microscopic observations? I have already read such questions in microscopy related forums and I think that this issue needs some clarification. After all, this question of microscopically testing food and body fluids for the presence of pathogenic (ie. disease causing) bacteria is not only limited to EHEC but was also asked in connection to Clostridium difficile and other bacteria. It is a question that reappears periodically and there seems to be a need for an explanation.

Here, for once, the answer is quite easy: It is not possible to use microscopic observations for identifying bacteria. I know that this may sound ironic, because, historically, microscopes were those devices that gave the field of microbiology and bacteriology a great push forward. In the following, I would like to outline a few points why it is not possible to use microscopes for testing for the presence of pathogenic bacteria.

Morphology

The shape of bacteria, their morphology, does not provide information on the danger of the organism. Bacteria are disease causing, if they possess so called “virulence factors” (this term has nothing to do with viruses). The virulence factors can either be toxins that are released, or structures on the surface of the cells that allow the bacteria to adhere to surfaces, such as the wall of the human intestine. These virulence factors can not be observed microscopically. Two bacteria that have exactly the same morphology, one can be pathogenic, the other one not, depending on whether they possess these factors or not. For diagnostic purposes morphology is pretty much irrelevant. To use an analogy: Just because two cars have the same shape does not mean that both of them are safe to drive, one of them could have a safety problem, which can not be seen from the outside. And just because your own car is a blue Ford does not mean that all blue cars are Fords. Just like morphology, the color of a car says nothing about its internal characteristics and model.

Density

The bacterial density on food is too low for microscopic observation. In the case of EHEC, eating about only one hundred bacterial cells are enough to cause an infection. If these bacteria are distributed over the whole food, then which part of the food should be microscoped? The chances are pretty good that there are thousands of other, non pathogenic, bacteria present as well, so how do you want to distinguish them? Generally, the bacteria have to be enriched by placing the suspected food sample into a growth medium, where they reproduce. The bacteria have to be cultured first, before they can be analyzed. Now this is something that I would definitely not recommend to do for safety reasons. It is even illegal for non authorized people to do this, after all, one does enrich potentially pathogenic organisms. Even if an enriched culture of bacteria is available, the problem would be the same as the problem mentioned above. The shape of a bacterium says nothing about its danger and its kind.

Testing for presence of bacteria

Some people may simply be interested to test for the presence of bacteria on their food, to assess whether it is safe for consumption or not. They simply want to check if “something is there”, disregarding if it is EHEC or not. If bacteria are present (regardless of the kind), then they would play it safe and not consume the food at all. No fancy tests would be needed in this case, and enrichment is also not necessary. Take a cotton swab, collect some bacteria from the surface of the vegetable, transfer them to a slide and observe them under the microscope. This should work? Or not?

There several false assumptions made. First, they assume that food is generally free of bacteria, which is not the case. The vast majority of them are not pathogenic, however. Bacteria are omnipresent and there are even more bacterial cells growing on and in our human body than we have body cells. They simply can not be avoided and are part of out natural environment. Unless one sterilizes the food, bacteria are certainly to be found.

The second false assumption is that often people think that most bacteria are pathogenic. Bacteria have a negative connotation, and are linked, in public perception, to the “three Ds”: dirt, disease and dismay. Bacteria truly need a lobby, and more positive PR, I may remark. Generally finding bacteria on food says nothing about the quality of the food product. It’s the type of bacteria that matter, and this, we know, can not be determined microscopically.

There is also a third false assumption. If one is not able to see bacteria under the microscope, it does not mean that none are present. Bacteria require more advanced optics (phase contrast) and a certain amount of skill to distinguish them from non-bacterial objects. They can be quite small as well. And the density of the bacteria can be quite low.

What do the labs then do? They selectively enrich the bacteria and analyze a range of biochemical, immunological and genetic parameters. At the end they make a nice false color a 3d image of EHEC using a scanning electron microscope so that they have something nice to show to the media. Pictures from a light microscope often do not look spectacular enough. Still, these pictures are not used for diagnosis or identification purposes.

What about Fluorescence Microscopy?

Now, it is possible to use labeled antibodies and mark the bacterial antigens to test for EHEC and other pathogens. This is a more advanced procedure and in this case it is not the bacterial morphology that is used for identification, but rather the ability of the antibody to interact with structures on the bacterial surface. I just wanted to briefly include this point for the sake of completion.

In summary

Many words, but simple message. One can observe bacteria microscopically as much as one wants, but one will not be able to assess their danger this way. Microscopic observation has no diagnostic relevance. One will not even be able to say if two bacteria of the same shape are genetically related or not. Without culturing the bacteria (don’t do this), chances are pretty good that the bacterial density is not even high enough for you to see anything under the microscope, unless your food sample was spoiled. But in this case you don’t even need a microscope to know that.

Questions? Comments?

There is a comment form below!

]]> http://www.microbehunter.com/2011/06/08/microscopic-observation-of-ehec/feed/ 2 http://www.microbehunter.com/2011/06/04/microbehunter-magazine-june-2011/ http://www.microbehunter.com/2011/06/04/microbehunter-magazine-june-2011/#comments Sat, 04 Jun 2011 20:27:59 +0000 Oliver http://www.microbehunter.com/?p=3308  
 
 
]]>
Volume 1, Number 6, June 2011

Download PDF: Microbehunter (June 2011) (912)

Paper version: Order printed version

Welcome to the 6th issue of MicrobeHunter magazine. You can download the PDF and order a printed version.

At this point I would also like to encourage all readers to participate by writing articles (short and long). Read the submissions page for more information.

If you encounter any errors or mistakes, please inform me, I will correct these and upload a new version.

 

 


Contents

The Ethics of Medical Homicide and Mutilation
In this episode, Joseph Wilhelm continues to build a Zeiss GFL compound and Spencer stereo microscope.
G. Joseph Wilhelm

Dissecting the Larva of a Beetle
Sometimes things are not as they appear. In this case we have a look at a suspected parasitic infection of a larvae, which turned out to be something quite different.
Rodney Brightwell

Making a Microtome
Here we have a look at the construction of a microtome by modifying a micrometer screw gauge.
César Guazzaroni

Making a no-nonsense Slide Box. Or two. Plain and Simple.
Why buy a slide box when it is so easy to make one yourself? A little corrugated cardboard and wood will do the trick.
Oliver Kim

Measuring Distances in Micrographs
The free program GIMP provides tools that can be used to determine the pixel distance between two points. This information can then be used to calibrate the system to be able to measure the size of structures in micrometers.
Oliver Kim

]]> http://www.microbehunter.com/2011/06/04/microbehunter-magazine-june-2011/feed/ 1 http://www.microbehunter.com/2011/05/21/muscle-fibers-in-the-tongue-of-a-rabbit/ http://www.microbehunter.com/2011/05/21/muscle-fibers-in-the-tongue-of-a-rabbit/#comments Sat, 21 May 2011 07:57:09 +0000 Oliver http://www.microbehunter.com/?p=3208  
]]>



The zoomable image shows the cross-section through the tongue of a rabbit. The muscle fibers are stained red-brown. The structures at the top of the image (bordering the white background) are the taste buds. The striations (light and dark bands) are not visible due to the lack of resolution.

]]> http://www.microbehunter.com/2011/05/21/muscle-fibers-in-the-tongue-of-a-rabbit/feed/ 0 http://www.microbehunter.com/2011/05/09/unpacking-the-leica-bm-e-microscope/ http://www.microbehunter.com/2011/05/09/unpacking-the-leica-bm-e-microscope/#comments Mon, 09 May 2011 19:05:02 +0000 Oliver http://www.microbehunter.com/?p=3268

Here we’re having a look at the Leica BM E educational microscope. This model is equipped with a 4x, 10x and 40x objective, with the possibility of adding a further objective. You can read more information about the BM E at the Leica Website.

]]> http://www.microbehunter.com/2011/05/09/unpacking-the-leica-bm-e-microscope/feed/ 9 http://www.microbehunter.com/2011/05/01/microbehunter-magazine-may-2011/ http://www.microbehunter.com/2011/05/01/microbehunter-magazine-may-2011/#comments Sun, 01 May 2011 11:09:48 +0000 Oliver http://www.microbehunter.com/?p=3240  
 
 
]]>
Volume 1, Number 5, May 2011

Download PDF: Microbehunter (May 2011) (820)

Paper version: Order printed version

Welcome to the fifth issue of MicrobeHunter magazine. You can download the PDF and order a printed version.

At this point I would also like to encourage all readers to participate by writing articles (short and long). Read the submissions page for more information.

If you encounter any errors or mistakes, please inform me, I will correct these and upload a new version.

Contents

The Novitiate’s Odyssey Episode 4: Mingling with organized humanity, specimen gathering and other forms of microscopy Zen
G. Joseph Wilhelm

Cherry Blossom – the Anther

Observ. II. Of the Edge of a Razor
Robert Hooke

Edge of a Razor – revisited

Some advice for making good micrographs

Making Zoomable Images with Zoomify Express

Step-By-Step guide to adding a remote control to a webcam for vibrationless photography
Yogeshkumar T. Patel

Buying Microscopes for Kids

]]> http://www.microbehunter.com/2011/05/01/microbehunter-magazine-may-2011/feed/ 0 http://www.microbehunter.com/2011/04/30/pumpkin-stem/ http://www.microbehunter.com/2011/04/30/pumpkin-stem/#comments Sat, 30 Apr 2011 10:00:48 +0000 Oliver http://www.microbehunter.com/?p=3204



The image shows a cross section through the stem of a pumpkin. Pumpkins belong to the dicotyledonous plants. The circular arrangement of the vascular tissue indicates this. Fifty five individual images were stitched together to make this zoomable image. The final size is about 100 megapixels (10000×10000 pixels). Happy exploring!

]]> http://www.microbehunter.com/2011/04/30/pumpkin-stem/feed/ 0 http://www.microbehunter.com/2011/04/23/artery-and-vein-cross-section/ http://www.microbehunter.com/2011/04/23/artery-and-vein-cross-section/#comments Sat, 23 Apr 2011 10:00:14 +0000 Oliver http://www.microbehunter.com/?p=3175



Use the horizontal slider to zoom into the image. On the left you can see a collapsed vein (the structure with the shape of a “2″), the oval structure on the right bottom is an artery. You can clearly see that the wall of the artery is substantially thicker. This is necessary to withstand the higher blood pressure.

]]> http://www.microbehunter.com/2011/04/23/artery-and-vein-cross-section/feed/ 0 http://www.microbehunter.com/2011/04/16/blood-cells-of-a-frog-and-of-a-human/ http://www.microbehunter.com/2011/04/16/blood-cells-of-a-frog-and-of-a-human/#comments Sat, 16 Apr 2011 10:00:09 +0000 Oliver http://www.microbehunter.com/?p=3129

Red blood cells of a frog at 400x. The darkly stained nuclei are visible.



Red blood cells of a human at 400x. Nuclei are not present.

The red blood cells of amphibians contain contain a nucleus, which is visible as a dark purple dot in the center of each cell. In contrast, the red blood cells of mammals do not possess a nucleus. The two pictures show both types of blood photographed with a 40x achromatic objective and a 2.5x photo projection ocular.

Further Reading

• Red blood cells of amphibinas in phase contrast
]]> http://www.microbehunter.com/2011/04/16/blood-cells-of-a-frog-and-of-a-human/feed/ 0 http://www.microbehunter.com/2011/04/11/starch-grains-of-a-potato/ http://www.microbehunter.com/2011/04/11/starch-grains-of-a-potato/#comments Mon, 11 Apr 2011 20:23:38 +0000 Oliver http://www.microbehunter.com/?p=3145

Potato starch grains (pink)

The pink oval structures are potato starch grains. One of the easiest and fastest way to observe starch grains is to scratch some sample off the surface of a potato with a sharp object and then adding some diluted iodine solution.

Further Reading

• Starch grains of a Potato
]]> http://www.microbehunter.com/2011/04/11/starch-grains-of-a-potato/feed/ 0 http://www.microbehunter.com/2011/04/09/epidermis-of-a-tulip-showing-stomates/ http://www.microbehunter.com/2011/04/09/epidermis-of-a-tulip-showing-stomates/#comments Sat, 09 Apr 2011 10:41:34 +0000 Oliver http://www.microbehunter.com/?p=3109

The stomates are interspersed in the lower epidermis of the leaf.


The nuclei are stained red, Chloroplasts are visible in the guard cells.

Stomates (or stomas) are openings on the underside of a leaf, which allow gases to pass in and out of the leaf. The pictures show these stomates as narrow gaps. Two guard cells control the size of the opening. The guard cells have chloroplasts, which are visible as dark dots in the cell. During the day the stomates open to allow carbon dioxide gas to enter the leaf. During night the stomates close to minimize the loss of water vapor from the leaf.

Further reading

• Wikipedia link
• Illustration of a stomate
]]> http://www.microbehunter.com/2011/04/09/epidermis-of-a-tulip-showing-stomates/feed/ 1 http://www.microbehunter.com/2011/04/01/microbehunter-magazine-april-2011/ http://www.microbehunter.com/2011/04/01/microbehunter-magazine-april-2011/#comments Fri, 01 Apr 2011 19:14:48 +0000 Oliver http://www.microbehunter.com/?p=3087  
 
 
]]>
Volume 1, Number 4, April 2011

Download PDF: Microbehunter (April 2011) (923)

Paper version: Order printed version

Welcome to the fourth issue of MicrobeHunter magazine. You can download the PDF and order a printed version.

At this point I would also like to encourage all readers to participate by writing articles (short and long). Read the submissions page for more information.

If you encounter any errors or mistakes, please inform me, I will correct these and upload a new version.

Contents

A Simple Centrifuge for Microscopists
Commercial centrifuges can be expensive, why not try to make your own?
Robert F. Hancock

The Novitiate’s Odyssey: Episode Three
Experiences and lessons of restoring antique microscopes.
Joseph G. Wilhelm

Introducing: The Hemocytometer
Haemocytometers are specialized specimen slides that are designed to quantify the cell density.
Oliver Kim

A Laser Refractometer
It is possible to make one yourself to measure the refractive index of different mounting media.
Robert F. Hancock

Photomicrography Course in Mexico
From Mexico, sharing our love of microscopy to young scientist aspirants.
Víctor Rafael Zárate-Ramírez, M.Sc.

Parasitoid wasps: A Photomicrographic Survey of their Life Cycle in the Laboratory
In this contribution we describe the life cycle of Aphidius colemani, a parasitoid wasp.
Víctor Rafael Zárate-Ramírez et al.

]]> http://www.microbehunter.com/2011/04/01/microbehunter-magazine-april-2011/feed/ 0 http://www.microbehunter.com/2011/02/23/microbehunter-magazine-march-2011/ http://www.microbehunter.com/2011/02/23/microbehunter-magazine-march-2011/#comments Wed, 23 Feb 2011 20:58:23 +0000 Oliver http://www.microbehunter.com/?p=3066  
 
 
]]>


Volume 1, Number 3, March 2011

Download PDF: Microbehunter (March 2011) (1383)

Paper version: Order printed version

Welcome to the third issue of MicrobeHunter magazine. You can download the PDF and order a printed version.

At this point I would also like to encourage all readers to participate by writing articles (short and long). Read the submissions page for more information.

If you encounter any errors or mistakes, please inform me, I will correct these and upload a new version.

Contents

Firewood Winter Microscopy: A Photo Essay
This photo essay documents sample extraction and microscopy of fungi found in fire wood.

The Novitiate’s Odyssey. Episode Two: Willie in Wonderland
Joseph Wilhelm shares experiences and lessons of restoring antique microscopes.

My Fascination with Microscopy
Michael Gibson introduces his microscopy equipment and presents some of his pictures

Do-it-yourself Microscope LED Illuminator
Don’t throw out your broken microscope just yet! It may be a good opportunity to try out LEDs.

A Review of some useful Books and Internet Sites on Diatoms
Are you interested in diatoms, but do not know where to start your research? Here is a collection of books and Websites.

Robert Hooke’s Micrographia: Of a Louse
An excerpt of Micrographia from Project Gutenberg

Predators in the micro world
Observing an attack of the ciliate Litonotus.

Staining Onion Cell Nuclei
A staining method using regular fountain pen ink.

Infinity vs. DIN Optics: a primer
In recent years many large microscope manufacturers moved away from the traditional DIN standard to so-called “infinity corrected” optics.

]]> http://www.microbehunter.com/2011/02/23/microbehunter-magazine-march-2011/feed/ 0 http://www.microbehunter.com/2011/01/21/microbehunter-magazine-february-2011/ http://www.microbehunter.com/2011/01/21/microbehunter-magazine-february-2011/#comments Fri, 21 Jan 2011 21:30:39 +0000 Oliver http://www.microbehunter.com/?p=3039  
 
 
]]>



Volume 1, Number 2, February 2011

Download PDF: Microbehunter (February 2011) (1505)

Paper version: Order printed version

Welcome to the second issue of MicrobeHunter magazine. You can download the PDF and order a printed version.

At this point I would also like to encourage all readers to participate by writing articles (short and long). Read the submissions page for more information.

If you encounter any errors or mistakes, please inform me, I will correct these and upload a new version.

]]> http://www.microbehunter.com/2011/01/21/microbehunter-magazine-february-2011/feed/ 1 http://www.microbehunter.com/2011/01/09/safe-sources-of-microorganisms-for-microscopy/ http://www.microbehunter.com/2011/01/09/safe-sources-of-microorganisms-for-microscopy/#comments Sun, 09 Jan 2011 12:19:42 +0000 Oliver http://www.microbehunter.com/?p=2964 On several occasions I’ve heard that people want to grow bacteria and other microorganisms so that they have something to observe under the microscope. I generally do not think that it is a good idea for novices to grow bacteria in petri dishes, for safety considerations. There are even laws that regulate this. Of course, one could start to grow ciliates by making a hay infusion (read: Making a Hay Infusion), but it may not even be necessary to go that far. A simple check of the refrigerator (or the super market) provides many safe sources for microorganisms to view. In any case, you should be always using fresh food. Breathing in the spores of molds (of rotten food) can cause an allergic reaction.

Yeast

You can either use fresh (wet) yeast or dried yeast. In either case, take a small amount and dissolve in a little bit of water, until the liquid becomes turbid. Use this suspension for microscopy. (read The hemocytometer (counting chamber) to sell how yeast cells look like in a counting chamber).

Yogurt

One of the more difficult specimens. Yogurt contains many bacteria, these are a bit difficult to see with bright-field microscopy. You can stain them (read ). Take a small sample (knife-tip) and dissolve in water. Then apply a drop to the slide, apply a cover glass, and observe under the microscope.

Cheese

Here you have to take the right kind of cheese. The toast-cheese (the one where each one is wrapped separately in plastic foil) won’t work. They don’t have any fungus growing on them (Do not let it rot, you may be growing poisonous fungi). I’m a cheese lover and I consider Camembert, Brie, Gorgonzola blue cheese not only good for eating but also a valuable source for the fungi Penicilium.

Honey

Some of them contain pollen. If the honey is turbid (opaque) then this may be due to sugar crystals or due to pollen. Clear honey won’t work.

]]> http://www.microbehunter.com/2011/01/09/safe-sources-of-microorganisms-for-microscopy/feed/ 0 http://www.microbehunter.com/2011/01/05/heat-fixing-and-staining-human-cheek-cells/ http://www.microbehunter.com/2011/01/05/heat-fixing-and-staining-human-cheek-cells/#comments Wed, 05 Jan 2011 12:50:07 +0000 Oliver http://www.microbehunter.com/?p=2796 Observing human cells is a good introductory activity to learn heat-fixing and staining. I will not waste many introductory words here. Here is the method:

Heat fixing

Heat fixing essentially “bakes” the cells to the glass slide much like a fried egg sticking to a frying pan. Heat fixing is absolutely essential before staining. Otherwise the staining procedure will wash away the cells.

• Take a cotton swab and rub the inside of your cheeks to collect epithelium cells.
• Smear these cells on a microscopy slide
• Completely air-dry the slide, without applying heat. This should not take long because there is not much liquid on the slide anyway. If you heat the slide before it is completely dry, then you end up “boiling apart” the cells. The vapor pressure inside the cells will burst them…
• Heat fix the dried slide by quickly pulling it through a Bunsen burner (2x), but in a way that the cells do not touch the flame. Pull it through the flame with the cells on top and the flame below. The slide should be pretty hot but you should still be capable of holding it in the palm of your hand without burning yourself. You should just be capable of holding the slide. Too high a temperature and you destroy the cells on the slide (and on your skin!). Too low a temperature and the cells will not stick to the glass slide.

Staining

Apply a drop of the stain (eg. methylene blue) to the heat fixed but cold specimen slide. Allow the stain to work for a few minutes and then carefully rinse away the stain with water. Do not apply the water stream to the cells directly. Allow the water to run over the cells from the top of the slide. Air dry the slide and observe in the microscope.

]]> http://www.microbehunter.com/2011/01/05/heat-fixing-and-staining-human-cheek-cells/feed/ 1 http://www.microbehunter.com/2011/01/03/cell-division-mitosis-in-the-apical-meristem-of-onion-root-tips/ http://www.microbehunter.com/2011/01/03/cell-division-mitosis-in-the-apical-meristem-of-onion-root-tips/#comments Mon, 03 Jan 2011 13:24:46 +0000 Oliver http://www.microbehunter.com/?p=2955


The apical meristem is the quickly growing part of the roots and shoots of plants. It is an ideal place to observe many dividing cells. The zoom image shows two onion root tips. Try to find the different stages of mitosis, in which the chromosomes are then clearly visible. Compare the cell division stages of the onion with those in the lily: Mitosis stages of the Lily

]]> http://www.microbehunter.com/2011/01/03/cell-division-mitosis-in-the-apical-meristem-of-onion-root-tips/feed/ 1 http://www.microbehunter.com/2011/01/01/artistic-applications-of-stereo-microscopes/ http://www.microbehunter.com/2011/01/01/artistic-applications-of-stereo-microscopes/#comments Sat, 01 Jan 2011 08:39:54 +0000 Oliver http://www.microbehunter.com/?p=2946 First of all: a Happy New Year 2011!
I’d like to start the new year by sharing a video that I found – a purely artistic application of stereo microscopes. Willard Wigan is an artist who creates microscopic sculptures.
]]> http://www.microbehunter.com/2011/01/01/artistic-applications-of-stereo-microscopes/feed/ 2 http://www.microbehunter.com/2010/12/30/500-magazine-downloads-in-3-weeks/ http://www.microbehunter.com/2010/12/30/500-magazine-downloads-in-3-weeks/#comments Thu, 30 Dec 2010 13:58:09 +0000 Oliver http://www.microbehunter.com/?p=2936 It’s time to write another short editorial and to summarize some of the trends of the past 3 weeks.

The Magazine

Today we celebrate the download of the 500th MicrobeHunter magazine, and I expect the download frequency to go up with every issue.

Currently I’m working on the next issue (for Feb. 2011), which will be released around January 20th, 2011. At this point, I would also like to thank those people who have sent in contributions. It is the active participation of the microscopy community which makes a magazine like this interesting and diverse (articles that I receive later than middle of January, I’ll include in the March issue – Read the submissions page for more info.)

Facebook page

Followers of the MicrobeHunter Facebook page will also have noticed that I’m now posting a microscopy-related link every day. I encourage everyone to post comments to the Facebook wall as well. If you want to receive and automatic status update, then click on the “like” button.

There is another newly established Microscopy Facebook page, that you may be interested in.

The Forum

As of now, there are 19 registered members in the forum, with a total of 82 posts. After a slow start, the forum is now also starting to take off, and I hope that it becomes a platform for interesting conversations.

The Website

I now added a microscope supplies page to the main menu. It contains a list of microscopy-related products that can be purchased over Amazon.com.

Guest Bloggers

If there is anyone interested also in contributing guest blogs, then please contact me. The blog and the magazine should be a platform for microscopy-fans of all levels to exchange their experience and enthusiasm.

]]> http://www.microbehunter.com/2010/12/30/500-magazine-downloads-in-3-weeks/feed/ 0 http://www.microbehunter.com/2010/12/27/a-digital-reticle-micrographs-with-an-iphone/ http://www.microbehunter.com/2010/12/27/a-digital-reticle-micrographs-with-an-iphone/#comments Mon, 27 Dec 2010 11:50:53 +0000 Oliver http://www.microbehunter.com/?p=2848 I found the following YouTube video, which illustrates the use of an iPhone applet for the taking of micrographs. I have tested this applet and plan a review article about it in the next issue of MicrobeHunter magazine (which will be released around Jan. 20th 2011).
]]> http://www.microbehunter.com/2010/12/27/a-digital-reticle-micrographs-with-an-iphone/feed/ 0 http://www.microbehunter.com/2010/12/17/how-many-cells-are-there-in-the-human-body/ http://www.microbehunter.com/2010/12/17/how-many-cells-are-there-in-the-human-body/#comments Fri, 17 Dec 2010 20:00:40 +0000 Oliver http://www.microbehunter.com/?p=2797 “How many cells are there in a 9-year old tree, in a flower and in an elephant?” – I was asked this question recently by an elementary school teacher, and I, as a biologist, should naturally know this answer. The students found out, by research, that the adult human body contains an estimated 10 trillion cells. Fascinated by this number, they asked the teacher on the number of cells of all sorts of organisms.

Estimating the number of cells should, mathematically, not be too difficult: We assume that an average eukaryotic cell is about 10 micro meters across. Further, we assume that a human cell is a cube. We calculate the volume, and then assume that the density of the cell is about like the density of water. This way we can compute the mass of a cell. You then simply weigh the organism, and multiply this mass by the number of cells in one kg, and voila: you have the number of cells in the body.

• Diameter of a cell: 10 micro meters (microns)
• Volume of a cell: 10x10x10 cubic microns = 1000 cubic microns
• If there are a billion (10⁹) cubic microns in a cubic mm, then this means that there are a million cells in a cubic mm.
• Consequently, there are a million million (10¹²) cells in a cubic decimeter (1dm³ = one liter). This happens to be one trillion cells in one liter of volume.
• We assume that 1 liter is about 1kg, assuming the density of water. There are therefore 10¹² cells in one kg.
• If we assume that the person has a mass of 80kg, then we obtain: 80×10¹² cells, this is 80 trillion cells.

This is a more than the estimated 10 trillion, but considering the wide range of cell sizes, I think it’s still an acceptable answer… But then again, what does a number like this really mean to 8 year-olds? What does 10 trillion mean to me? We simply lack every day experience with numbers of this scale.

Back to the original question: “How many cells are there in a 9-year old tree, in a flower and in an elephant?” The elephant one is easy to answer, if one knows the mass of an elephant. An elephant masses 7.5 tons (7500 kg), and is therefore 100 times heavier than a person. We therefore assume that it also contains 100 times more cells. If a human has 10 trillion cells (depending on the estimate), then this would account for 1000 trillion cells in an elephant.This is one quadrillion cells (10¹⁵). Maybe the kids are more interested in the names of these numbers than in the actual cell count…

But don’t forget that there are more prokaryotes (bacteria) growing on your body and in the digestive system than we have body cells. After all, they are about 1000x smaller in volume. Now things are really starting to become interesting. Just my 2 cents.

]]> http://www.microbehunter.com/2010/12/17/how-many-cells-are-there-in-the-human-body/feed/ 0 http://www.microbehunter.com/2010/12/09/microbehunter-magazine-january-2011/ http://www.microbehunter.com/2010/12/09/microbehunter-magazine-january-2011/#comments Thu, 09 Dec 2010 21:06:44 +0000 Oliver http://www.microbehunter.com/?p=2684
Welcome to the first issue of MicrobeHunter magazine. You can also order a printed version! At this point I would also like to encourage all readers to participate by writing articles.
 
 
 
]]>


Volume 1, Number 1, January 2011

Download PDF: Microbehunter (January 2011) (1831)
Paper version: Order printed version

Welcome to the first issue of MicrobeHunter magazine. You can download the PDF and order a printed version.

At this point I would also like to encourage all readers to participate by writing articles (short and long). Read the submissions page for more information.

The February issue will be released around Jan 20, 2011.

]]> http://www.microbehunter.com/2010/12/09/microbehunter-magazine-january-2011/feed/ 11 http://www.microbehunter.com/2010/10/30/some-humor-microscopy-cartoons-and-comics/ http://www.microbehunter.com/2010/10/30/some-humor-microscopy-cartoons-and-comics/#comments Sat, 30 Oct 2010 12:07:48 +0000 Oliver http://www.microbehunter.com/?p=2621 Today something different! I found some cartoons and comics relating to microscopy. Here are the links:
Cartoons 1 | Cartoons 2

]]> http://www.microbehunter.com/2010/10/30/some-humor-microscopy-cartoons-and-comics/feed/ 0 http://www.microbehunter.com/2010/10/20/setting-up-a-home-laboratory-for-microscopy/ http://www.microbehunter.com/2010/10/20/setting-up-a-home-laboratory-for-microscopy/#comments Wed, 20 Oct 2010 06:32:18 +0000 Oliver http://www.microbehunter.com/?p=2574 Why a home lab?

For someone who wants to observe ready-made permanent slides or an occasional pond water sample, a fully equipped home laboratory may not be necessary and somewhat of an overkill. In this case it is sufficient to find a reasonably dust-free place to store and operate the microscope. The microscope can then be unpacked as required. For someone wants to prepare slides, perform microtoming and staining procedures, the issue may be somewhat different and space as well as equipment requirements are higher. As so often the case, it depends very much on the type of work that needs to be done.

The advantages of a dedicated lab can be summarized in a few points:

• Safe working environment – You need to protect family members, furniture and your own health from the chemicals that you use.
• Convenience and comfort – A dedicated work place does not require you to pack and unpack the chemicals and equipment that you use.
• Equipment safety – Microscopes should not be moved around too much – there is the danger that you drop them on your toes. This may hurt your microscope…
• Specimen quality – A proper work place makes it easier to produce (nearly) dust-free specimens. There is also less hassle.
• Fun – It’s simply more fun to work in an environment which has been designed accordingly. After all, it’s a hobby.

Be cautious about growing bacteria

There are several legal issues that you must be aware of if you intend to furnish a “wet” laboratory for microbiological work. If you want to grow (unidentified) bacteria in Petri dishes and culture medium, then you are already working in an elevated Biohazard Level 2 (out of 4 levels). You simply do not know if you are growing a pathogen or not. Even Level 1 laboratories must adhere to certain safety standards and decontamination procedures. Level 2 is even more stringent.

Now, what does this mean for the amateur microscopist? The answer is: do not enrich and grow unidentified bacteria. Even the enrichment and growth of bacteria that belong to the lowest Biohazard Level (level 1), such as E. coli and B. subtilis, may not be permitted, because a home is (legally) not considered a laboratory. And how do you want to obtain these known microorganisms? Cell culture collections such as the DSMZ (Deutsche Sammlung für Mikroorganismen und Zellkulturen) in Germany or the ATCC (American Type Culture Collection) may not even send samples to private individuals. Microbiological work may be prohibited even in school laboratories, because they do not possess the appropriate license to conduct microbiological work. They generally also do not possess the appropriate equipment in order to conduct safe work. The legal situation may differ from country to country, naturally, but I would not take the risk. Proper microbiological work also requires you to use a gas Bunsen burner, an additional hazard source.

As a side note: properly observing bacteria requires you to use a phase contrast microscope, something that not all amateur microscopists have available. Personally I also think that there are more interesting samples to observe than bacteria.

Microorganisms to observe

The amateur microscopist should not despair, there are many safe microorganisms, including bacteria that can be observed. My advice: go for microorganisms that can be found growing on fresh food:

• Joghurt - This is a good source of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus.
• Cheese - Roquefort cheese, including other blue cheeses, can serve as a source for molds. Camembert cheese is a source for the moulds Penicillium candidum and Penicillium camemberti.
• Pond water samples and water from a home aquarium - These are good sources for a wide variety of ciliates, water fleas and algae. What about safety? Can you take a swim in the pond? Be aware that keeping pond water samples for extended periods of time in a jar may result in the water to turn foul. Unfriendly microorganisms may start to grow and I would be more cautious.
• Yeast - Also safe. Can be grown in a petri dish.

The requirements of setting up a microscopy work place

• Place for the microscope - The scope should have its own place and ideally it should not be necessary to pack and unpack the instrument. The table should be extremely stable to minimize vibrations. It should be easily cleanable with water to remove dust. There should be drawers for storing microscopic tools, slides and mounting media.
• Place for chemicals - You need a safe place to store the chemicals. You must be able to lock away the substances to protect them from kids. The place should also allow for containment and easy cleaning, in case there are spills. I once dropped a small bottle of iodine solution on our wood floor. The top layer of the wood floor had to be polished away because the solution ate its way into the wood, staining it red.
• The “WAF” - This one is often overlooked: the “Woman Acceptance Factor”. I once got into trouble because I wanted to store fly maggots and earth worms for dissection in the kitchen refrigerator. I did not even dare to ask if it is OK to modify the living room to accommodate a work bench for the microscope. The living room cupboards are also taboo for chemicals, also due to safety considerations.
• Dust-free environment - Often a difficult thing to achieve. Electronic equipment likes to attract dust due to static electricity. This dust can be quite interesting to observe under the microscope, but in most cases it is a serious nuisance, greatly decreasing the quality of microscopic images.
• A place for storing water samples - Pond water samples should not be stored in direct sunlight. This may cause overheating and (if there are few algae in the sample) a reduction in oxygen. The water can turn foul.
• Running water and sink - This is needed for cleaning the equipment and for disposing (permitted) solutions. Note, that some wastes must be collected and disposed separately.
• Work bench - You need some space for staining and preparing the slides. Some stains can be very aggressive and will irreversibly stain wood and other organic materials. Make sure that the work bench is easily cleanable.
• Ventilation - You need fresh air if you work with volatile solvents such as alcohol.

Equipment of a microbiology lab

Some amateurs (or teachers) may be interested in growing safe microorganisms such as yeast. It still needs to be mentioned that contaminations of the culture medium can be a health hazard. For people who want to equip a wet lab, the following equipment is necessary. You may also want to read the post: What accessories should be bought?.

• An autoclave - This is a pressure cooker. Used for sterilizing equipment and nutrient media. It is also used to kill off microorganisms on petri dishes before they are discarded.
• An incubator - This device allows for the control of the temperature. Petridishes with microorganisms can be placed into the incubator. This one is not always necessary. If the room temperature is too low, microorganisms may simply take longer to grow.
• Flowing water and a sink - Used for cleaning and washing. This one is pretty self-explanatory.
• Gas - The gas flame is used for sterilization and to minimize the risk of contamination when making the agar plates. It is also used to heat-fix the microorganisms on the slide.
• A shaker - This one is only needed if one intends to grow microorganisms in liquid medium. The shaking ensures that the liquid medium is supplied with oxygen from the air.
• Inoculation loop - For picking up colonies of microorganisms
• Nutrient media and agar - They supply the food to the microorganisms. The agar is used to solidify the medium.
• Petridishes - It contains the agar nutrient media.
• Parafilm - For sealing off the petri dishes.
• Various stains and reagents - These are used for fixing and staining the specimens.
• Miscellaneous - Materials such as gloves, alcohol for disinfection etc. are also needed
]]> http://www.microbehunter.com/2010/10/20/setting-up-a-home-laboratory-for-microscopy/feed/ 1 http://www.microbehunter.com/2010/10/10/phase-contrast-vs-bright-field-microscopy/ http://www.microbehunter.com/2010/10/10/phase-contrast-vs-bright-field-microscopy/#comments Sun, 10 Oct 2010 10:00:32 +0000 Oliver http://www.microbehunter.com/?p=2482


An amplitude specimen decreases the intensity (i.e. the amplitude) of the light. Phase specimens cause a phase shift of the light. This phase shift can not be detected with the unaided eye and requires a phase contrast microscope.

Air is completely transparent, I hope you agree. And water is transparent. If this is indeed the case, then why is it possible to see air bubbles in water? The answer is, that the bubbles have a different refractive index than the surrounding medium, the water. Phase contrast microscopy is now capable of converting a difference in refractive index into a difference in brightness. The optics of the phase contrast microscope would make objects appear brighter or darker (depending on the optics used), thereby increasing their color contrast with the surrounding mounting medium.

Phase Specimens and Amplitude Specimens

Specimens that do not possess much color but a different refractive index that the surrounding mounting medium can be referred to as phase specimens as they cause a phase-shift in of the light. The unaided human eye is not capable of detecting this phase shift. This phase shift is then converted into a brightness difference by the optics of the phase-contrast microscope. Bacteria are a good example here. They are nearly completely transparent but nevertheless appear darker or brighter (depending on the optics) than the background.

Amplitude specimens possess a color and are able to decrease the brightness of the passing light all on their own. These specimens are best observed using bright-field microscopy. Pigmented structures (such as chloroplasts) and specimens that are selectively stained are examples.

Many specimens are a combination of these two. Here the choice of the right kind of microscope is important in order to see that what one wants to see. Phase contrast microscopes will optically darken certain structures to the extent that it is not possible to see the natural color of the structure. In this case it is probably better to use bright field microscopy. Stained bacteria, for example, should be observed in bright field.

Advantages and disadvantages of bright field and phase contrast microscopy

Advantages of bright-field microscopy:

• The optics do not change the color of the observed structures. Sometimes stains are used to make certain structures visible. The optics of a bright field microscope do not change these colors.
• Bright-field optics is generally cheaper than phase contrast optics
• Bright-field microscopy requires fewer adjustments before one is able to observe the specimens.

Advantages of phase contrast microscopy:

• It is possible to visualize certain structures that are otherwise invisible. This includes certain cell organelles which can not be seen well in bright field.
• Sometimes the phase contrast image subjectively looks better than a bright field image due to the details visible.

To see pictures of phase contrast specimens, read this post: Bacteria in phase contrast

]]> http://www.microbehunter.com/2010/10/10/phase-contrast-vs-bright-field-microscopy/feed/ 2 http://www.microbehunter.com/2010/10/03/some-thoughts-on-recreational-amateur-microscopy-part-2/ http://www.microbehunter.com/2010/10/03/some-thoughts-on-recreational-amateur-microscopy-part-2/#comments Sun, 03 Oct 2010 06:31:45 +0000 Oliver http://www.microbehunter.com/?p=2450 Today, I’d like to continue my thoughts on microscopy as a hobby. I tried to brainstorm a list of strengths and opportunities as well as areas of improvements. In a previous article I already mentioned that (in my personal view), recreational microscopy as not as well established as other recreational sciences. In particular, I compared amateur microscopy with amateur astronomy, which seems to be enjoy a much stronger foundation. In this post I want to explore some of the strengths and weaknesses of microscopy as a hobby (and encourage anyone to comment).

Strengths and opportunities of microscopy

• School labs: Many biology labs of schools already possess microscopes. How many schools, in comparison, possess telescopes? Microscopes are more accessible to students. The question is now what should be done to foster and retain the interest of the students?
• Comparatively low cost: Reasonable microscopes can be bought for a quite reasonable price. The financial entry barrier into recreational microscopy is not high.
• Location, weather and time independence: There is no need for a clear sky and microscopic specimens can be observed around the clock.
• Many samples: A nearly unlimited number of samples that can be observed. Specimen preparation can be very simple ranging to quite complex. This offers many opportunities for the hobbyist.
• Photography: The observations can be documented using cameras and shared over the Internet. While photography can also be done in astronomy, the equipment costs and experience required can be much higher.

Areas of improvement

• Lack of awareness of stereo microscopes: Many beginning microscopists will think of compound microscopes when they think of a microscope. Stereo microscopes pose an even lower entry-barrier, especially for children. Stereo microscopes are often cheaper and elaborate sample preparation is not necessary.
• Problems of discoveries: An amateur astronomer who discovers a new comet (or other astronomical event) will receive credit for this discovery. Microscopy alone is rarely sufficient to justify the new discovery of a species. Genetic and biochemical tests are also necessary and this is often outside the scope of an amateur. For this reason, I think that amateur microscopy somewhat lacks competitiveness. Many hobbies are supported by the fact that people are able to “build up” something, collect awards and are able to participate in competitions. While this competitive aspect may not be in everyone’s interest, I think that competitiveness can still carry forward and support a hobby.
• Possible negative associations: Microscopy may be negatively associated with germs and pathogens. Microscopes may have the “hospital taste” attached to them.
• Amateur microscope making and technical tinkering: There are not many possibilities to “tune” a microscope. Microscopy is therefore mostly an observing activity, of using a ready-made technical device. Flying model airplanes, for example, contains both aspects the technical construction and then the flying of the model. Also amateur telescope making is able to combine both aspects. In microscopy it is possible to prepare specimens, but this activity is largely non-technical and routine.
• Toxic chemicals: many substances used for specimen preparation are toxic, expensive, or sold only to qualified laboratories. There is a need for safe microscopic methods.

What (Biology) teachers need

• Student-proof methods: Many specimen preparation techniques use methods and chemicals that are not suitable for classroom use. The reagents may be toxic, the methods too complex or time-consuming, or they may require sophisticated equipment. How should a teacher teach a class of 20+ students to use use a microtome, if there are only 1 or 2 of these available? What about the associated dangers? Additionally, some methods may require substantial experience and trial-and-error until a satisfactory specimen is obtained for observation. This time is often simply not available in schools. Student motivation may also be at risk, if a certain preparatory step has to be repeated several times until a satisfactory result is obtained. Being a teacher myself, I found it easiest to work with ready made permanent slides.
• Observation and project ideas: Teachers need straight-forward observation ideas. One reason why the microscopy of onion cells (and onion cell plasmolysis) is so popular in schools is, that the preparation is simple, relatively safe and can be completed and observed within one class period. Teachers need more observation ideas.
• Integration into the curriculum: Practical microscopy work must/should fit into the Biology curriculum. What specimens should/could be observed for the curriculum topic digestion? For the topic nervous system? Some commercial permanent slide sets for schools already contain specimens from a variety of different sources, so that it becomes easier to find appropriate specimens for the different curriculum topics.

What advanced recreational microscopists need

• Validation methods and integration into mainstream research science: An amateur astronomer, who discovers an asteroid can easily validate the discovery against a database and then receives credit for the discovery. What should an amateur microscopist do with his or her observations? Microscopic images are rarely enough to justify the discovery of a new species Amateur astonomers can hunt for supernovae in distant galaxies and search for near-earth asteroids. In my opinion (please correct me), amateur microscopists do not seem to be integrated into mainstream research science to the same extent. A possible reason could be that modem biological research does not rely as much on the more qualitative observations as it once used to. There was a shift towards molecular and biochemical analyses in the bio sciences.
• Competitiveness: To some extent this already exists in micrograph competitions. While competitions are not something for everyone, some amateur microscopists may still be motivated by matching their skills with others. At this point, I do want to recommend Nikon Small World, which is a step into the right direction.

What beginning recreational microscopists need

• Information: Buying a new microscope is not easy, if one does not know what to look out for. Beginners need accessible and non-technical information. Regrettably there are not many amateur microscopy magazines around that contain advertisements for suitable microscopes or other general information for beginners.
• Source of reagents: Stains and other chemicals may need to be obtained from chemical supply companies. These companies often do not target amateurs and may refuse to send these substances to private individuals.
• Networks and clubs: In many areas it seems to be easier to find an astronomy club compared to microscopy clubs. I wonder why considering the fact that a decent telescope can cost substantially more than a microscope.

  Do you have any suggestions? Write a comment!

  ]]> http://www.microbehunter.com/2010/10/03/some-thoughts-on-recreational-amateur-microscopy-part-2/feed/ 2 http://www.microbehunter.com/2010/09/26/introducing-the-microscopy-forum/ http://www.microbehunter.com/2010/09/26/introducing-the-microscopy-forum/#comments Sun, 26 Sep 2010 10:00:26 +0000 Oliver http://www.microbehunter.com/?p=2570 I’ve now installed a microscopy forum which adds an extra level of organization by pre-defining certain categories. It is also not necessary to register in order to use the forum.

  While mailing list services (such as the amateur microscopy group in Yahoo Groups) is a great installment, I found it difficult to find things that I’m looking for. This is due to the organization of the mailing list. There are individual threads and as soon as the discussion of a topic is finished, the thread starts to move into the background and quickly disappears out of sight.

  It can take time until there are enough forum posts. I therefore encourage everyone to contribute to the forum, to make it an active platform for enthusiast microscopists.

  ]]> http://www.microbehunter.com/2010/09/26/introducing-the-microscopy-forum/feed/ 1 http://www.microbehunter.com/2010/09/22/using-a-hemocytometer-to-calculate-cell-size/ http://www.microbehunter.com/2010/09/22/using-a-hemocytometer-to-calculate-cell-size/#comments Wed, 22 Sep 2010 10:00:01 +0000 Oliver http://www.microbehunter.com/?p=2538 I already illustrated how to calculate cell size (Determining Size in Microscopic Images). The method required you to take a picture of a ruler and then use this as a reference for cell size calculation. This system had several disadvantages: first, it only works for low magnifications (you have to be able to see 1mm of the ruler on the image), and was generally rather imprecise.

  I would now like to show you a much better method of determining the size of microscopic structures. You do need a hemocytometer (counting chamber), however. These specialized slides are designed to determine the concentration of cells but they can also be used to determine size. The disadvantage is, that hemocytometers do cost quite a bit more than regular slides. There are different types of hemocytometers around, it does not matter which one you use, as long as you know the real-life size of the engraved squares.

  In this case, we use the side length of one of the squares of the hemocytometer as a reference. These lines are very fine and therefore permit you to make very precise measurements and size calculations (in comparison to the picture of a ruler, see above link). The math is easy, but be careful that you use the same units.

  • Step 1: Take a picture of a square of the hemocytomer with associated cells. There are squares of different sizes, so make sure that you know the dimensions of the square that you are looking at. Read this post for more information on the different square sizes of the Neubauer improved haemocytomerter: The hemocytometer (counting chamber). Make sure that one complete side length of a square is visible.
  • Step 2: Print the micrograph. The square of the hemocytometer is out internal reference. You do not have to worry about the size of the print out. The larger the print out, the more precise the result, however.
  • Step 3: Measure the length of the side of one square and the diameter of a cell. Use the same units (generally mm is appropriate).
  • Step 4: Calculate the real-life size of a cell: You know the real-life side length of a square and the length of the square on the print out. You also know the diameter of the magnified cell. This data is enough for you to calculate the real-life size of the cell.
  • Step 5: Divide the length of a square of the print-out with the real-life side length. This gives you the magnification on paper. This magnification has nothing to do with the magnification of the objective and eye piece. We’re talking about magnification on the paper.
  • Step 6: Divide the size of the cell on paper with the magnification to obtain the real-life cell size. If you mix units, (cm, mm), then you won’t get the right result. You need to convert to the same units first. E.g. do not divide the square size in cm with the real life size in mm.

  Things to watch out for:

  • Do be careful when observing cells that have a vastly different refractive index to the surrounding. In this case the cells will appear to have a thick “wall” around them, which is actually nothing more than a diffraction pattern. This may make obscure the true size of the cell. Open the condenser aperture diaphragm to minimize this artifact.
  • Counting chambers have squares of different sizes. Read the manual first so that you know the true size of the square that you are looking at.
  ]]> http://www.microbehunter.com/2010/09/22/using-a-hemocytometer-to-calculate-cell-size/feed/ 0 http://www.microbehunter.com/2010/09/19/online-virtual-microscopes/ http://www.microbehunter.com/2010/09/19/online-virtual-microscopes/#comments Sun, 19 Sep 2010 10:00:06 +0000 Oliver http://www.microbehunter.com/?p=2549 I’d like to give you a quick evaluation of some online virtual microscopes and microscope simulations that I found. Write a comment, if I overlooked something and if you have further recommendations.

  Online Digital Microscope
  This virtual microscope allows the user to choose from a variety of plant, animal and microbe specimens. It is not a comprehensive simulation of a microscope, but still useful for educational purposes because of the description of the different specimens.
  I liked: The descriptions of the specimens were very complete (including staining information etc.). There are numerous specimens to look at and to explore.
  I did not like: In order to switch to a higher magnification, it is necessary to click into a small rectangle. It is therefore possible to only magnify pre-selected areas.
  Use it for: giving an overview of different staining techniques and for showing the differences in cell shapes.

  Bob Ketcham’s Virtual Microscope
  This interactive site is a true simulation of a microscope. The users even have to switch the micoscope “on” in order to see something. An instruction (with audio!) is included as well. This is a true instructional interactive site which allows students to experiment with the microscope. Here is the project homepage.
  I liked: The instructional audio is great. It is a full simulation of the microscope. If the slide is not centered, you won’t be able to see anything. There is much attention to detail.
  I did not like: There are only 4 specimens to choose from. For this reason there is not much for students to explore (the focus of the site is more on operation of the microscope).
  Use it for: instructing students on how to use a compound microscope.

  Kbears Virtual Microscope
  The Kbears Virtual Microscope is a site for younger students (elementary school). It’s not a true microscope simulation, but rather a collection of 11 microscopic images.
  I liked: Children-friendly design, short explanations of the specimens.
  I did not like: low resolution of some images, no possibility to zoom in and to magnify.
  Use it for: showing students different pictures of specimens (“What is this?” type of activity).

  Virtual Petrological Microscope
  This software simulates a petrological microscope. There are 7 rock samples to choose from. It is suitable for teaching students how to make measurements under the microscope.
  I liked: It also allows users to switch to polarized light. It’s one of the rare non-biological “microscopes”.
  I did not like: I could not figure out the function of the two circles at the bottom of the site. The documentation could be better.
  Use it for: Showing students how to make measurements and magnification calculations.

  Virtual Cell Staining Tool
  Here the users can see the effect of different antibody-based (immunological) stains. Users can choose the different stain types and colors.
  I liked: The different parts of a cell can be visualized very well.
  I did not like: It does not say which cell it is. There are many different stains to choose from, but there is no visual difference for some of these stains.
  Use it for: showing students how different cell organelles and structures look using fluorescent stains.

  MicrobeHunter Virtual Microscope
  Ha! And you thought I’ve forgotten! And because I’m fair, I’ll also critically evaluate my own virtual microscopy project.
  I liked: The large images. There is plenty to explore. It’s also possible to seamlessly zoom into the specimens.
  I did not like: No information concerning magnification is given. There is no reference point concerning magnification.
  Use it for: Enjoyment and entertainment and to marvel about the beauty of nature! Why does science always have to be educational and “serious”?

  ]]> http://www.microbehunter.com/2010/09/19/online-virtual-microscopes/feed/ 1 http://www.microbehunter.com/2010/09/15/answering-reader-questions-2/ http://www.microbehunter.com/2010/09/15/answering-reader-questions-2/#comments Wed, 15 Sep 2010 10:00:26 +0000 Oliver http://www.microbehunter.com/?p=2470 Why is refractive index of mounting media important? The refractive index is important for several reasons. First, it influences the resolution of the image. Second, if the refractive index of the specimen is too similar to the refractive index of the mounting medium, then it may be difficult to see the specimen if it is not stained. Phase contrast microscopy relies on a different refractive index between medium and specimen. If the refractive index is too different (eg. mounting the specimen in air), then the specimen may appear to be too dark.

  What are some differences electron microscope and light microscope concerning cost and skills required? Generally, electron microscopes require substantially more sample preparation time than light microscopes. However, this is only a generalization. Some staining techniques in light microscopy are also highly elaborate and time consuming. It depends much on the actual preparation technique used. These two types of microscopes can hardly be compared. Due to the elaborate sample preparation techniques which are required by electron microscopy, the chances are much higher to introduce artifacts. It requires much skill in identifying these. Compound light microscopes are sufficiently simple to be used in schools and allow for a fast observation of specimens.

  What are some disadvantages of permanent slides? Permanent slides contain specimens that are fixed, dehydrated and possibly also microtomed (sliced into thin sections). The organisms are therefore not moving. Over time the specimen may also start to lose color. Generally, permanent slides require much more elaborate preparation. The advantage is, however, that once prepared the slide can be used over and over again and can be stored for longer time periods.

  Why is a mounting medium important? The mounting medium physically supports the specimen, conserves it, and provides the correct refractive index in order to see the relevant details.

  Why is it important to apply a coverslip (cover glass) at a 45 degree angle when making a wet mount? This reduces the possibility of air-bubble formation. It does not have to be exactly 45 degrees. The point is, that one should not drop the cover glass horizontally on the water droplet on the slide. By lowering the cover glass at an angle, the water slowly replaces the air from one side. Read the following post (and video) on how to correctly make a wet mount: Making a wet mount microscope slide

  Why are unstained bacteria difficult to see? They are difficult to see in bright-field microscopes, because they are small, transparent and lack color. Beginners also have problems distinguishing bacteria from dust and debris. Phase contrast microscopes are much better for viewing of unstained bacteria.

  Why is water added when mounting tissue onto a microscope slide? The water is the mounting medium. It supports the specimen, and most importantly, improves the resolution of the image by providing the correct refractive index. Try it out yourself. Look at some dry specimens (insect wings, pollen) with and without water. Read the following post for more information and pictures: The effect of the mounting medium on specimen and image quality

  ]]> http://www.microbehunter.com/2010/09/15/answering-reader-questions-2/feed/ 0 http://www.microbehunter.com/2010/09/12/some-thoughts-on-recreational-amateur-microscopy/ http://www.microbehunter.com/2010/09/12/some-thoughts-on-recreational-amateur-microscopy/#comments Sun, 12 Sep 2010 10:00:26 +0000 Oliver http://www.microbehunter.com/?p=1519 Amatuer Microscopy and Amateur Astronomy

  I don’t know how many of you still remember comet Hale-Bopp, which became visible back in 1996-97. The comet was the incentive for me to become involved in amateur astronomy. The Internet, at that time, was still young and for this reason I obtained most of the info about the comet from astronomy magazines that I bought at the magazine store located in the local train station. I remember, that there were about 5-7 different astronomy magazines (in several languages) in the shop. The range was quite impressive: Some magazines were quite advanced and scientific. Others were much more down-to-earth, targeting readers interested in a more casual and popular approach. I scrutinized them in detail, and found one which had the right mix between science and hands-on advice and one which was just right for beginners. I subscribed to the magazine, looked at the ads, and mail-ordered a small telescope. I became a star-gazer. Together with two other friends, I spent many hours in the cold night looking at the stars and trying to identify them with the help of a star chart. We had a great time. And I remember that I was even crazy enough to keep a diary of my observations.

  Just one year later, in 1998, I also got involved with amateur microscopy and spent a little fortune on a compound microscope. This was not the first time for me to use microscopes, I did quite a bit of bright-field and phase-contrast work during my university studies, mostly in the field of bacteriology. At that time I used different staining techniques to characterize the bacteria that we isolated, to help us in the identification process. At the end of my studies, I decided to continue microscopy as a hobby and to broaden my microscopic endeavors into exploring my surroundings. I obtained water samples, looked at the algae, purified diatoms, photographed the crystallization of Vitamin C and citric acid. I also made video recordings of these events and had some nice educational material. But this is a different story.

  I quickly realized that the life of a recreational microscopist resembles the life of a “lone wolf”. Microscopy magazines with a recreational approach are scarce, at least I could find none of them in the magazine shop. Many science journals from the university library did address a range of microscopy-related issues, but the topics were far too specific and not understandable to someone without a scientific background in the particular research area. In many cases the topics revolved around more advanced electron microscopic techniques – interesting, for certain, provided that you understood the science behind it. Very few magazines had the “amateur-approach” of astronomy magazines. In particular, nice colorful pictures were missing.

  Naturally I do understand that there is a fundamental difference between true science journals and “recreational” magazines, which can be bought in a magazine shop. A notable exception is the German microscopy magazine Mikrokosmos, which tries to place a stronger focus on the “observation” aspect. The online magazine Micscape also goes into this direction. Just a pity that they don’t also offer a nicely formatted PDF version (I’d even be willing to pay for one, if it’s not too expensive).

  Now why is this? Why is there such an imbalance between amateur microscopy and astronomy? Does it have historic reasons? Maybe the entry barrier into amateur astronomy is higher (equipment costs, knowledge required etc.) and therefore the support by astronomy clubs and organizations may be more important than for microscopy. Maybe these organizations helped to promote and support amateur astronomy over many years and there is a stronger tradition and community. Or could it be, that there are indeed many amateur microscopists around, but that they are simply not organized into clubs and therefore not visible to the public? But even if this were the case, there should be many amatuer microscopy magazines around, which is not the case.

  Maybe microscopes simply do not offer as many possibilities for the technical tinkerer as telescopes. Amateur telescope making seems to be so popular that this activity even has its own acronym: “ATM”. Try to Google “amateur microscope making”, and you will be awarded with an astonishing 2 hits (yes this is two). The search string “amateur telescope making” gives you 30900 hits (August 2010). OK, maybe this comparison is a bit unfair, considering the fact that quite usable microscopes can already be obtained for a comparatively low price, but the discrepancy is startling nevertheless. Still, I recommend this link here, it’s pretty good http://www.funsci.com/fun3_en/ucomp1/ucomp1.htm

  In any case, I’ll continue to publish my thoughts on this issue and encourage you to write a comment.

  ]]> http://www.microbehunter.com/2010/09/12/some-thoughts-on-recreational-amateur-microscopy/feed/ 2 http://www.microbehunter.com/2010/09/08/life-in-the-flower-pot-water-or-comparing-the-size-of-prokaryotes-and-eukaryotes/ http://www.microbehunter.com/2010/09/08/life-in-the-flower-pot-water-or-comparing-the-size-of-prokaryotes-and-eukaryotes/#comments Wed, 08 Sep 2010 10:00:43 +0000 Oliver http://www.microbehunter.com/?p=2524
  

  Large green algae (eukaryotes) and bacteria (prokaryotes).

  Over the past few years, I’ve met several students who wanted to see bacteria through the microscopes that we have at school. Unfortunately, these devices are not equipped with phase contrast optics. The transparent bacteria are therefore difficult (but not impossible) to see. The biggest draw-back, however, comes from a different source: some of the optics are a bit dirty, and the resolution and contrast therefore low. Some of the microscopes have been in operation for about 30 years, and actually I’m a bit surprised that they still work as well as they do. Regular maintenance is a must, but this is a different issue.

  I therefore found it necessary to take a picture of some bacteria using my own microscope. It too does not have phase contrast optics. I’ve taken a water sample from the dish of a flower pot. The dish collected the excess water from the pot to prevent it from creating a mess. The pot and dish has been standing in the sun for a few days and was green, a clear sign that algae and other photosynthetic organisms are thriving. I think that I do not need to mention that the water which filtered through the soil is an ideal growth medium for algae (rich in nutrients etc.). I took some of the green slime and under the microscope, I could clearly see both green algae and bacteria, nicely next to each other (see picture).

  Size of Bacteria and Algae

  Bacteria and Algae belong to two different categories. Bacteria are prokaryotes, algae are eukaryotes. Prokaryotes are generally much smaller, about 1 micrometer (1/100 mm) in diameter, while eukaryotes can have a diameter of about 10-100 micrometers. These are averages and generalizations, of course. Some prokaryotes can also grow into long filaments, it really depends much on the type of organism. The picture illustrates this difference in size quite nicely with both cell types next to each other.

  The picture should also make something else clear: Due to their small size, it is difficult to bring the bacteria into focus. They tend to either actively swim away, wiggle around or drift away as more and more water evaporates from the slide. Due to their green color, algae can also be much more easily identified. If you look closely, you can see that unlike the green algae, the bacteria lack structure on the inside and appear blurred. We are already at the limits of the microscope’s resolution. Other images show that the bacteria are brighter on the inside and darker on the outside. This is due to the refraction of light and an optical artifact. Opening the condenser aperture diaphragm would make the dark fringes disappear, but also more difficult to see the bacterial cells.

  ]]> http://www.microbehunter.com/2010/09/08/life-in-the-flower-pot-water-or-comparing-the-size-of-prokaryotes-and-eukaryotes/feed/ 0 http://www.microbehunter.com/2010/09/05/time-lapse-video-of-vitamin-c-crystallization/ http://www.microbehunter.com/2010/09/05/time-lapse-video-of-vitamin-c-crystallization/#comments Sun, 05 Sep 2010 10:00:38 +0000 Oliver http://www.microbehunter.com/?p=2547

  The video shows the crystallization of Vitamin C (dissolved in water) in time lapse, under crossed polarizing filters.

  Some technical information:

  • The frames in the video were taken with a time interval of 2 sec.
  • The colors were not adjusted at all. No contrast enhancement, no levels were corrected, no white balance.
  • The pictures were taken with a digital SLR camera at low magnification (4x objective and 2.5x photo projection ocular).
  • The original 3:2 aspect ratio image was cropped to 16:9. Software used: PHATCH
  • Exposure time: 0.5 sec or 1 sec. The relatively long exposure time was used to compensate for vibrations (this allowed the vibrations to swing out).
  ]]> http://www.microbehunter.com/2010/09/05/time-lapse-video-of-vitamin-c-crystallization/feed/ 3 http://www.microbehunter.com/2010/09/01/determining-size-in-microscopic-images/ http://www.microbehunter.com/2010/09/01/determining-size-in-microscopic-images/#comments Wed, 01 Sep 2010 10:00:26 +0000 Oliver http://www.microbehunter.com/?p=2514
  

  Measure the length of 1mm using a ruler or caliper. In this case, 1mm is magnified to 108.5mm.
  
  

  Then measure the size of the structure on paper. In this case, we look at stomates from the bottom of a leaf. The guard cells are 3.6mm long.
  
  Our Biology curriculum in school requires students to be able to calculate the size of cells and other structures from light micrographs, which have a scale bar. It’s probably more interesting for students to actually take the light micrographs themselves. It is not difficult to determine the size of cells and other structures in light micrographs, provided that one has a size standard. It is possible to take a picture of a structure of known size and use this as a basis to calculate the size of other structures. I admit that this is a somewhat improvised method, but it does work for lower magnifications.
  • Place a ruler on the stage and take a picture. A full unit (1 mm) should be visible. Transparent ruler are better, otherwise it’s not possible to see the markings. Take a digital photograph of the ruler.
  • Print the micrograph of the ruler.
  • Take a picture of the specimen. Make sure that you use the same magnification.
  • Print the picture of the specimen and be sure that the size of the picture is the same as the size of the picture of the ruler. Do not change the size of the print out.
  • Now it’s time for a little math. Use the ruler and measure out the size of the 1mm on the print out. Measure it out in mm. Let’s call this “r”.
  • Measure out the size of the structure that you want to determine. This is “s”. Make sure that you use the same units (mm)!
  • The real size of the structure in mm can be calculated as follows: size = (1mm * s) / r

  Let’s use the example in the pictures on the left:
  size = (1mm * 3.6mm) / 108.5mm
  size = 0.03mm = 30 micrometers

  ]]> http://www.microbehunter.com/2010/09/01/determining-size-in-microscopic-images/feed/ 0 http://www.microbehunter.com/2010/08/29/how-to-prevent-air-bubbles-in-wet-mounts/ http://www.microbehunter.com/2010/08/29/how-to-prevent-air-bubbles-in-wet-mounts/#comments Sun, 29 Aug 2010 10:00:31 +0000 Oliver http://www.microbehunter.com/?p=2508
  

  The air bubbles possess a different refractive index than the surrounding medium (water). This makes the bubbles appear to have a thick dark border. The shape of the bubble focuses the light in such a way that the center of the bubble appears bright.

  The statistics feature of my blogging software allows me to see what readers are searching for, and one of the questions that keeps reappearing over and over again is the question on how to prevent air bubbles in wet mounts. I have already published a video on how to correctly make a wet mount (temporary mount), but now I think it’s time to address the issue of air bubbles in more detail. Here is the video on how to make a wet mount: Making a wet mount microscope slide

  Samples that are prone to form air bubbles

  Not all specimens are the same. Some specimens can be the cause for more air bubbles than others. This depends on a variety of factors. The following characteristics may result in more bubbles:

  • Large sheet-like specimens (e.g. onion skin): These specimens may catch air bubbles underneath them and prevent them from escaping. Push out the air bubbles before adding a cover slip.
  • Specimens with many fine hair: The hair catch much air and prevent the water from reaching all the parts of the specimen. The surface tension of the water is too high, and the water therefore does not “flow” into all parts of the specimen. This is comparable to the “Lotus Effect”, where the water does not wet the surface of the lotus leaf.
  • Fatty and hydrophobic specimens: These too do not accept water well, especially if the surface area of the specimen is large (many fine hair, etc). It may help to treat the specimen in alcohol or an alcohol-water mixture to remove the fatty surface.
  • Porous specimens: The pores of the specimen may be filled with air, which can be difficult to remove. The cells of plant stems, the vascular tissue, for example, are able to hold air. It is possible to remove the air by placing the specimen into a vacuum while it is submerged in the fixing solution. Aspirators (eductor-jet pumps) can be mounted to a water tap to produce a vacuum.

  Why air bubbles should generally be avoided

  Some air bubbles are certainly tolerable and unless one wants to produce high-quality pictures it is often not worth the effort to make a completely bubble-free specimen. It is easily possible to simply move the slide and observe a different part of the specimen. Generally, air bubbles should be avoided, especially by beginning microscopists, who may have a problem distinguishing bubbles from the real specimen. The reasons why air bubbles can be problematic are:

  • Bubbles hinder the free movement of organisms, such as ciliates
  • The bubbles cause optical artifacts at the place where the air meets the water. The air bubble appears to be surrounded by a dark ring. This dark ring covers some parts of the specimen and makes observation more difficult.
  • The microscope optics are designed to give optimum resolution for a specimen which is surrounded by water. If the bubble is large and the specimen completely surrounded by air, then the resolution is lower.

  Are there cases when air bubbles are beneficial?

  Under some rare circumstances, air bubbles can even be beneficial. The bubbles can serve as a source of oxygen for some organisms, such as paramecia and other ciliates. It is possible to see them collect around the bubbles. Air bubbles are also easily viewable and can therefore help beginners to more easily find the correct focus. Naturally, the bubbles should not be confused with the actual specimen, something that beginners sometimes do because the bubbles are so prominent and can be seen even if the specimen itself is not in focus.

  How to minimize air bubbles in wet mounts

  Needless to say, the preferred method depends on the characteristics of the specimen. Try out the following:

  • Cover slip placement: Lower the cover slip on the water droplet with an angle. This permits air to escape on one side.
  • Water placement: If the specimen is not fully submerged in the water droplet, add another droplet on top of the specimen before lowering the cover slip.
  • Immersion oil: Use a medium other than water. Try immersion oil, which is hydrophobic. Some specimens prefer water, others oil.
  • Break the surface tension: Add a small amount of detergent, such as soap. This will break the surface tension of the water. The water will therefore adhere better to some specimens, thus preventing bubbles. The soap may also harm some water organisms, however.
  • Apply a vacuum: This speeds up the movement of the fixing solution or water into the specimen.
  • Dehydrate the specimen: Place the specimen into alcohol. Some specimens will shrink and lose water and air. By placing the specimen into water again, the specimen will take up the water.
  • Remove oil and fat: Wash the specimen in alcohol.
  • Add water: If the air bubble is large and reaches the side of the cover glass, you can add more water from the side of the cover glass.
  ]]> http://www.microbehunter.com/2010/08/29/how-to-prevent-air-bubbles-in-wet-mounts/feed/ 0 http://www.microbehunter.com/2010/08/26/answering-reader-questions-3/ http://www.microbehunter.com/2010/08/26/answering-reader-questions-3/#comments Thu, 26 Aug 2010 05:16:11 +0000 Oliver http://www.microbehunter.com/?p=2512 Why use phosphate buffer when making a permanent slide of cheek cells? Assuming, that the buffer refers to phosphate buffered saline, the advantages are that the pH is stable and that the solution is isotonic to the cells. The cells, therefore, do not change shape. The solution is used to make dilutions of the cells and to separate the cells from each other. The buffer contains sodium chloride, sodium phosphate. Other recipes also contain potassium chloride and potassium phosphate.

  How should you remove extra water from a wet mount slide? Remove excess water with a piece of filter paper or tissue paper. Have a look at the following video for an explanation on how to make a wet-mount slide: Making a wet mount microscope slide

  How can I make a sample stick to a microscope slide? This depends on the sample. Bacterial suspensions should be first dried on the slide and then heat-fixed. The slide should be briefly heated over a Bunsen burner. This will immobilize the cells.

  How to permanent mount algae? They are frequently mounted in a water-based mounting medium, such as glycerin gelatin.

  Is Spirogyra microscopic? The individual cells can only be seen with the microscope, the whole algae can naturally be seen with the unaided eye as thin cotton-like filaments.

  If you observed the same organism on a prepared slide and a wet mount how would the images compare? This depends on the refractive index of mounting medium. Wet mounted specimens can move. Because they were generally not fixed in alcohol, they also do not show artifacts and (possible) shrinkage.

  ]]> http://www.microbehunter.com/2010/08/26/answering-reader-questions-3/feed/ 0 http://www.microbehunter.com/2010/08/25/testing-the-hand-microtome/ http://www.microbehunter.com/2010/08/25/testing-the-hand-microtome/#comments Wed, 25 Aug 2010 10:00:38 +0000 Oliver http://www.microbehunter.com/?p=2510

  A few days ago I ordered a microtome. Here is a video showing you the different parts: Parts of a Microtome

  Now it’s time to test the device. The first sample is a carrot. It can be cut into the right shape to fit into the specimen holder of the microtome and it is sufficiently solid to allow for easy cutting, but not too hard. Carrots can also be used to hold other specimens. In this case the “carrot cylinder” is cut in half and the specimen can be inserted between the carrot halves. The carrot acts as a support.

  ]]> http://www.microbehunter.com/2010/08/25/testing-the-hand-microtome/feed/ 12 http://www.microbehunter.com/2010/08/22/euparal-mounting-medium/ http://www.microbehunter.com/2010/08/22/euparal-mounting-medium/#comments Sun, 22 Aug 2010 06:14:23 +0000 Oliver http://www.microbehunter.com/?p=2498 Euparal is a semi-synthetic mounting medium used in microscopy. It is slightly yellowish in color, flows well and cures after a few days. After curing, it becomes very hard but not brittle, keeping elasticity. Euparal also adheres strongly to glass. It has a refractive index of 1.5174.

  Compared to other mounting media, Euparal has a significant advantage: Specimens can be directly transferred from alcohol into Euparal. Other mounting media, such as Canada Balsam, require the specimen to be transferred to xylene (toxic) prior embedding.

  Euparal was first described by G. Gilson (prof. of Zoology at Louvain University, Louvain, Belgium) in 1906, and is liked for its ease of use and stability.

  I wanted to find out more about this mounting medium and conducted a quick Web search, only to find out that the information is scarce. I wanted to know more about the composition of Euparal, also because of safety considerations. Many non-aqueous mounting media contain toxic organic solvents, which I try to avoid.

  After some time I was indeed able to find the original publication by G. Gilson [1], published in French. An article published in the Journal of the Royal Microscopical Society [2] summarized parts of this his and together with a translation software, I was able to extract some meaning of Gilson’s article.

  Composition of Euparal

  Gilson found out that Sandarac (or sandarach) is a suitable resin for mounting. It is obtained from Callitris quadrivalvis, a tree belonging to the cedar family. The resin has been previously used to make varnish and protective coatings for paintings. Alcohol could disolve the sandarac well, but the resulting medium was not suitavle for microscopic work. During the curing process the sandarac started to crystalize and crack.

  Gilson then mixed the sandarac with camsal, a mixture of phenyl salicylate (salol) and camphor. The camsal was not a good solvent for the sandarac, but prevented the formations of crystals and cracks. He added either isobutylic or propylic alcohol to further dissolve the sandarac. Especially isobutylic alcohol was considered suitable, as it was commonly used to dehydrate microscopic specimens. The specimens could then be directly transferred into the mounting medium (containing the same solvent).

  While this was aldready a step into the right direction, the added alcohol was a substantial disadvantage. Stained specimens could not be mounted with this medium, as the alcohol dissolved many the dyes which are commonly used in microscopy, such as eosin, safranin, methyl green were affected by the alcohol. Camsal alone was not able to sufficiently dissolve the resin.

  Gilson, therefore, searched for replacements for the alcohol. He discovered that a combination of eucalyptol and paraldehyde was able to substitute for the alcohols, without harming pigmentation. He thus gave the mixture containing sandarc, salol, EUcalyptol and PARAldehyde the name Euparal.

  • Sandarac: A resin which solidifies in air. Originally used as a varnish for furniture.
  • Paraldehyde: Preservative and solvent.
  • Eucalyptol: Solvent of Euparal. dominant portion of Eucalyptus globulus oil. eucalyptol is used as an insecticide.
  • Phenyl salicylate (salol): Antiseptic substance, was introduced in 1886 under the name Salol, a desinfectant.
  • Camphor: An antimicrobial substance, previously used for embalming. Obtained from the evergreen tree camphor laurel (Cinnamomum camphora).
  • Camsal: A mixture (1:1) of camphor and Phenyl salicylate (salol).

  Advantages of Euparal

  Towards the end of the article, Gilson lists several advantages of Euparal. These advantages are now briefly summarized:

  • It is possible to directly transfer specimens which were stored in 70% alcohol into Euparal for permanent mounting. It is not necessary to completely dehydrate the object by placing it into absolute alcohol.
  • It has a low refractive index (1.481), which can be an advantage to observe certain structures. Other publications consider this low refractive index a disadvantage, however.
  • Euparal can be colored green (“Euparal vert”) by adding some copper salt. This can further increase the contrast of specimens stained with hematoxylin.
  • Euparal possesses reducing properties and therefore prevents oxidation of some dyes (such as hematoxylin).
  • Last, Euparal possesses good fluidity, does not pull strings and handles easily.

  I now would like to mention a few advantages of Euparal:

  • Unlike other non-water-based mounting media, Euparal does not use the harmful solvent xylene
  • Euparal cures relativley quickly
  • And on the less serious side: Euparal smells nicely (but don’t inhale, nevertheless!!! Irritant and flammable!).

  Euparal does possess some disadvnatages as well:

  • Acid sensitive dyes do not keep well, when embedded in Euparal.
  • Euparal does have the tendency to shrink a bit. This can introduce air bubbles.
  • It is flammable and an irritant. Eye and skin contact must be avoided.

  References

  [1] Gilson, G. (1906). La Cellule, Vol. 23, pp. 425-432. http://www.archive.org/details/lacellule23lier
  [2] Hebb, RG., ed., (1907). Journal of the Royal Microscopical Society. p.501. http://www.archive.org/details/journalofroyalmic1907roya

  ]]> http://www.microbehunter.com/2010/08/22/euparal-mounting-medium/feed/ 5 http://www.microbehunter.com/2010/08/18/parts-of-a-microtome/ http://www.microbehunter.com/2010/08/18/parts-of-a-microtome/#comments Wed, 18 Aug 2010 10:00:19 +0000 Oliver http://www.microbehunter.com/?p=2503

  A hand microtome (or cylinder microtome) is a device used to make thin cuts of a specimen for microscopic observations. In the video I am unpacking a new hand microtome and showing the different parts:

  • The clamp: This one is optional, but very useful. It holds the microtome to a table. It adds stability and convenience, thereby making the microtome cuts more reproducible.
  • The knife: This one looks like an old fashioned razor knife.
  • The microtome: It has a central hole into which to place the specimen. A screw at the opposite end moves a piston up, which in turn pushes the specimen up. The plate of the microtome acts as a guide for the knife.
  • The mold: A small brass cylinder serves as a mold for making paraffin blocks containing the specimen. This paraffin block is then inserted into the hole of the microtome.
  ]]> http://www.microbehunter.com/2010/08/18/parts-of-a-microtome/feed/ 0 http://www.microbehunter.com/2010/08/13/making-a-wet-mount-microscope-slide/ http://www.microbehunter.com/2010/08/13/making-a-wet-mount-microscope-slide/#comments Fri, 13 Aug 2010 12:19:53 +0000 Oliver http://www.microbehunter.com/?p=2500

  What is a wet mount?

  In a wet mount, the specimen is suspended in a drop of liquid (usually water) located between slide and cover glass. The water refractive index of the water improves the image quality and also supports the specimen. In contrast to permanently mounted slides, wet mounts can not be stored over extended time periods, as the water evaporates. For this reason, a wet mount is sometimes also referred to as a “temporary mount” to contrast it from the “permanent mounts”, which can be stored over longer times. The permanently mounted slides use a solidifying mounting medium, which holds the cover glass in place. The naming can be a bit problematic, because it is also possible to make wet mounts that can store over extended time periods. These are special cases, however.

  Different types of wet mounts

  Wet mounts can be made using several different kinds of liquids. Water, immersion oil and glycerin (glycerol) can be used, with water probably being the most commonly used. The source of the water is quite important, especially when observing living specimens. If you use water with a wrong osmotic potential (ie. too much or too little salt and mineral content), then there is the danger of damaging the specimen. A too high salt content can result in the specimen to lose too much water. Too low a salt content, and the specimen may swell and burst.

  • Using water from the natural habitat of the organism: In the case of water organisms, such as algae or ciliates, the liquid water should come directly from the sample. In this case the organism is immersed in its own natural environment. The microscopist uses a dropper to place a drop of pond water directly on the microscope slide.
  • Using 0.9% salt water: In some cases water from the natural habitat may not be available. This is the case when observing bacteria or molds grown on petri-dishes. Yoghurt bacteria, for example, need to be diluted a lot before being able to observe them, otherwise they are too dense to be observed as single cells. In this case it is necessary to mix some salt (NaCl) into some water to ensure an optimal osmotic potential. This “physiological saline”, as it is called, can be made by dissolving 9 grams of table salt (NaCl) in 1 liter of water (or 0.9g Nacl in 100ml of water).
  • Using tap water: If one wants to observe non-living specimens, such as dust samples, sand grains, or thin section cuts of plant material, then it is also possible to use regular tap water. These specimens are not osmotically sensitive. If the specimen is observed without water, in a dry condition, then the resolution and image quality may not be sufficiently high. I advise you to try out both to see the difference. The following post includes images of pollen grains mounted in air and water, for comparison: The effect of the mounting medium on specimen and image quality
  • Using immersion oil: Some wet mounts are not made with water, but by using immersion oil. Immersion oil is usually placed on top of the cover glass. In this case the specimen does not get into contact with the oil. It is also possible to submerge the specimen in the oil, however. Heat-fixed bacteria can be observed directly by placing a drop of immersion oil on the specimen, without cover glass. The oil-immersion objective is then rotated directly into the oil for observation. It goes without saying, that this procedure can only be used for specimens that do not contain water (and are, therefore, not living). It also only works for specimens that stick to the glass slide – there is no cover glas. If you need to observe these specimens with a lower magnification (ie. no immersion objective), then one needs to use a cover glass, of course. Other specimens, such as synthetic textile fibers, are hydrophobic in nature, and do not like to be mixed with water. They tend to float on top of the water drop and this can be cause for air bubbles. In this case I also recommend to use immersion oil and a cover glass to keep the sample flat.
  • Pure glycerin or glycerin-water mixtures: Glycerin has a strong tendency to withdraw water from the sample. For this reason it also acts as a preservative. On the down side, the glycerin may therefore cause the specimen to shrink and deform. Especially algae and other water organisms are sensitive to dehydration. Other specimens, such as sectioned or microtomed plant material are not as sensitive. The reason why glycerin is used is because of its high refractive index. This may be necessary to see certain structures. If a lower refractive index is needed, then one should mix some water into the glycerin. It is possible to seal the glycerin mount by applying nail polish to the sides of the cover glass. This will hold the cover glass in place for longer time periods. This is then an example of a wet mount, which was made into a permanent mount.

  Advantages and disadvantage of a wet mount

  Compared to permanently mounted slides, wet mounts do have certain advantages:

  • Quick preparation: specimen fixation, dehydration and staining are not necessary (but possible, if required). For this reason, wet mounts are the first kind of mounts that students learn to make.
  • Few artifacts: If there is no chemical and physical processing of the specimens before observation (no fixation), there are little artifacts and the specimens appear in their natural condition.
  • Living and moving: It is possible to observe living and moving organisms. It is also possible to observe certain processes of life, such as feeding, cell division etc. (for water-based mounts)
  • Natural colors: The colors are natural and not faded. The colors of permanently mounted specimens may fade over time.

  Disadvantages of wet mounts include:

  • Movement: The advantage of observing movement can also be a disadvantage. Due to the movement of the organisms it may be more difficult to take pictures or to make drawings. There is a solution to this problem: one can slow down ciliates and other protozoa by adding a solution such as ProtoSlo, which increases the viscosity of the water.
  • Evaporation: The heat of the lamp causes the water to evaporate more quickly. More water must be added under the cover glass from time to time.
  • Focus: Some organisms may swim vertically in the water and therefore move in and out of focus. Here it is important not to use too much or too little water. Too little water may squeeze the specimen between cover glass and slide.
  • Storage: Wet mounts can not be stored over a longer time.

  Materials and Method

  For making a wet mount you need these materials:

  • Microscope slides
  • Cover glasses
  • The specimen to be observed: make sure that the specimen is sufficiently small and thin. Thick specimens must either be cut (microtomed) into sections, be squeezed or torn apart.
  • Water: take care that the osmotic potential of the water is compatible with the specimen. For example, do not use fresh water with marine specimens, and vice versa. Use pond water (and not tap water) for observing pond organisms.
  • Droppers, pipette: these are for transferring the water
  • Tweezers: for handling the specimen, the cover glass and for adding water

  If the specimen is already in water (algae, ciliates etc.) then you can proceed the following way:

  1. Place a small drop of sample fluid (containing the specimen) in the center of the microscope slide.
  2. Hold the cover glass on one side with the help of tweezers. Lower the cover glass onto the water drop at an angle.
  3. Then slowly lower the cover glass into the liquid. This will minimize disturbing air bubbles.
  4. Remove excess water with filter paper or tissue paper. The cover glass should not float freely. The surface tension of the water should hold it in place. Alternatively you can add more water using a pipette or tweezers.

  If the specimen is not in water:

  1. Place a small drop of water (without specimen) in the center of the microscope slide.
  2. Place the specimen into the water.
  3. Add some more water on top of the specimen and make sure that the specimen is completely submerged. Otherwise there is the possibility for air bubbles forming between cover glass and specimen. The remaining steps are the same as above.
  4. Hold the cover glass on one side with the help of tweezers. Lower the cover glass onto the water drop at an angle.
  5. Then slowly lower the cover glass into the liquid. This will minimize disturbing air bubbles.
  6. Remove excess water with filter paper or tissue paper. The cover glass should not float freely. The surface tension of the water should hold it in place. Alternatively you can add more water using a pipette or tweezers.

  If you are using a dry specimen (dust, insect parts, etc.), then place a small drop of tap water

  How to prevent drying out

  The heat of the microscope light will evaporate the water relatively quickly. There are several possibilities to counteract this:

  • Keep adding more water from the side of the cover glass. Surface tension will pull the water in.
  • Seal the sides of the cover glass with a thick layer of Vaseline (petroleum jelly). Press the cover glass against the slide so that the vaseline is able to seal off the water from the outside.
  • Use nail polish to seal off the cover glass. This is used when making wet mounts with glycerin. Keep the glycerin drop very small. The nail polish will not stick to those parts of the cover glass and slide which came into contact with the glycerin.
  • Use slides that have an indentation (concave) and are therefore able to hold more fluid. This only works for some samples because the liquid layer may be to thick. These slides are more expensive.
  • Use two additional cover glasses to support a third cover glass left and right. These two cover glasses serve as a distance holder for the third cover glass. This way the third cover glass does not float freely on the liquid but is held in place by the two supporting glasses. More fluid can be stored in a stable manner.
  ]]> http://www.microbehunter.com/2010/08/13/making-a-wet-mount-microscope-slide/feed/ 8 http://www.microbehunter.com/2010/08/05/fixing-specimens-for-making-permanent-slides/ http://www.microbehunter.com/2010/08/05/fixing-specimens-for-making-permanent-slides/#comments Thu, 05 Aug 2010 14:18:36 +0000 Oliver http://www.microbehunter.com/?p=2496 Before specimens can be processed for making permanent slides, they may need to be fixed. This step kills the specimen and preserves the structures. It also prepares the specimen for staining. There is no one single method to fix a specimen, too much depends on the nature of the specimen itself and on the subsequent preparation steps.
  

  Characteristics of a chemical fixative

  A good fixing agent should fulfill several criteria:

  • It must kill the specimen quickly: But be careful, some chemical fixing agents are toxic and are also harmful to the health of a person.
  • It must preserve the structures of the specimen, without introducing deformations or other artifacts. Insects may pull together their appendages, making them more difficult to see. The structures should then be sufficiently stable to withstand the dehydration and mounting.
  • It must enter the specimen well to react with all parts: This can be problematic with some specimens. Make sure that the specimen is sufficiently small. Alternatively it is possible to puncture the specimen (insects) so that the fixing agent can enter more easily. Some specimens may contain air bubbles which prevent the fixing agent to reach all parts. In this case it may be necessary to apply a vacuum to remove the air.

  Types of fixing agents

  Chemical fixing agents can be categorized into the following 4 groups:

  • Alcohol and acetic acid: This combination denatures proteins. The alcohol also removes some lipids. This is probably the preferred fixing agent for hobbyists, because it is less toxic than some other fixatives.
  • Aldehydes (such as formaldehyde – toxic!): these react with amino groups in the specimen.
  • Oxidation agents: these react with lipids.
  • Tanning agents: react with proteins and with amino groups.

  The choice of the fixing agent must be carefully matched with the specimen. Some fixing agents (eg. alcohol) may result in the shrinking of the specimen and therefore introduces artifacts. Sometimes it may be necessary to gradually increase the concentration of the fixing agent in order to prevent the formation of artifacts, but this depends much on the type of specimen used. I can not give general advice here, and recommend that one consults specific laboratory manuals.

  Using alcohol

  For the hobbyist who wants to prepare a slide every now and then, keeping a whole set of different chemical fixatives is probably an overkill (and not healthy either). I keep a small bottle of 96% rubbing alcohol on my shelf, into which I drop the specimens, usually small insects, as they arrive. They will store nearly indefinitely in this solution. When For making permanent slides, I directly transfer them into Euparal mounting medium.

  Pure alcohol (ethanol) is also suitable for fixing and storing plant specimens, without cell contents. The alcohol has the tendency to shrink the cytoplasm, but does not affect the cell walls. The alcohol also hardens the plant material, making it easier to cut with a microtome (which often removes the cell contents anyway).

  Alcohol/acetic acid solution

  Acetic acid (acetate) compensates the shrinking effect of the alcohol. The Carnoy Clarke solution uses 3 parts 92% rubbing alcohol mixed with one part pure acetic acid. The correct alcohol:acetate ratio should be fine-tuned experimentally. If the cytoplasm still shrinks too much, the recipe according to Farmer may be tried out (2:1 alcohol:acetate ratio). Fixing should take place for about 24 hours.

  After fixing

  There are two more steps necessary: the fixing agent has to be removed (washing) and the specimen has to be dehydrated. Several fixing agents are water-based and this water has be be removed before mounting them in a non-water based mounting medium. Dehydration is not necessary when mounting in a water-based mounting medium such as glycerin gelatin. Dehydration is commonly done by placing the specimen in successively higher concentrations of ethanol. Afterwards the specimen is transferred into a solvent which is compatible to the mounting medium. Some mounting media require the specimen to be submerged in xylene (toxic). Other mounting media are able to directly accept the specimen from the alcohol (Euparal). If one sees a clouding of the slide, then this can be an indication that there was still some water in the specimen.

  Heat-fixing of bacteria

  Bacteria are treated differently. They must not only be killed, but also physically fixed to the glass slide. Otherwise they will be washed off during the staining process. This method also works with cells collected from the inside of the cheek and water samples.

  • Place a bacterial suspension on the slide and let dry. Dry gently, dry completely but do not heat, otherwise the cells may pop open.
  • Pull the glass slide through the flame of a Bunsen burner (1-2 times). The specimen should not come into contact with the flame (specimen on top, flame on the bottom). This step is called “heat fixing”. It kills of the bacteria and binds them to the glass slide much like an egg to a frying pan. The glass slide should be so hot that you are just able to hold it in the palm of your hands without causing burns. Heat the slide too much and you end up burning the bacteria 8and destroying their structure).
  • The bacteria can now be stained. Place a drop of the staining solution on the cold slide. Rinse off with water and dry it in air. Do not dry-wipe, you will remove the fixed bacteria. You can then observe the bacteria directly in oil immersion even without a cover glass. Place the immersion oil directly on the fixed and stained bacteria.
  ]]> http://www.microbehunter.com/2010/08/05/fixing-specimens-for-making-permanent-slides/feed/ 5 http://www.microbehunter.com/2010/06/27/the-hemocytometer-counting-chamber/ http://www.microbehunter.com/2010/06/27/the-hemocytometer-counting-chamber/#comments Sun, 27 Jun 2010 08:35:24 +0000 Oliver http://www.microbehunter.com/?p=2459
  

  Counting chamber: This one is called the Neubauer improved. There are other standards with different grids available as well.

  

  Yeast cells in the hemocytometer. The grid is clearly visible.

  

  Yeast cell suspension applied to the chamber. Notice that some of the cell suspension has gone into the overflow area.

  

  One counting chambers has grids of different sizes. Consult the manual to find out the size.

  

  Do not count cells on the top and right lines. Here it's necessary to count the in the big square because there are too few cells in individual small squares.

  

  Counting chamber seen from the side.

  

  Grid layout of the Neubauer Improved hemocytometer.

  
  

  Purpose of the hemocytometer

  The hemocytometer (or haemocytometer or counting chamber) is a specimen slide which is used to determine the concentration of cells in a liquid sample. It is frequently used to determine the concentration of blood cells (hence the name “hemo-”) but also the concentration of sperm cells in a sample. The cover glass, which is placed on the sample, does not simply float on the liquid, but is held in place at a specified height (usually 0.1mm). Additionally, a grid is etched into the glass of the hemocytometer. This grid, an arrangement of squares of different sizes, allows for an easy counting of cells. This way it is possible to determine the number of cells in a specified volume.

  Preparing the sample

  The fluid containing the cells must be appropriately prepared before applying it to the hemocytometer.

  • Proper mixing: The fluid should be a homogenous suspension. Cells that stick together in clumps are difficult to count and they are not evenly distributed.
  • Appropriate concentration: The concentration of the cells should neither be too high or too low. If the concentration is too high, then the cells overlap and are difficult to count. A low concentration of only a few cells per square results in a higher statistical error and it is then necessary to count more squares (which takes time). Suspensions that have a too high concentration should be diluted 1:10, 1:100 and 1:1000. A 1:10 dilution can be made by taking 1 part of the sample and mixing it with 9 parts water (or better saline of correct concentration to prevent bursting of the cells). The dilution must later be considered when calculating the final concentration.

  Counting the cells

  • Counting cells that are on a line: Cells that are on the line of a grid require special attention. Cells that touch the top and right lines of a square should not be counted, cells on the bottom and left side should be counted.
  • Number of squares to count: The lower the concentration, the more squares should be counted. Otherwise one introduces statistical errors. How many squares? To find out one could calculate the cell concentration per ml based on the numbers obtained from 2 different squares. If the final result is very different, then this can be an indication of sampling error.

  Calculating the cell density

  Here it is necessary to do some simple math. The following numbers are needed: number of cells counted in a square, area of the square, height of the sample, dilution factor. The objective is to find the number of cells in 1ml of original solution.

  • Step 1 – Averaging: If one did not count all of the cells in a large square (1mmx1mm) then it is necessary to average the results first before proceeding. For the purpose of this example, I use an average cell count of 123.456 cells.
  • Step 2 – Computing the volume: It is necessary to determine the volume represented by the square. The width and height of the square (e.g. 0.25mm x 0.25mm) must be multiplied by the height of the sample (often printed on the hemocytometer, in this example it is 0.1mm): v = 0.25mm x 0.25mm x 0.1mm = 0.00625mm³ = 0.00625ul (where ul is microliters).
  • Step 3 – Calculating the number of cells in 1 ml: if there are 123.456 cells in 0.00625ul, then how many cells are there in 1ml (=1000ul)? We do simple direct proportion:

    123.456cells/0.00625ul = X/1000ul
    (123.456cells*1000ul)/0.00625ul = X (the ul cancel out)
    X = 19 752 960 cells

  • Step 4 – Correcting for dilution: If the sample was diluted before counting, then this must be taking into consideration as well. We assume that the sample was diluted 1:10. The final result is therefore 19 752 960 cells x 10 = 197 529 600 cells in 1 ml. That a lot of cells.

  Things to watch out for

  • Type of counting chambers: There are different types of counting chambers available, with different grid sizes. One counting chamber also has grids of different sizes. Take care that that you know the grid size and height (read the instruction manual) otherwise you’ll make calculation errors.
  • Use the provided cover glasses: They are thicker than the standard 0.15mm cover glasses. They are therefore less flexible and the surface tension of the fluid will not deform them. This way the height of the fluid is standardized.
  • Moving cells: Moving cells (such as sperm cells) are difficult to count. These cells must first be immobilized.
  • Objective The hemocytometer is much thicker than a regular slide. Be careful that you do not crash the objective into the hemocytometer when focusing.
  Disclaimer: This page is intended purely for educational purposes. Do not use this information for medical diagnosis. No guarantee is given for the correctness of the information published in this site.
  ]]> http://www.microbehunter.com/2010/06/27/the-hemocytometer-counting-chamber/feed/ 10 http://www.microbehunter.com/2010/06/19/how-to-obtain-the-best-resolution-with-your-microscope/ http://www.microbehunter.com/2010/06/19/how-to-obtain-the-best-resolution-with-your-microscope/#comments Sat, 19 Jun 2010 18:44:09 +0000 Oliver http://www.microbehunter.com/?p=2467 The resolution that a microscope is capable of achieving is probably the single most important factor that determines the quality of a microscopic image. Without a sufficiently high resolution, magnification is not possible without loss of quality. Read the following introductory post: Magnification and Resolution.

  There are a variety of different factors that determine the achievable resolution. Some of these factors can not be actively influenced by the microscopist, others can. Some of the factors play a larger role, others a smaller one. In the following post, I want to summarize some of these factors.

  Objective-related factors

  • Correction of lens errors: In contrast to achromatic objectives, apochromatic objectives focus more colors of the spectrum to one point. This results in a sharper image.
  • The numerical aperture of the objective: This value is printed on the objective. The higher the value, the higher the resolution. The numerical aperture is a dimension less value which represents the cone of light that can be caught by the objective.

  Lighting system

  • General color of light: The shorter the wavelength, the higher the resolution. If your microscope uses halogen or tungsten lamps (instead of LEDs), then the color of the light will shift towards the red end of the spectrum with increasing age. This will reduce the resolution. The color of the light also changes with its intensity. If you turn up the light to maximum intensity, then the color of the light will be more towards the blue end of the spectrum (shorter wavelength and higher resolution). LEDs do not change their color with age or brightness.
  • Light spectrum (color range): The color range may also impact on resolution. In the case of monochromatic light, chromatic aberration does not play a role and the light can be focused on one point.

  Specimen-related factors

  • The correct thickness of the cover glass: The correct cover glass thickness is extremely important for high numerical-aperture objectives. For other objectives, the effect may not be noticeable.
  • The correct refractive index of the cover glass: This is something that you do not have to worry about, this is the task of the cover glass manufacturer.
  • The correct refractive index of the mounting medium: This one should be as close to the refractive index of glass as possible.
  • Thickness of the mounting medium: the thinner the better.
  • The presence of immersion oil: Objectives that carry the label “OIL” need the correct immersion oil for best resolution.

  Adjustments of the microscope

  • The correct condenser diaphragm setting: This setting must match the numerical aperture of the microscope in use.
  • The correct setting of the correction collar: Some objectives have a correction collar (a turnable ring) to adjust to the cover glass thickness. Most objectives do not have one, however.

  Maintenance-related factors

  • The cleanness of the optical parts: Dust and dirt generally decrease image quality and are a big annoyance, especially if one uses dark-field microscopy.

  Stability of the photomicrographic system

  • Moving objects: Moving cells naturally cause a blurring when long exposure times are used. This decreases resolution of the moving object.
  • Stability: A shaky photographic system generally decreases resolution of the image.

  The checlkist: how to obtain the best image quality

  • Use new light bulbs and turn up the light. This will reduce the wavelength of the light. Alternatively, use a blue filter.
  • Use cover glasses of the correct thickness and make sure that the mounting medium has a refractive index which is close to the refractive index of glass.
  • Adjust the condenser aperture diaphragm to the numerical aperture of the objective
  • If you use oil immersion, make sure that the oil has the correct refractive index
  • Use fresh light bulbs (low in red light, high in blue light)
  • Keep the microscope free of dust
  • Make sure that the objectives, eye pieces are clean
  ]]> http://www.microbehunter.com/2010/06/19/how-to-obtain-the-best-resolution-with-your-microscope/feed/ 2 http://www.microbehunter.com/2010/06/12/cover-glass-thickness-and-resolution/ http://www.microbehunter.com/2010/06/12/cover-glass-thickness-and-resolution/#comments Sat, 12 Jun 2010 07:21:06 +0000 Oliver http://www.microbehunter.com/?p=2455 The thickness of the cover glass can have a significant impact on the resolution. The effect is highest with high-numeric aperture aperture (high magnification) objectives, and barely noticeable when using objectives of a low numeric aperture.

  Types of cover glasses

  Cover glasses come in all sorts of different sizes. I already wrote a post about cover glass size: Microscope Slides and Cover Glasses. In this post, we’ll now have a look at the importance of cover glass thicknesses. The table gives a summary of available thicknesses:
  
   

  Number	Thickness (mm)
  #0	0.08 – 0.13
  #1	0.13 – 0.16
  #1.5	0.16 – 0.19
  #2	0.19 – 0.25
  #3	0.25 – 0.35
  #4	0.43 – 0.64

  Why cover glass thickness is important

  Most microscope objectives have the optimum cover glass thickness engraved into them. For most objectives this is 0.17mm. Read the following post for more information on the engravings: About the numbers on the Objective. The correct cover glass thickness is important to achieve the best resolution with a given objective. But do not go out to buy the more expensive 0.17mm cover glasses, get the thinner and cheaper ones (will be explained below).

  Generally speaking, the higher the numeric aperture of the objective, the more serious the loss in resolution if the wrong cover glass thickness is used. For some high-aperture objectives, a cover glass thickness of only a few micrometers can significantly reduce resolution. Therefore, some more advanced objectives possess a correction collar. This is an adjustment ring which can be turned to adjust the objective to the actual cover glass thickness which is in use.

  Importance of the mounting medium

  The optimum cover glass thickness of many objectives is 0.17mm. Now, why is it that the most commonly available cover glasses are of category 1 (0.13-0.16mm), which is thinner than the calculated optimum? The answer is a bit more complex: The thickness of the cover glass is not the only parameter which is important. The specimen is embedded in mounting medium. The thickness of this medium must be added to the thickness of the cover glass. A specimen which is located deep in the medium will have a larger “effective” cover glass thickness than a specimen which is located right beneath the cover glass. A calculated (ideal) cover glass thickness 0.17mm is therefore a good compromise, even if the “real” cover glass is thinner. And yes, the refractive index of the mounting medium also plays a role.

  How to determine the thickness of a cover glass

  Cheap cover glasses which are used for uncritical routine observations will show a statistical spread of different thicknesses. There are also assorted cover glasses available that show a much more narrow spread of thicknesses. Some people buy cheap cover glasses (with a larger spread) and then manually measure their thickness using a caliper to sort them. Is it worth the effort? When using low-magnification objectives with a low numeric aperture, the difference in cover glass thickness may not even be noticeable and the more expensive pre-selected cover glasses may only be necessary for specific applications where a high resolution is necessary and the objectives do not possess a correction collar. One should not forget that the thickness and refractive index of the mounting medium also has an impact on the resolution, and mounting medium thickness may be much more difficult to standardize.

  ]]> http://www.microbehunter.com/2010/06/12/cover-glass-thickness-and-resolution/feed/ 7 http://www.microbehunter.com/2010/06/05/answering-reader-questions/ http://www.microbehunter.com/2010/06/05/answering-reader-questions/#comments Sat, 05 Jun 2010 10:00:28 +0000 Oliver http://www.microbehunter.com/?p=2452 And yet again it’s time to answer some reader questions

  What are the things that all types of microscopes have in common?
  Microscopes can be very different (see Different types of microscopes. I therefore limit the answer to light microscopes. Things that optical microscopes have in common include:
  Objectives, Oculars/eyepieces, stage (carries specimens), light source, focusing system.

  Does glycerol mounting cure?
  It will dry only up to a point, but will not (and should not) completely dry out. The glycerol will prevent the complete drying. This makes sure that a certain amount of water remains in the sample. A complete drying of the glycerol mounting medium could result in a shrinking and deforming of the specimen. Algae and other water organisms are especially sensitive to this. It is possible to protect the permanent slide by sealing the edges of the cover slip with nail polish.

  Why do electron microscopes produce black and white images?
  They produce B/W images because electrons do not have a color. Different wavelengths of light, in contrast, do possess colors that we can perceive. It is possible to artificially color electron microscopic images, however. But this does not reflect the “true” colors of the object.

  Is pollen a microbe?
  No, pollen are not considered microorganisms (microbes), because they are not capable of reproduction. Pollen do not divide to form more pollen. They form sperm cells for fertilizing the plant’s egg cell.

  Why does the smell of hay infusions decrease over time?
  As a hay infusion ages, different microorganisms start to grow (and others start to die out). Different microorganisms produce different substances which are responsible for the smell.

  Is a bacterium too small to be seen under a compound microscope?
  No, most bacterial can be seen with compound light microscopes from magnification of 400x up. If the resolution of the microscope optics is not very good, then it will be difficult to see them. You need phase contrast optics to be able to see bacterial well. They may be difficult to see using regular bright-field optics, because bacteria are transparent. Alternatively one may need to stain them. Beginners may have problems distinguishing bacteria from small specks of dirt and dust.

  Which type of microscope would be best to use if you wanted a 3-Dimensional view of a virus?
  Compound light microscope: It is not possible to see viruses with these microscopes. Resolution and magnification are not large enough.
  Transmission electron microscope (TEM): It is possible to see viruses with TEMs, but they provide 2D views.
  Scanning electron microscope (SEM): These are the ones that are able to visualize viruses in 3D

  Why is it important to apply a cover slip at a 45 degree angle when making a wet mount?
  Applying the cover slip at an angle (instead of dropping it down flat on the specimen) pushes the air to the side and therefore minimizes the risk of air bubbles.

  ]]> http://www.microbehunter.com/2010/06/05/answering-reader-questions/feed/ 2 http://www.microbehunter.com/2010/05/29/how-to-make-macro-images/ http://www.microbehunter.com/2010/05/29/how-to-make-macro-images/#comments Sat, 29 May 2010 10:00:47 +0000 Oliver http://www.microbehunter.com/?p=2443
  
  Aperture: f/19.9, Exposure time: 10sec.
  
  
  Aperture:f/22.6, Exposure time: 20sec.
  
  This time I’d like to talk about a topic which is only indirectly related to microscopy: macro imaging. Taking high-quality macro images can be quite a challenge and can involve quite a bit of trial and error until one has found the ideal conditions. The pictures of the rose have been taken with a Sigma 70-300mm DG APO objective and a Canon EOS 450D camera. No artificial light was used, the exposure times were therefore quite long.

  In order to obtain good looking macro images one has to take several measures:

  Technical set-up

  • A solid, stable tripod: A heavy and stable tripod is absolutely necessary. Even the slightest vibrations (people walking, wind, shutter vibrations, etc.) will translate into a blurry image. I used a tripod designed for heavier video cameras.
  • High f-stop values: Macro images generally have a low depth of field. In order to increase the depth of field it is necessary to increase the f-stop values. This again results in longer exposure times.
  • Reducing shutter vibrations: Shutter vibrations can be minimized by using the mirror-lock up function of the camera. When the live-view feature of the camera is enabled, the mirror is in the “up” position. During release, the mirror does not have to swing up (because it is already up) and this significantly reduces vibrations. Not every SLR camera has a mirror lock-up feature, however. In this case, one can use a trick to completely eliminate shutter vibrations. Adjust the camera to have a long exposure time (let’s say 20 seconds). Cover the objective with a black cardboard, without touching the objective. Then release the shutter and wait 1 second for the system to stop vibrating. Then remove the black cardboard, this starts the exposure. One second before closing of the shutter cover the objective again. The opening and closing of the shutter (and mirror swing) will then take place while you have the black cardboard in front of the objective. Amateur astronomers use the same technique for taking vibration-free images of the night sky. They cover the telescope aperture with a dark cloth (without touching the telescope, of course).
  • Long exposure times: This may come as a surprise for some. Long exposure times can result in more steady images because the duration of the vibrating camera/tripod system (after release) are much shorter than the total exposure time during which a steady image reaches the sensor of the camera.

  Composition, artistic aspects etc.

  • Sufficient ambient light: To prevent hard shadows, I decided to use the natural, indirect light of the room to take the picture of the rose. This results in longer exposure times, which makes a very stable tripod a necessity.
  • Contrast: Make sure that the main object is set off from its background. This can be achieved either by blurring the background, or by making sure that the background has a distinctly different color.
  • Exposure time: If you use a black background, then the camera may expose longer than necessary. The black background “fools” the camera into thinking that the image is too dark. This may result in the object being overexposed. Underexpose by 1-2 stops if the main object is too bright.
  • Post processing: Crop the image and adjust the colors so that the background is completely black (if you use a black background). This will increase the impact of the picture.
  ]]> http://www.microbehunter.com/2010/05/29/how-to-make-macro-images/feed/ 0 http://www.microbehunter.com/2010/05/22/volvox/ http://www.microbehunter.com/2010/05/22/volvox/#comments Sat, 22 May 2010 10:00:18 +0000 Oliver http://www.microbehunter.com/?p=2445
  
  
  
  Volvox is a fresh water green algae and a member of the Chlorophyta. The picture shows a spherical volvox colony, each ball can contain hundreds, if not thousands of individual cells. The picture shows six daughter colonies inside the main colony. The main colony disintegrates and the daughter colonies are then released. Volvox is a nice example of an organism which shows first signs of multicellularity. Larger colonies can be up to 1mm in diameter and can be seen with the unaided eye.

  Reproduction

  Volvox reproduces both sexually and asexually. During asexual reproduction cells from the equator of the colony move to the inside and divide to form daughter colonies. The daughter colonies will grow and multiply. The mother colony will then rupture and release the offspring.

  During sexual reproduction, Volvox colonies form sperm and egg cells (ova). The sperm cells will swarm out to find ova in other colonies. The fertilized egg cells will then form new colonies.

  Growing and observing Volvox

  Microscopists who are interested in observing Volvox should try to investigate water samples from ponds and puddles. It is also possible to grow Volvox at home. Volvox likes to grow in nutrient-rich water. Dilute some plant fertilizer in water and add some pond water containing Volvox (or other green algae that you want to grow). Place the container on the window sill for several days but prevent direct sunlight as this may cause overheating, and drives out the CO2 for photosynthesis from the water. Alternatively, you can also use a plankton net to catch the colonies.

  For making permanent mounts, it’s probably best to use a water-based mounting medium such as glycerin gelatin. Solvent based media may dissolve the chlorophyll of the chloroplasts and may cause the cells to lose water and shrink.

  ]]> http://www.microbehunter.com/2010/05/22/volvox/feed/ 0 http://www.microbehunter.com/2010/05/15/dandelion-parachute-ball-up-close/ http://www.microbehunter.com/2010/05/15/dandelion-parachute-ball-up-close/#comments Fri, 14 May 2010 22:00:18 +0000 Oliver http://www.microbehunter.com/?p=2442
  
  Dandelion macro image.
  
  This is one of the first tries taking pictures with my new Sigma objective, and I have to admit that I’m very satisfied with the lens. The lens does not include image stabilization, a steady tripod is therefore a must. Contrast was slightly enhanced to make the background (my computer screen!) completely black. Mirror lock-up was used to further minimize vibrations. At a focal length of 300mm even the smallest vibrations cause a blurry image.

  Lens: Sigma 20-300mm APO DG MACRO (macro up to 1:2)
  Camera: Canon EOS 450D
  Exposure Time: 2 sec. (long time to minimize vibrations)
  Aperture Value: 6.62 EV (f/9.9)
  ISO Speed Rating: 100
  Focal Length: 300.0 mm

  ]]> http://www.microbehunter.com/2010/05/15/dandelion-parachute-ball-up-close/feed/ 0 http://www.microbehunter.com/2010/05/13/the-effect-of-the-mounting-medium-on-image-quality/ http://www.microbehunter.com/2010/05/13/the-effect-of-the-mounting-medium-on-image-quality/#comments Thu, 13 May 2010 10:55:07 +0000 Oliver http://www.microbehunter.com/?p=2426
  
  Ranunculus pollen mounted in air, no cover glass.
  
  
  Ranunculus pollen mounted in air with cover glass.
  
  
  Ranunculus pollen mounted in water.
  
  
  Ranunculus pollen mounted in Euparal. The pollen grains started to shrink.
  
  
  Ranunculus pollen mounted in clear nail polish. The pollen grains show signs of significant shrinkage.
  

  The mounting medium can have a significant effect both on the image quality and on the specimen itself. I tried a little experiment by observing pollen from a plant (in this case the buttercup, Ranunculus), mounted in five different ways:

  • Air-mounted, with no cover glass
  • Air-mounted, with a cover glass
  • Mounted in water (temporary mount)
  • Mounted in Euparal medium (permanent mount)
  • Mounted in nail polish (permanent mount)

  All observations were made using a 20x achromatic objective.

  Results

  The images on the right show that the mounting method has a significant impact on the way that the pollen grains appeared. The results can be summarized as follows:

  • Air-mounted specimens show the least details. The pollen grains show a thick dark fringe, which covers much of the details. This is due to the large difference in refractive index between the pollen grains and the surrounding air. Opening the condenser diaphragm reduces the dark fringes, but also lowers contrast and depth of field. The cover glass presses the pollen against the slide, so that more of them are in focus. Otherwise the cover glass did not seem to make much difference.
  • The water-mounted sample provides a much better image. The dark fringes are now gone, due to the similar refractive index of the pollen and the medium. The pollen appear spherical, because the water causes them to swell up.
  • Pollen mounted in Euparal started to shrink and therefore appear smaller in size. Kinks and folds are also visible. These artifacts are produced because the (non-water based) Euparal has withdrawn moisture from the pollen.
  • Clear nail polish showed a similar, but more pronounced effect as Euparal. The deformations of the pollen are very clearly visible. Evidently the solvent of the nail polish also removed significant amounts of water from the specimen. The nail polish itself lost some of its volume during drying and started to shrink as well. Air bubbles also became visible in the nail polish. Irregular drying of the mounting medium and a change in the shape of the mounting medium during drying can lead to shear-forces, which may distort the shape of the specimen.

  What about Glycerin Gelatin (glycerol gelatin, jelly)?

  Glycerin Gelatin is a water-based mounting medium. Glycerin Gelatin according to Kisser is one of several Glycerin Gelatin variations. It is a common medium for mounting pollen. Due to its water-based nature it does not cause the pollen to shrink. I’ll add a picture of this, when I have some of this mounting medium available. An alternative water-based mounting medium is fructose syrup. Both Glycerin Jelly and fructose syrup do not dry completely and therefore require a sealing of the sides of the cover slip with nail polish (but the pollen do not touch the nail polish).

  Lessons learned

  What can we learn from these observations?

  • First, permanently mounting a specimen is not only important for slide storage. The mounting medium significantly influences the transparency, resolution and shape of the specimen.
  • Second, the choice of the mounting medium depends on the type of specimen to be observed and on the type of microscopic technique to be used. For phase-contrast work the refractive index of the mounting medium should be different from the refractive index of the specimen. For bright-field work the refractive indexes should be similar. Large differences in refractive index can lead to the dark fringes as seen in the air-mounted specimens.

  Some philosophy

  So which mounting medium now results in pollen grains with a “true” or “correct” shape? The problem now is: what is the “correct” shape? Biological specimens may change their appearance depending on the environment. After a rain shower, the pollen may have a more roundish appearance, after having osmotically absorbed much liquid. Pollen that has dried in the air may resemble more the shape of the Euparal and nail polish samples. The choice of the mounting medium may therefore even include these considerations.

  External Links, References

  • An introduction to pollen analysis
  • Aqueous Mounting Medium Protocols
  • Making and Using Aqueous Mounting Media
  ]]> http://www.microbehunter.com/2010/05/13/the-effect-of-the-mounting-medium-on-image-quality/feed/ 1 http://www.microbehunter.com/2010/05/09/q-a-what-people-searched-for/ http://www.microbehunter.com/2010/05/09/q-a-what-people-searched-for/#comments Sun, 09 May 2010 21:20:27 +0000 Oliver http://www.microbehunter.com/?p=2429 In this post I’d like to address some of the search queries that people typed to find this web site. Naturally people typed many, many more search queries, most of the queries are made of 1 or 2 words. I selected the longer ones for this post.

  Q: What is the principal advantage of an electron microscope over an optical microscope?
  A: Electron microscopes have a far greater resolution compared to optical microscopes. Consequently, a much higher magnification is possible. Optical microscopes can magnify up to about 1000x, electron microscopes up to about 1 000 000x.

  Q: How to increase resolution of image?
  A: The resolution of am image can not simply be increased, once a picture has been taken through the microscope. Information which is not present in the first place can not simply be created. When taking pictures with the microscope, one should make sure that all the parameters are optimized to reach the maximum theoretical resolution. This includes a steady camera-microscope connection, the correct condenser diaphragm setting, the optimum mounting medium, etc.

  Q: Parts of the microscope and their functions?
  A: This question can not simply be answered in a line or two. I would recommend to watch the video, or read the post: Parts of a Compound Microscope

  Q: What are some microbes that you can see under a microscope?
  A: Ultimately you can see all types of microbes, provided you have the right type of microscope and use the appropriate technique. Viruses can be seen with electron microscopes, but not with light microscopes. Bacteria can best be seen with light microscopes that use phase contrast optics. Single celled eukaryotes (ciliates, algae etc.) as well as multicellular microorganisms can be seen with bight-field compound microscopes and also with stereo microscopes.

  Q: How many different types of microscopes are there?
  A: It depends on what system of classification you use and how many subdivisions you include. One common way to classify microscopes is into optical and non-optical microscopes. I already wrote a post on different types of microscopes:

  Q: Which type of microscope would be best to use if you wanted a 3-dimensional view of a bacteria cell?
  A: Here you have to be careful, the question can be misinterpreted. For true 3D, stereoscopic views two different images are needed.

  There are two types of microscopes that provide 3-D (stereoscopic) views:

  • Scanning electron microscopes: These devices scan the surface of the object. One single image is produced, which appears 3D (including “shadows” and surface texture). An example image can be found in this article:
  • Confocal laser microscopes: These are highly specialized optical microscopes, in which a computer computes a final. In this case it is possible to compute two different pictures, one for the left and one for the right eye. The image is then truly stereoscopic

  Q: Compare the kind of image obtained with scanning electron microscope with that obtained using transmission electron microscopy.
  A: In short, scanning electron microscope (SEMs) produce images that have a 3D appearance, Transmission electron microscopes (TEMs) produce 2D images.

  Q: Why is it desirable that microscope objectives be parfocal?
  A: Parfocal objectives are not only desirable, but (in my humble view) a necessity for efficient microscopic work. Parfocal objectives manufactured in such a way that a change in objective will not result in a significant loss of focus. If the image is in focus using a 4x objective, then the image is also in focus when a 10x objective is used. Significant refocussing is not necessary with parfocal objectives.

  Q: Part of the microscope that contains the ocular lens
  A: One word answer: the eyepiece. Sometimes the terms “eyepiece” and “ocular lens” are used interchangeably, but the eye piece contains more than one lens element.

  Q: Different types of microbes
  A: The term “microbe” is a colloquial term which refers to organisms (living things) that are too small to be seen with the unaided eye. The term is somewhat unclear, because microscopic insects (and other multicellular organisms) generally are not included. Viruses are not alive and therefore do not quality as microorganisms. Without going into too much detail, microorganisms include prokaryotes (Bacteria, Archaea), microscopic fungi, single-celled algae and protozoa (ciliates and amoeba belong to this category, among others).

  Q: Who invented the microscope?
  A: Which microscope? There are many kinds. In 1931, Ernst Ruska and Max Knoll constructed the first prototype of an electron microscope. Optical microscopes as we know them today evolved over a longer time period. Many people contributed to the developments. Two notable figures are Antonie van Leeuwenhoek (1632 – 1723) and Robert Hook (1635 – 1703). Leeuwenhoek made single-lens microscopes with which he discovered bacteria. Hook constructed compound microscopes (composed of objective and ocular lenses) and coined the term “cell”.

  ]]> http://www.microbehunter.com/2010/05/09/q-a-what-people-searched-for/feed/ 0 http://www.microbehunter.com/2010/05/01/ranunculus-buttercup-pollen/ http://www.microbehunter.com/2010/05/01/ranunculus-buttercup-pollen/#comments Sat, 01 May 2010 19:28:12 +0000 Oliver http://www.microbehunter.com/?p=2423
  
  Ranunculus repens pollen in dark field
  
  
  Ranunculus repens pollen in bright field
  
  
  Ranunculus repens
  
  Spring time is pollen time! Here are two images of Ranunculus repens (the Creeping Buttercup or Creeping Crowfoot) pollen, the top one in dark field, the bottom one in bright field. This plant is poisonous and can cause skin irritation. The name “Crowfoot” comes from the shape of the leaves, which resemble the claws of a crow.

  Now a few words concerning sample preparation. The pollen was collected by dusting the flower over a microscopic glass slide. The pollen was briefly dried in open air (about 1 hour) and then permanently mounted in Euparal mounting medium. The standard mounting medium for pollen is Glycerin gelatin, which is water based. I assume that the drying and the Euparal caused the pollen to shrink somewhat, but I yet have to make a comparison with fresh pollen.

   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   
   

  ]]> http://www.microbehunter.com/2010/05/01/ranunculus-buttercup-pollen/feed/ 0 http://www.microbehunter.com/2010/04/30/common-defects-of-old-microscopes/ http://www.microbehunter.com/2010/04/30/common-defects-of-old-microscopes/#comments Fri, 30 Apr 2010 05:21:53 +0000 Oliver http://www.microbehunter.com/?p=2420 During the past couple of years I’ve seen numerous microscopes that required maintenance due to heavy use. Here are some of the problems that I observed. It may pay off to also consider these problems when shopping for a used microscope.

  Problems with the optics

  • Dirty lens: This is due to immersion oil on the optical surfaces, which have collected dust and have hardened.
  • Lens kit dissolving: Some lenses are glued together. Flower-like bubbles forming in the lens are an indication that the lens kit is coming loose.
  • Fungi on the optical surfaces: This is a problem with microscopes which have been in use in areas of high humidity (such as the tropics). An anti-fungal coating of the lenses may prevent this.
  • Scratches or cracks: These can occur if the objective is rotated into the specimen. You can see an extreme example of this in the following post: Dirty microscope objective: Its effect on image quality
  • Loss of coating: Excessive rubbing or a wrong cleaning solution may remove the anti-reflective coating of the lenses.

  Problems with the mechanics

  • Stage drift: In this case, the stage slowly lowers due to its own weight. This can be fixed by tightening some screws.
    Focus difficult to turn: In this case the oil in the gears has solidified due to age and accumulated dust. Do not use force, it may increase the wear on the gears. It’s better to get the device cleaned.
  • Mechanical stage difficult to move: Like with the focus knobs a solidified oil makes the stage difficult to move.
  • Too much slack: Sometimes there is too much tolerance and turning the focus knobs. There may be too much slack in the gears, possibly due to too much wear.

  Problems with electricity

  • Old lamp: An old lamp will have a spectrum shifted towards the red. This is a disadvantage for digital photography. The sensors of the camera are very red-sensitive. Use a blue filter.
  • Brittle insulation: Old power cables may become brittle and be a hazard.
  ]]> http://www.microbehunter.com/2010/04/30/common-defects-of-old-microscopes/feed/ 0 http://www.microbehunter.com/2010/04/17/virtual-microscope-male-flower-of-a-pine-tree-pinus/ http://www.microbehunter.com/2010/04/17/virtual-microscope-male-flower-of-a-pine-tree-pinus/#comments Sat, 17 Apr 2010 19:51:40 +0000 Oliver http://www.microbehunter.com/?p=1521
  

  
  The male pine cone (or flower) is responsible for forming pollen. These pollen grains are also visible in the image above. For a zoom-able image of a female pine cone, visit the following link: Virtual microscope: female pine cone (Pinus)

  ]]> http://www.microbehunter.com/2010/04/17/virtual-microscope-male-flower-of-a-pine-tree-pinus/feed/ 0 http://www.microbehunter.com/2010/02/28/virtual-microscope-cross-section-of-the-earth-worm-lumbricus-terrestris/ http://www.microbehunter.com/2010/02/28/virtual-microscope-cross-section-of-the-earth-worm-lumbricus-terrestris/#comments Sun, 28 Feb 2010 13:55:12 +0000 Oliver http://www.microbehunter.com/?p=1520
  

  The image above shows Lumbricus terrestris, the earth worm, in cross section. The red part in the center is the digestive system. You can zoom into the image. The only adjustment done to the image was a color correction. The image was not sharpened.

  ]]> http://www.microbehunter.com/2010/02/28/virtual-microscope-cross-section-of-the-earth-worm-lumbricus-terrestris/feed/ 0 http://www.microbehunter.com/2010/02/23/human-hair-under-the-microscope/ http://www.microbehunter.com/2010/02/23/human-hair-under-the-microscope/#comments Tue, 23 Feb 2010 11:00:38 +0000 Oliver http://www.microbehunter.com/?p=1516
  
  Two intertwined human hair under the microscope. The original image. The field iris diaphragm is still visible at the corners.
  
  
  The power of image clean-up: dust and dirt removed.
  
  Today I’d like to show you a nice microscopic picture, which I took several years ago of two human hair. The pictures on the right show you the original, unprocessed image at the top, and a second cleaned-up image with a nice background on the bottom. The top image shows darkened corers from the field diaphragm (from Köhler illumination). I closed the diaphragm quite a bit in order to increase image contrast (I intended to crop the image anyway). Then I did some processing in PhotoShop (or was it GIMP?). I selected the hair to remove dust and dirt and placed it on a nice blue gradient background. The most difficult part of the project was the making the hair knot and finding of an appropriate focus of the relatively thick sample. I did not do any image stacking (combining several images into one to get one final image which is sharp throughout).
   
   
   
   
   
   
   
   
  ]]> http://www.microbehunter.com/2010/02/23/human-hair-under-the-microscope/feed/ 1 http://www.microbehunter.com/2010/02/21/kohler-illumination-to-reduce-reflections/ http://www.microbehunter.com/2010/02/21/kohler-illumination-to-reduce-reflections/#comments Sun, 21 Feb 2010 13:50:28 +0000 Oliver http://www.microbehunter.com/?p=1513
  
  Field diaphragm is wide open. Reflections from the side of the tube are very strong.
  
  
  Field diaphragm is half open. The reflections are less.
  
  
  Field diaphragm is closed. Only the direct light is able to reach the camera.
  
  
  Taking a picture of the tube with a webcam. Any camera with a small lens would also have done the job.
  
  The Köhler (or Koehler or Kohler) field diaphragm is located above the light source. It is responsible for controlling the width of the light beam (but not its intensity). The light source of a microscope without Köhler illumination will illuminate the whole specimen, which may be the source of stray light and excessive heating of the specimen. By closing the field diaphragm, it is possible to limit the beam of light only to the part of the specimen which is actually observed.

  Advantages of Köhler illumination for photography

  Köhler illumination increases the contrast of a photomicrograph because it reduces stray light and glare caused by reflections inside the microscope. On the right side you can see images taken through a trinocular head with a web cam. The more that the field diaphragm is closed, the less the reflections coming from the side of the tube. The bright spot in the center is the light which comes directly (unreflected) from the light source. In order to see a picture, it would be necessary to remove the lens from the webcam and project the image directly on the sensor of the webcam. In this case, the lens was left on to be able to see the side of the tube.

  For more background info on Köhler illumination, you may be interested in the following two posts:
  

  • Advantages of Koehler Illumination
  • Adjusting Koehler Illumination

   
  
   
  
   
  
   
  
   
  
   
  
   
  
   
  
   
  
   
  
   

  ]]> http://www.microbehunter.com/2010/02/21/kohler-illumination-to-reduce-reflections/feed/ 0 http://www.microbehunter.com/2010/02/16/5-rules-of-buying-a-microscope/ http://www.microbehunter.com/2010/02/16/5-rules-of-buying-a-microscope/#comments Tue, 16 Feb 2010 20:07:57 +0000 Oliver http://www.microbehunter.com/?p=1508 I’ve been repeatedly asked for advice concerning the purchase of microscopes of hobby and amateur purposes. The following rules should help you in your choice.

  Rule 1: Be weary about “department store” microscopes

  Enthusiasts who want to pick up the hobby frequently encounter their first microscopes in department stores and toy shops. If you are serious about microscopy as a hobby, then I have to disadvise you from purchasing these devices. Microscopes are precision technical instruments and the low cost of toy microscopes simply does not allow them to keep up with the demands of the more serious enthusiast. The resolution of the optics is lower. Stability can also be an isue. It’s better to invest a bit more. You have to contact a retailer which is specialized for microscopes and who sells microscopes to hospitals, schools or research organizations.

  Rule 2: Consider carefully if you want a stereo microscope or a compound microscope

  Consider your areas of applications. Do you want to observe large or opaque specimens (stereo microscope) or are you more interested in observing small, transparent objects (compound microscope). If you want to do microscopy work with young children, then I would recommend stereo microscopes. See the other post for more info: Which Microscope for Children?. Compound microscopes allow you to observe much smaller specimens, but require you to engage in sample preparation (unless you purchase ready-made specimens).

  Rule 3: The magnification is one of the least important criteria

  Resolution, stability, extensibility, light intensity etc. also play a significant role. Get the big picture and look at the whole device. Do not get bogged down simply on magnification. Getting a high magnification is the easiest thing to achieve. Simply add a stronger eyepiece, or take a picture and enlarge it on the monitor. Magnification without resolution is meaningless. And a shaky plastic microscope will produce such an unsteady picture that you won’t be able to see much anyway.

  Rule 4: Go for standards

  Make sure that the microscope has exchangeable objective lenses manufactured according to the “160mm” standard. In this case you have a wide selection of different objectives available from different manufacturers. Infinity corrected optics are an alternative, but there is no universal standard. Some microscopes are not modular in design (“closed system”) and it is not possible to exchange parts later on. When choosing the microscope make sure that you also consider possible future interests and uses.

  Rule 5: Consider your current interests

  Microscopy does not have to be an entirely new hobby, it can also be a valuable extension of one of your existing pastimes. You may want to evaluate your current hobbies to see which type of microscope fits best.

  • Choose a stereo microscope if you are collecting stamps, minerals, rocks, coins, trading cards, smaller antiquities, insects or other objects that are small enough to be placed directly on the stage. Also choose a stereo microscope if younger children should have access to the device.
  • Choose a compound microscope of you are keeping a home aquarium, if you want to make specimen preparation (microtoming, staining, etc.) as part of your hobby.
  ]]> http://www.microbehunter.com/2010/02/16/5-rules-of-buying-a-microscope/feed/ 1 http://www.microbehunter.com/2010/02/12/magazine-articles-on-microscopy/ http://www.microbehunter.com/2010/02/12/magazine-articles-on-microscopy/#comments Fri, 12 Feb 2010 11:00:45 +0000 Oliver http://www.microbehunter.com/?p=1505 Popular Science magazine published many different articles dealing with microscopy. These articles can be accessed over Google Books:
  Popular Science articles on microscopy. Even though some of the articles are quite dated (going back into the 1930s), they still can contain valuable information and tips which remain valid up to today. Be aware that not all presented methods may be suitable for the use in schools and with children.
  ]]> http://www.microbehunter.com/2010/02/12/magazine-articles-on-microscopy/feed/ 0 http://www.microbehunter.com/2010/02/10/how-to-make-microscope-filters/ http://www.microbehunter.com/2010/02/10/how-to-make-microscope-filters/#comments Wed, 10 Feb 2010 11:00:20 +0000 Oliver http://www.microbehunter.com/?p=1502
  
  Different filters (patch stops) printed on overhead foil. The blue filter on the left is a commercial blue glass filter, on the bottom: the condenser with the 2 centering screws.
  
  Commercial microscope filters are usually made of stained glass. In the case of patch stops (as used in dark-field illumination), they may be made of aluminum. The dark-field patch stops block some of the light and the specimen will appear bright on dark background. The traditional way of DIY patch stops is cutting them out from black cardboard, but I consider this somewhat difficult to do, and it’s not the most elegant way. In this post I’d like to show you a method of making patch stop and color filters using a printer. Needless to say, if you use a color printer, then you can even make color filters. You do need a condenser with a filter holder, of course.

  In a previous post, I already mentioned the making of patch stops from cardboard. For some background information (and more pictures) try these articles:

  • Oblique Illumination
  • Increasing Contrast using Optical Methods

  Making Patch stops for dark-field illumination.

  • Measure the diameter of the filter holder of your condenser.
  • Using a program, such as PowerPoint or OpenOffice Impress to draw a circle, fill-color white, of the same diameter as the filter holder. You can adjust the size of the circle in the context menu.
  • Draw a smaller black circle into the center. Copy-paste both circles and then change the size of the inner smaller circle. You want to make several filters to find the one that works best.
  • Print the filters on overhead foil. Print with a laser printer. The overhead foils for laser printers are more heat resistant.
  • Cut out the filters with a scissor
  • Take a black marker and darken the black inner circle.
  • For microscopy work, take two of these filters and place them on top of each other. This ensures that the central circle is completely black.
  • Place the filter into the filter holder, completely open the condenser aperture diaphragm and the field diaphram (should you have one).
  • Try out the different objectives and find the suitable filter/objective combination.

  Making patch stops of oblique illumunation

  The method is very similar to making patch stops for dark filed. In this case, light is only allowed to hit the specimen from one side only. This will produce a relief-like image.

  • Draw a black and a white circle of the diameter of the condenser filter holder.
  • Overlap the two circles, so that the white circle covers part of the black circle. The white circle should not reach the center of the black circle.
  • Cut out and proceed as described for making a dark field patch stop.
  • Again it is necessary to experiment to find the appropriate filter/objective combination.

  Making Rheinberg filters

  Maybe you want to show yellow specimens on a blue background. Take the dimensions of the dark-field patch stop and color the center yellow and the periphery blue (color printer!). You have to use intensive colors to achieve an effect. Try different color combinations.

  ]]> http://www.microbehunter.com/2010/02/10/how-to-make-microscope-filters/feed/ 3 http://www.microbehunter.com/2010/02/08/stereo-microscope-projects/ http://www.microbehunter.com/2010/02/08/stereo-microscope-projects/#comments Mon, 08 Feb 2010 11:00:15 +0000 Oliver http://www.microbehunter.com/?p=1489 You’ve bought your kid a stereo microscope as a birthday present and now wonder what to look at. Or maybe you are teacher and want to give your class an introduction into (stereo) microscopy and need some specimens to look at (or maybe you bought yourself one, and now want to start out observing…)

  Requirements of the specimen

  When microscoping with children, I recomend the

  • Not too abstract: The specimen should not be too abstract for the children. I mean, YOU may be interested in the circuitry of computer electronics parts under the microscope (and they DO look interesting), but for kids I’d suggest something more tangible.
  • Flat: A flat object makes it easier to adjust the depth of field. Most of the object will then be in focus.
  • Contrast: A high contrast makes it easier to see structures and details.

  Specimens to look at

  • Safe: This is self-explanatory. Do not use organisms or substances that are hazardous.
  • Rocks: Collect some smooth rocks, wash and clean them in running water. Either observe the rocks while they are wet (and still shiny) or make them shiny by polishing them with a drop of oil. Shiny rocks have more contrast and simply look better than dull ones.
  • Leaves: They are flat and transparent. They can be observed both with the light source from the top and from the bottom.
  • Insects: This can be problematic. The insects should be dead, otherwise they are too difficult to observe, moving around all the time. Be aware that catching insects (such as butterflies) may not be allowed, as some of them are protected.
  • Foods: Cornflakes, cut open fruits, seeds, mushrooms can make very educational samples. Place the cut surface is horizontally under the stereo microscope, and you won’t have a depth of field problem.
  • Money: Count the scratches on the coins! The highly reflective surface of the coins make them an easy specimen.
  • Pictures: This is the first specimen that we use in school when teaching the students how to use the stereo microscope. Printed pictures are made of many dots, which can be observed. Many kids did not know this. This way the children learn how to focus properly and how to change magnification. Later we give them specimens with a thickness.
  • Own fingers: Here it is important to instuct the children to rest their fingers on the platform of the microscope. Many children will attempt to view their fingers by holding them mid-air beneath the objective. It is nearly impossible to find a proper focus this way.
  • Own handwriting: This is a good possibility to estimate size and magnification.
  • Textiles: stretch them flat and observe how they look different in epi- and trans- illumination.

  Specimens not to look at

  • Dust: some kids may have a dust allergy (mites), but it depends on the type of dust.
  • Body fluids (blood): for hygienic reasons. And they are not interesting anyway at a low magnification.
  • Spoiled food: fungal spores are not healthy to breath in, and bacteria on food are not good…
  • Anything else which can be considered dangerous
  ]]> http://www.microbehunter.com/2010/02/08/stereo-microscope-projects/feed/ 0 http://www.microbehunter.com/2010/02/06/bacteria-in-phase-contrast/ http://www.microbehunter.com/2010/02/06/bacteria-in-phase-contrast/#comments Sat, 06 Feb 2010 09:00:44 +0000 Oliver http://www.microbehunter.com/?p=1501

  Cocci in packets

  

  Cocci in pairs and in packets

  

  Short rods

  

  Rods, slightly curved

  About phase contrast

  Bacteria are transparent and therefore difficult to see using regular bright-field microscopy. The bacterial cells will appear just as bright as the surounding medium and there is no color contrast. Phase contrast optics provides a solution. Phase contrast optics convert the differences in optical density (i.e. the refractive index) of the bacterial cells into different shades of brightness. The optics achieves this by interference of the light which passes through the specimen (the bacteria) with the light that goes around the medium. Phase contrast optics therefore work only if the cells have a different refractive index compared to the medium.

  How the bacteria were prepared

  The bacteria were grown in pure culture in an appropriate microbiology laboratory. A colony was then suspended in saline (salt water) of right concentration and then microscoped with a 1000x magnification in oil immersion (using a 100x oil objective).

  If one takes too much liquid, then the cells start to float in and out of focus and it is not easily possible to capture the shape of the individual cells. A similar problem can occur if the cells are much smaller than the film of liquid between the slide and cover slip. The evaporation of the liquid from the edges of the cover slip will cause a constant movement of the cells and make it difficult to take a steady picture. In this case it is necessary to heat-fix the bacteria. A colony was then suspended in saline and dried at room temperature. The slide was briefly pulled through the flame of a bunsen burner, with the bacteria on the opposite side of the the flame. This heating process fixed the bacteria to the glass slide. Immersion oil was then directly applied to the slide and the bacteria were observed without cover glass. One disadvantage of heat fixing is, that during the drying process the bacteria may aggregate (as the volume of liquid decreases) and it may become more difficult to see individual cells.

  About the photographs

  The pictures were taken on analog B/W film and then digitized with a camera and an adapter (see the following post for more info on the set-up: Digitizing photographic slides with a digital camera ). The negative was then inverted and the contrast levels adjusted. The soft, slightly blurry appearance of the pictures shows that we are already at the limits of the resolution. The images were not sharpened. Notice the bright halo around the bacterial cells. This is typical for phase contrast microscopy.

  ]]> http://www.microbehunter.com/2010/02/06/bacteria-in-phase-contrast/feed/ 4 http://www.microbehunter.com/2010/02/04/trichinella-spiralis-the-pork-worm/ http://www.microbehunter.com/2010/02/04/trichinella-spiralis-the-pork-worm/#comments Thu, 04 Feb 2010 11:00:33 +0000 Oliver http://www.microbehunter.com/?p=1494
  
  Encapsuled Trichinella spiralis larva in muscle. The larva is cut diagonally.
  
  
  Longitudinal cross-section.
  
  
  The larva (circular patterns) in cross-section.
  
  Trichinella spiralis is the smallest nematode parasite in humans. It causes the disease trichinosis. It is also one of the most wide spread parasites of the world. It can be contracted by eating raw or half-cooked pork or wild game animals.

  Life Cycle of Trichinella spiralis

  • T. spiralis larva are encapsuled in the muscle of the host animal. The pictures on the right show different cross-sections of this stage.
  • A person who eats raw or undercooked meat will take these larva up into the body. The larvae are released by the stomach acid and pass into the intestine, where they mature and start to reproduce.
  • The offspring larvae travel in the circulatory system throughout the body and settle in the muscles, where they encyst again.

   
   
   
   
   
   
   
   
   
   
   
   
   
   
   

  ]]> http://www.microbehunter.com/2010/02/04/trichinella-spiralis-the-pork-worm/feed/ 0 http://www.microbehunter.com/2010/02/02/digital-methods-for-improving-microscopic-photographs/ http://www.microbehunter.com/2010/02/02/digital-methods-for-improving-microscopic-photographs/#comments Tue, 02 Feb 2010 11:00:48 +0000 Oliver http://www.microbehunter.com/?p=1487 Digital photography gives the users many new possibilities in improving photographs taken through the microscope. This post gives an overview of the different image processing functions that can be applied to microscopic images. This post places a focus on what is possible, but does not explain the “how” part. This is something that I plan to include later posts.

  Stacking

  Microscopic images generally have a low depth of field. It is possible to take several images of different depth of fields and to combine them in such a way that the final image is sharp throughout. By carefully turning the fine-focus knob a specified amount, it is possible to section through a complete specimen. Care should be taken, however: If too many parts of a transparent specimen are in focus in the final image, then these parts may cover each other, thus reducing the information content of the final image. Two cell organelles which are located behind each other, both being in focus, will cover up each other, and it is not possible to say which part belongs to what organelle.

  Stitching

  In this method, different overlapping images are assembled together into a larger final picture. While stacking combines the images “vertically”, stitching produces a larger final image by “horizontally” combining them. By stitching, it is possible to overcome the limited field of view. Stitching can be accomplished by using a panorama software. When choosing the software, one should take care that it allows for the combining of images both horizontally and vertically (some only permit for horizontal combination). Microscopic images often do not offer much image complexity. For this reason, the software may have problems assembling the images automatically. It pays off to do a little planning beforehand.

  • How large will the final image be? The processing requirements increase significantly with increased image size.
  • What camera resolution should be used? The choice of camera resolution has a significant impact on the final image size and required processing power. One should first test, if a high camera resolution is indeed necessary of if it is not simply results in empty magnification. Read: Required camera resolution for photography through the microscope
  • How much image overlap should be used? More overlap may make it easier for the software to automatically assemble the pictures, but at the same time more pictures are needed to cover the whole specimen (which again increases work time).

  All of the images of the category Virtual Microscope were stitched together.

  Background clean up

  The optical surfaces (especially the lighting system and condenser optics) are rarely completely free of dust. These disturbances will be present in the image, whether or not a specimen slide is present. It is now possibly to mathematically subtract these disturbances from the image. A picture with and without a specimen has to be taken at the same magnification and using the same exposure time. The empty image (without specimen) is then subtracted from the image containing the specimen.

  Alternatively, it is possible to clean the background by selecting the specimen without background and copying it to a new clean background. This system was employed when taking a photograph of the tick (See: Virtual microscope: The Tick). An automatic selection only works well if the specimen’s color or brightness is significantly different from the background.

  Increasing contrast

  Contrast enhancement is one of the methods which, when done correctly, does not result in any loss of image information content, provided that the image does not use the full brightness spectrum from white to black in the first place. Nearly all photo editing programs contain a “levels” or “histogram” function, with which one can adjust the contrast.

  Sharpening

  Sharpening the image may subjectively increase image quality, but it will not result in a higher information content. Excessive sharpening introduces artifacts, it may enhance image noise and may enhance irrelevant image components, such as dust and dirt. Before the image is sharpened, it is probably better to increase the contrast. This will sometimes also give an impression of a sharper and more pleasing image.

  White balance adjustment

  This is a critical adjustment if one wants to obtain reproducible results. Microscopic light will show a different color temperature, based on the intensity level. Turning up the light to a high intensity will also shift the color temperature towards the blue end of the spectrum. A lower intensity setting will increase the red components. The age of the light bulb also shifts the color temperature towards the red. Digital cameras can adjust the white balance automatically, but this may not be a reliable setting, as the camera uses a predefined standard. A specimen which contains many red components, for example, may fool the camera into thinking that the light source is too red. The camera will then shift the color balance toward the blue, which does not reflect the real nature of the specimen. This is a particular problem of colorful images of crystals and specimens which cover the full field of view, without a visible background from the lamp. Some cameras also have a custom white balance function. In this case an empty reference image without specimen is taken. The camera will then use this image as a basis for correcting the white balance of all subsequent images.

  Photo editing software also permits users to automatically or manually adjust the white balance. An automatic setting will also take the specimen itself into consideration (just like in the automatic camera white balance setting described above), and the results may not be pleasing. I generally make white balance adjustments manually. In this case, one has to click on those parts of the image that should be considered white, usually the background.

  ]]> http://www.microbehunter.com/2010/02/02/digital-methods-for-improving-microscopic-photographs/feed/ 2 http://www.microbehunter.com/2010/01/31/observing-bacteria-under-the-light-microscope/ http://www.microbehunter.com/2010/01/31/observing-bacteria-under-the-light-microscope/#comments Sun, 31 Jan 2010 11:00:08 +0000 Oliver http://microscopy.okim.info/?p=1403
  Can one see bacteria using a compound microscope? The answer is a careful “yes, but”.
  Generally speaking, it is theoretically and practically possible to see living and unstained bacteria with compound light microscopes, including those microscopes which are used for educational purposes in schools. There are several issues to consider, however.

  Why bacteria are difficult to see

  Bacteria are difficult to see with a bright-field compound microscope for several reasons:

  • They are small: In order to see their shape, it is necessary to use a magnification of about 400x to 1000x. The optics must be good in order to resolve them properly at this magnification.
  • Difficult to focus: At a high magnification, the bacterial cells will float in and out of focus, especially if the layer of water between the cover glass and the slide is too thick.
  • They are transparent: Bacteria will show their color only if they are present in a colony. Individual cells present on the slide are clear. Regular bright-field optics will only show the bacteria if one closes the condenser iris diaphragm. This is due to the difference in the refractive index between the water and the bacterial cells.
  • Difficult to recognize: An untrained eye may have problems differentiating bacteria from small dust and dirt which is present on the slide. Some bacteria also form clumps and therefore it is difficult to see the individual cells.

  Research organizations and advances amateurs use phase contrast optics to see bacteria. This system converts the differences of the refractive index of the bacteria into brightness. The transparent bacteria can then be seen dark on bright background. In bright-field, closing the condenser iris diaphragm will also make the bacteria appear darker, but at the same time one also introduces artifacts (“fringes”) around the individual cells. One possibility is to stain the bacteria, but in this case there fixing and staining process may introduce artifacts.

  What is a safe source of bacteria? For recreational or educational purposes, one should never use spoiled food or (heaven forbid!) use bacteria obtained from the human body and grown on agar plates. The risks involved are simply not worth it, especially when working with students. Other sources, such as soil or humus have other disadvantages. The impurities make it difficult to keep bacteria from other particles apart, especially if one uses bright-field optics. Rather I recommend the use of yogurt. It should be possible to see small circular cells (cocci), which may also occur in pairs. It is also possible to scratch some bacterial cells off from certain kinds of cheese. Brevibacterium can be found on Limburger cheese, for example. One has to be aware that some cheeses use a combination of bacteria and fungi, however, and that the larger fungal cells may outweigh the bacteria.

  In summary, there are easier (and maybe also more interesting) specimens to observe than bacteria. I you want to see individual cells, then I do recommend that you start out with yeast suspensions. These eukaryotic cells are much larger and can be more easily identified.

  For pictures of bacteria in phase contrast read the following post: Bacteria in phase contrast

  ]]> http://www.microbehunter.com/2010/01/31/observing-bacteria-under-the-light-microscope/feed/ 0 http://www.microbehunter.com/2010/01/29/making-a-wet-mount-for-microscopy/ http://www.microbehunter.com/2010/01/29/making-a-wet-mount-for-microscopy/#comments Fri, 29 Jan 2010 11:00:59 +0000 Oliver http://microscopy.okim.info/?p=1402 A wet mount (or temporary mount) is one of the most common ways of observing specimens under the microscope. The sample to be viewed floats in a layer of water which is between the slide and the cover glass. The water performs an important optical function. Without it, the resolution is lower.

  The general procedure of making a wet mount

  1. Place a drop of water on the center of the slide. It is also possible to first place the specimen on the slide, but small specimens usually separate more easily from the tweezers or needle if dipped into the drop of water.
  2. Place the specimen into the drop of water and if the specimen floats, add another drop of water on top of it. This reduces the possibilities of air bubbles forming.
  3. Carefully lower the cover glass so that it touches with one side the drop of water. The cover slip should form an angle of about 45 degrees with the slide. Touch the cover glass on the sides only to prevent finger prints. Alternatively, use tweezers to hold the cover glass.
  4. Then lower the cover slip completely. Placing the cover slip at an angle prevents the formation of air-bubbles.
  5. Remove excess water with a filter paper or tissue paper

  Possible problems of making a wet mount

  • The cover glass floats and moves: This is due to too much water. Remove water with the help of a tissue paper. Under no circumstances should there be water droplets on top of the cover glass. This water may get into contact with the objectives.
  • The liquid streams and does not settle: This could be due to evaporation. Add more water between coverslip and slide.
  • Air bubbles start to become visible: If bubbles were not present before and start to form, then this could be an indication of oxygen production due to photosynthesis. This depends on the oxygen saturation of the water and the amount of photosynthetic algae present.
  • Air bubbles are present: Often the cover glass was not lowered from the side at an angle, but placed horizontally on the water drop. It may also be that the the specimen is hydrophobic (fatty) and /or fluffy. In this case, the the water may have problems reaching all of the areas of the speciemen and there is much air caught by the fine structures. Wet the specimen briefly in alcohol and then transfer directly from the alcohol to water. Alternatively you can try to break the surface tension of the water by adding a small amount of surfactant, such as soap or shampoo. Be aware that alcohol or soap may have adverse effects on living organisms.
  ]]> http://www.microbehunter.com/2010/01/29/making-a-wet-mount-for-microscopy/feed/ 0 http://www.microbehunter.com/2010/01/27/making-mounts-of-pollen-grains/ http://www.microbehunter.com/2010/01/27/making-mounts-of-pollen-grains/#comments Wed, 27 Jan 2010 11:00:14 +0000 Oliver http://www.microbehunter.com/?p=1480 Permanent slides of pollen grains can be used as a reference for identifying unknown pollen samples. It is therefore important, that the pollen grains remain in an authentic, natural shape. The preparation and mounting of the pollen can introduce artifacts: the pollen may lose some of its pigment, start to shrink and shrivel or absorb water and swell. A careful preparation is therefore necessary.

  There are several methods of preparing pollen grains, each one offers advantages and disadvantages. I can not give a general rule, it simply depends on the goal of the investigation and on the sample investigated. Pollen from wind-pollinated plants taken from a dry environment are probably best left in a dry condition, and not mixed with a water-based mountant, which may cause them to swell (depends on the osmotic potential of the medium, however). On the other hand, the obtained image quality and resolution may not be satisfactory in such a dry mount. It is a compromise, in which several factors have to be taken into consideration. A microscopy enthusiast, who does not need the slides for identification purposes, will again set different standards (such as avoidance of toxic solvents). People who want to publish their results, in turn, may have to rely on the preparatory technique which is customary in their field of research, for reasons of comparison. I recommend that the different methods are tried out.

  Mounting techniques

  Glycerol wet mount: Place a small drop of glycerol on a clean slide and tap the anthers of the plant so that the pollen falls into the glycerol. If necessary, carefully separate large chunks of pollen grains by stirring. Place a cover slip on top and seal the sides of the cover slip with nail polish. Use a very small amount of glycerol to make sure that the nail polish has enough area to stick the coverslip to the slide. Glycerol wet mounted slides can be stored for months if there is no leakage. The glycerol will withdraw water from the pollen. If the pollen is not dry, then there is a possibility of the pollen to shrink.

  Air mounts (dry mounts): In this case, no liquid mounting medium is used. A cover slip is placed on top of the pollen grains and sealed on the side, either with nail polish or with tape. Nail polish may flow very quickly between cover slip and slide, so it may be best to use a nail polish of low viscosity (by letting some solvent evaporate first).

  Glycerol jelly (according to Kisser): This is a very popular mounting medium for pollen. It is phenol-free (antiseptic additive) and therefore non-hazardous. It contains 10g of gelatin, 35ml distilled water and 30ml of glycerol (glycerin). After mounting, the sides of the cover slip need to be sealed. Due to the lack of an antiseptic, it is also necessary to work in a sterile manner, otherwise there is the risk of fungal growth in the medium. Maybe it is a good idea to treat the pollen grains first in alcohol to reduce the chance of fungal contamination by spores. Alternatively, one could experiment by increasing the concentration of glycerol.

  Non-water-based mounting media: Euparal is a mounting medium which is not water based. Specimens which are present in alcohol can be directly transferred to Euparal. Place a pollen suspension on the slide and let the alcohol evaporate. Before mounting pollen in Euparal, I recommend that the pollen are first washed in alcohol and then compared to the original shape. Does washing in alcohol result in an unacceptable shrinking of the pollen or unacceptable loss of pigments? If not, then mounting the pollen in Euparal may be an alternative.

  Reading materal

  I found the following article: Marvels of pollen shown by your microscope (Popular Science, September 1939)
  (The article recommends the use of organic solvents (such as xylol/xylene and others) to remove oil from the pollen. I do not recommend this due to health reasons, especially when preparing samples for educational purposes. Still, it gives a nice overview of the topic.)

  ]]> http://www.microbehunter.com/2010/01/27/making-mounts-of-pollen-grains/feed/ 0 http://www.microbehunter.com/2010/01/25/required-camera-resolution-for-photography-through-the-microscope/ http://www.microbehunter.com/2010/01/25/required-camera-resolution-for-photography-through-the-microscope/#comments Mon, 25 Jan 2010 11:00:40 +0000 Oliver http://www.microbehunter.com/?p=1482
  
  Comparison of resolution. There is practically no visible difference between 3MP and 12 MP. The limiting factor is therefore the microscope or specimen, and not the camera resolution.
  
  My digital camera (a Canon EOS 450d) is capable of taking pictures at 3MP, 6 MP and 12 MP (MP=megapixels). Which setting should one choose to obtain the optimum results? If the camera resolution is too low, then this will result in the loss of image detail (but a small file). A resolution setting which is too high will result in a large file, but of low detail and empty magnification.

  The method of determining the optimum resolution is quite simple:

  • Take a low-resolution and a high-resolution picture of the same specimen. I took two pictures each, one with a setting of about 3MP and one with the maximum resolution of 12MP. In both cases, the file compression was low (and JPG image quality high).
  • Enlarge the low-resolution image to the size of the high-resolution image. In my case the low resolution (3MP) image had 2256 by 1504 pixels. It was enlarged to 4272 by 2848 pixels (12MP).
  • Compare the two images. If the high-resolution image shows details that are not present in the low-resolution image, then this indicates a loss of information when taking a low-res photograph. Otherwise one can safely use the low-res setting of the camera. In this latter case the limiting factor for image quality is not the resolution of the camera, but rather the optics or the specimen.

  The result? There was barely any discernible difference between the images. With the specimens that I used, it is perfectly OK to use the smallest camera resolution of 3MP. The limiting factor, so to say, is the optical system of the microscope and/or the specimen itself. For this reason, it is not necessary to choose a high resolution camera setting. Other specimens may deliver finer images, so the differences may become evident then, but from what I found, a small image resolution of 3MP is sufficient.

  ]]> http://www.microbehunter.com/2010/01/25/required-camera-resolution-for-photography-through-the-microscope/feed/ 2 http://www.microbehunter.com/2010/01/23/an-overview-of-mounting-media-for-microscopy/ http://www.microbehunter.com/2010/01/23/an-overview-of-mounting-media-for-microscopy/#comments Sat, 23 Jan 2010 11:00:46 +0000 Oliver http://www.microbehunter.com/?p=1473
  This post gives an overview of different water-based and non-water-based mounting media and their advantages and disadvantages.
  Mounting media are needed for making permanent slides. The mounting medium holds the specimens in place between the cover slip and the slide. The choice of the right mounting medium is a separate topic all on its own. There are countless commercial and home-made mounting media available. Which ones should one use? In many cases the microscopist has no choice: some specimens simply require the use of a specific mounting medium, otherwise the structure that one wants to observe is not properly visible. Alternatively, not all specimen types are chemically compatible with the solvents of the medium.

  Generally, mounting media for permanent slides can be categorized into water-based and organic solvent based mounting media. While many water-based mounting media for permanent slides solidify and hold the specimen firmly in place, some others remain in a liquid state. In this latter case, it is necessary to prevent the liquid from flowing out by sealing the four sides of the cover slip. Nail polish can be used for this. In the following paragraphs, I’d like to give you an overview of the different types of mounting media.

  Water-insoluble mounting media that solidify

  Euparal: This mounting medium was invented in 1904 by Prof. G. Gilson, Professor of Zoology at Louvain University, Belgium. It contains the substances sandarac, eucalyptol, paraldehyde, camphor, and phenyl salicylate. Euparal possesses a nice odor (but don’t smell it anyway), due to the natural oils that are included. Euparal is commonly used to mount histological specimens and insects. One big advantage of Euparal is, that the specimens can be transferred directly from the alcohol in which they are stored. Do not embed specimens which contain water, this may result in a clouding of the mounting medium.

  Summary: Advantages of Euparal include the possibility to directly transfer specimens from alcohol to Euparal without the need of toxic solvents. A disadvantage is the relatively long drying time of a few days.

  Canada Balsam: This is a natural mounting medium obtained from the e balsam fir tree (Abies balsamea). The optical properties are nearly identical with those of glass. For this reason, Canada Balsam was used for many years as a kit to hold optical lenses in place. Meanwhile, synthetic lens kits have replaced Canada Balsam, it is still used as a mounting medium for microscopy, however. Canada Balsam has the advantage that its optical properties do not deteriorate with age. Permanent slides mounted with Canada Balsam have been stored for a century and are still useful.

  The disadvantage of Canada balsam is, that the specimen must be placed into xylene (toxic!) before embedding. Wet specimens must first be dehydrated in alcohol and then transferred to xylene. Transferring specimens directly from alcohol to Canada balsam won’t work, because the alcohol won’t dissolve the Canada balsam.

  Summary: The advantage of Canada balsam is the long storage ability of the slides. Other, modern, mounting media may have a similar storage ability, but with Canada balsam there is historic experience. A disadvantage is the need for toxic solvents when preparing the specimen. Apparently, it is also not very cheap to obtain.

  Eukitt and other resin-based media: Eukitt is a very fast drying general-purpose resin-based mounting medium. Eukitt will solidify within about 20 minutes. The specimens must be free of water and placed first in alcohol and then in xylene prior to mounting. The use of xylene is a disadvantage, as it is harmful when inhaled. Eukitt itself can also be diluted by xylene to adjust it viscosity.

  Besides Eukitt, a range of other resin-based mounting media are commercially available, such as Diatex, Entellan, Malinol, Rhenohistol and Depex. They differ in their refractive index. All of these mounting media require the specimen to be first dehydrated in alcohol and then transferred to xylene. Some of these resins shrink significantly during the drying process.

  Summary: The advantage of Eukitt is that it is a fast drying mounting medium. The disadvantage is the need for toxic solvents to prepare the specimen.

  Clear nail polish: Nail polish can be used to seal the sides of the coverslip when using aqueous mounting media. It can also be used directly as a mounting medium. The specimens must first be dehydrated in alcohol and can then be directly mounted (without xylene) in nail polish.

  Summary: The advantage of nail polish is, that it is readily available and that it avoids the use of toxic organic solvents to treat the specimens. One disadvantage is, that it seems to shrink a lot when making very thick mounts (such as whole insects).

  Water-insoluble mounting media that remain liquid

  While it is possible to use various oils (immersion oil and paraffin oil) as a mounting medium, they are generally not used to make permanent slides. The specimen must be dehydrated with alcohol and then transferred to xylene so that the liquid mounting medium (the oil) is able to reach all the parts of the specimen. I can imagine that it is this xylene which causes a problem with the sealing of the cover slip, by preventing hardening of the nail polish used for sealing.

  Water-soluble mounting media that solidify

  Glycerol jelly: This is a water-based (aqueous) mounting medium. There are several variations to the recipe, fine tuned for specific mounting applications. The classical recipe according to Kaiser (1880) includes Phenol as an antiseptic, so it hazardous for the use in schools and at home. The handling of this mounting medium, is also not too easy. The bottle with the solid glycerol jelly must first be warmed in a water bath to make it liquid. Do not make it too hot, otherwise it will not solidify any more. The specimen is submerged in the warm jelly and the cover glass is placed on top. Bubbles are a problem with this medium. The edges of the cover glass now must be sealed with nail polish to prevent drying out.

  Glycerol jelly is one of the most difficult mounting mediums to use, but sometimes there is no other satisfactory alternative to an aqueous mounting medium. Water-based mounting media are useful for making permanent mounts of water organisms, algae, protozoa, etc. Glycerol jelly according to Kisser (not Kaiser) is commonly used to preserve pollen samples. Treating some specimens with organic solvent-based mounting media would cause them to shrink or change their shape in other unacceptable ways. Solvent-base media may also dissolve some of the pigments, such as chlorophyll, from the specimen, which does not happen when using aqueous media such as glycerol jelly.

  Summary: The advantage of Glycerol jelly is that it s water-based and that this avoids the need of alcohol dehydration (which possibly deforms the specimens), and other toxic organic solvents. Some specimens can only be satisfactorily mounted in Glycerol jelly. It also does not shrink. The disadvantages include the need for a potentially toxic antiseptic in the jelly, the difficulty of mounting the specimens and the need to seal the cover slip with nail polish.

  Water-soluble mounting media that remain liquid

  Glycerol: It is possible to make a permanent mounts by embedding the specimen either in pure liquid glycerol or a specified glycerol-water mixture. The glycerol-water mixture can be adjusted to an appropriate refractive index. Adding more water lowers the refractive index. It is also possible to use pure water alone (for some delicate algae, for example).

  Algae and other water organisms can be embedded this way. Algae that are embedded in pure glycerol may shrink because the glycerol withdraws water from the cells. If the algae shrink too much, then the glycerol should be more diluted with water. A high concentration of glycerol should be maintained, however, otherwise there is a risk of fungal growth in the medium.

  Making liquid permanent slides is somewhat more advanced. The drop of glycerol must be very small so that it will not touch the sides of the cover slip. On all sides, there should be a few mm of air between the sides of the cover slip and the glycerol. The sides of the cover slip are then sealed with nail polish two or three times to prevent glycerol from leaking out. Here it is very important that the glass surfaces are completely clean and have not been in contact with glycerol, otherwise the nail polish will not hold.

  Summary: The advantage of glycerol is, that fungi and algae do not shrink as much as with other mounting media. It is also not necessary to treat the specimens with alcohol or organic solvents, which may introduce artifacts and remove pigments. The disadvantage is, that it is difficult to prepare slides that are truly permanent in nature. A proper sealing of the cover slip corners is absolutely necessary if one wants to store the slides over extended periods.

  ]]> http://www.microbehunter.com/2010/01/23/an-overview-of-mounting-media-for-microscopy/feed/ 2 http://www.microbehunter.com/2010/01/21/choosing-the-right-mounting-medium-for-making-permanent-slides/ http://www.microbehunter.com/2010/01/21/choosing-the-right-mounting-medium-for-making-permanent-slides/#comments Thu, 21 Jan 2010 11:00:18 +0000 Oliver http://www.microbehunter.com/?p=1478
  Here I will give an overview of the different factors that may be used to decide on which mounting medium to choose.
  There are numerous different mounting media available for making permanent slides. What factors determine the choice of the mounting medium? Here are some possible points to consider.

  Toxicity: Solvent-based mounting media (such as Eukitt and Canada Balsam) require the specimen to be in xylene prior to embedding. This substance is toxic. Other mounting media, such as Glycerol jelly, may contain hazardous antiseptics. This aspect of toxicity is something to consider when making permanent mounts either as a hobby or for educational purposes in schools. One should ask oneself, if one should not use other alternatives.

  Refractive index: The correct refractive index (RI) of the mounting medium can be critical for seeing details of the structure. If one uses phase contrast microscopy, then the RI of the mounting medium should be very different from the RI of the specimen. For regular bright-field work with pigmented specimens, the RI should be the same. In an ideal world, the mounting medium should be matched with the type of specimen. For amateur or educational work, this may be of less relevancy, however. Some high-end microscope objectives are calibrated to be used for a specific RI of the mounting medium, otherwise the resolution is reduced.

  Compatibility with specimen: Specimes which are kept in water should be transferred into a water-based mounting medium. Transferring them into a solvent-based mounting medium may result in a clouding of the resin. Likewise, specimens which are kept in alcohol should be transferred to xylene and then embedded in a solvent-containing mounting medium. Euparal allows the specimen to be present in alcohol.

  Pigment stability: Some mounting media cause a fading of pigments and stains over time. If pigment stability is of relevancy, then one should use mounting media which do not react with the pigments of the specimen. In some cases a fading of pigments is desirable, however. This brightens the specimen and makes it more easy to observe.

  Shrinkage: Some mounting media shrink when they dry. The effect is particularly noticeable when thick specimens (e.g. whole insects) are embedded. Non-water based mounting media are known to do this. Glycerol jelly, which is water-based, does not shrink, however.

  Durability: How long should the permanent slides be stored? Non-solidifying mounting media may not hold the specimen in place very well and there is the risk of running out if not sealed properly. Other mounting media may start to crystallize over the years. Still others may adversely react with the pigments of the specimens. Canada balsam is known for its good durability.

  Cost: Some mounting media (such as Canada Balsam) are quite expensive. Others can be made in the kitchen from readily available materials (Glycerol jelly).

  Ease of use: Here we have to consider two aspects, the preparation of the specimen prior to mounting and the actual mounting process. Some mounting media require the specimens to be dehydrated and fixed before mounting (for resin-based media). This can be a time consuming process. During the mounting process, some media are more prone to form air bubbles (Glycerol jelly).

  ]]> http://www.microbehunter.com/2010/01/21/choosing-the-right-mounting-medium-for-making-permanent-slides/feed/ 0 http://www.microbehunter.com/2010/01/19/different-types-of-microscopes/ http://www.microbehunter.com/2010/01/19/different-types-of-microscopes/#comments Tue, 19 Jan 2010 09:57:50 +0000 Oliver http://www.microbehunter.com/?p=1468 How many different types of microscopes are there? More than you probably thought. I tried to research a list of different types, based on the physical principle used to make an image. Of course, one could also classify the microscopes based on their area of application, their cost, their versatility or any other aspect. These classification systems do have a problem: In this case one one type of microscope can be allocated to several groups, and the system becomes “messy”.

  Optical Microscopes: These microscopes use visible light (or UV light in the case of fluorescence microscopy) to make an image. The light is refracted with optical lenses. The first microscopes that were invented belong to this category. The price of optical microscopes varies from very cheap to nearly unfordable (for the private person, at least). Optical microscopes can be further subdivided into several categories:

  • Compound Microscope: These microscopes are composed of two lens systems, an objective and an ocular (eye piece). The maximum useful magnification of a compound microscope is about 1000x.
  • Stereo Microscope (dissecting microscope): These microscopes magnify up to about maximum 100x and supply a 3-dimensional view of the specimen. They are useful for observing opaque objects.
  • Confocal Laser scanning microscope: Unlike compound and stereo microscopes, these devices are reserved for research organizations. They are able to scan a sample also in depth. A computer is then able to assemble the data to make a 3D image.

  X-ray Microscope: As the name suggests, these microscopes use a beam of x-rays to create an image. Due to the small wavelength, the image resolution is higher than in optical microscopes. The maximum useful magnification is therefore also higher and is between the optical microscopes and electron microscopes. One advantage of x-ray microscopes over electron microscopes is, that it is possible to observe living cells.

  Scanning acoustic microscope (SAM): These devices use focused sound waves to generate an image. They are used in materials science to detect small cracks or tensions in materials. SAMs can also be used in biology where they help to uncover tensions, stress and elasticity inside biological structure.

  Scanning Helium Ion Microscope (SHIM or HeIM): As the name suggests, these devices use a beam of Helium ions to generate an image. There are several advantages to electron microscopes, one being that the sample is left mostly intact (due to the low energy requirements) and that it provides a high resolution. It is a relatively new technology and the first commercial systems were released in 2007.

  Neutron Microscope: These microscopes are still in an experimental stage. They have a high resolution and may offer better contrast than other forms of microscopy.

  Electron Microscopes: Modern electron microscopes can magnify up to 2 million times. This is possible, because the wavelength of high energy electrons is very small. At the same time, the high energy electrons are pretty tough on the sample being observed. It may take a long time to completely dehydrate and prepare the specimen. Some biological specimens also need to be coated with a very thin layer of a metal before they can be observed.

  • Transmission electron microscopy (TEM): In this case, the electron beam is passed through the sample. The result is a two dimensional image.
  • Scanning electron microscopy (SEM): Here the electron beam is projected on the sample. The electrons do not go through the sample but bounce off. This way it is possible to visualize the surface structure of the specimen. The image appears 3 dimensional.

  Scanning Probe Microscopes: It is possible to visualize individual atoms with these microscopes. The image of the atom is computer-generated, however. A small tip measures the surface structure of the sample by rastering over the surface. If an atom projects out of the surface, then a higher electrical current will flow through the tip. The amount of current is proportional to the height of the structure. A computer will then assemble the position data of the tip and the current to generate an image.

  Conclusion: Microscopes can be classified based on the physical principle that is used to generate an image. Different microscopes visualize different physical characteristics of the sample (eg. elasticity can be visualized with acoustic microscopes). Image contrast, resolution (which determines magnification) and destructiveness of the sample are other relevant parameters.

  ]]> http://www.microbehunter.com/2010/01/19/different-types-of-microscopes/feed/ 2 http://www.microbehunter.com/2010/01/16/taking-stable-photographs-with-a-microscope/ http://www.microbehunter.com/2010/01/16/taking-stable-photographs-with-a-microscope/#comments Sat, 16 Jan 2010 11:00:16 +0000 Oliver http://www.microbehunter.com/?p=1470 Specimens which are suspended in water are not completely immobilized. Small objects will start to vibrate when one is tapping on the table on which the microscope stands. The optics of the microscope will magnify even the smallest vibrations. These vibrations become problematic when taking pictures with a photo camera which is mounted directly to the microscope. Both pressing and releasing the shutter button as well as the shutter mechanics itself can produce so much vibration that the image quality suffers. One can, of course, use a cable release or self-timer to improve the situation, but the shutter mechanics of the camera still cause considerable shaking. The effect of the vibrations are, naturally, more pronounced the higher the magnification in use. Permanent mounts (in which the specimens are immobilized) are somewhat less sensitive to vibrations, but the effect of a shutter release vibration is still there at higher magnifications.

  There are several possibilities to reduce the vibrations:

  Long exposure time: The microscope-camera system vibrates for the fraction of a second after shutter release. One should therefore use a long exposure time (2-5 sec.). The camera will therefore collect most of the light when the system is steady. Of course, this does not work for moving objects.

  Very short exposure time: Alternatively the exposure time can be significantly reduced (about 1/250sec). This is so fast that the vibration will hardly be recorded. This requires much light, however.

  Micro flashing: One can also use a flash system mounted above the light source. With experimentation, it is also possible to make a system like this oneself.

  The cardboard technique: A similar technique is used in astronomy to make steady images. The camera is set to “bulb” (B) and the shutter is opened. The light intensity has already been adjusted, but the light source is covered by a dark piece of cardboard. The cardboard is then removed for exposure. This method is indeed free of vibrations but requires long exposure times to be practical.

  Mirror lock up: Some digital SLR cameras have a mirror-lock-up function. The integrated mirror of the camera can be moved into an “up” position. This reduces the vibrations significantly because the mirror of the camera does not have to swing up during exposure.

  Flexible camera-microscope connection: Here, the microscope does not carry the weight of the camera. Rather, the camera is mounted either on a tripod or on another separate system. A vibration of the camera is therefore not passed on to the microscope.

  So what methods do I use? I use a combination of several measures: I use a mirror lock-up function, a self-timer (2 sec) and a “long” exposure time of about 2 sec. During the mirror lock-up, it is not possible to look through the viewfinder to focus. Focusing has to be done already beforehand, or one could use the live-view feature to focus and evaluate the picture on the LCD screen before exposure.

  ]]> http://www.microbehunter.com/2010/01/16/taking-stable-photographs-with-a-microscope/feed/ 0 http://www.microbehunter.com/2010/01/15/staining-bacteria/ http://www.microbehunter.com/2010/01/15/staining-bacteria/#comments Fri, 15 Jan 2010 11:00:31 +0000 Oliver http://www.microbehunter.com/?p=1466 Here is yet another link to an article from Popular Science magazine. It deals with the isolation, fixing and staining of bacteria. I would not recommend the use of some of the solvents that they use (such as xylol) with children, however. They also describe a blood smear preparation, what I do not recommend for schools (it may not even be allowed in some countries). Still, the article gives a very nice introduction into several preparatory techniques. The article stretches over several pages, click the link at the end of the pages to continue reading. The fact that the article was published 75 years ago, in 1934, does not matter. The preparatory method stayed the same.

  Link to the article: Microb hunting with your Microscope (Popular Science, Sept 1934)

  ]]> http://www.microbehunter.com/2010/01/15/staining-bacteria/feed/ 0 http://www.microbehunter.com/2010/01/14/virtual-microscope-dandelion-seed/ http://www.microbehunter.com/2010/01/14/virtual-microscope-dandelion-seed/#comments Thu, 14 Jan 2010 11:00:04 +0000 Oliver http://www.microbehunter.com/?p=1465
  

  
  This the the parachute of a dandelion seed. The seed is not shown, it is attached to the long extension on the right. The leaves of the plant are toothed. The name “dandelion” comes from the French “dent-de-lion” meaning “lion’s tooth”. The microscopic observation reveals that the leaves are not the only part of the plant that have teeth. The fine hair of the parachute also show a tooth-like appearance.

  Quite noticeable is the chromatic aberration, which can be seen as a blueish fringe around some of the hair.

  ]]> http://www.microbehunter.com/2010/01/14/virtual-microscope-dandelion-seed/feed/ 0 http://www.microbehunter.com/2010/01/13/virtual-microscope-the-tick/ http://www.microbehunter.com/2010/01/13/virtual-microscope-the-tick/#comments Wed, 13 Jan 2010 11:00:23 +0000 Oliver http://www.microbehunter.com/?p=1463
  

  
  This is a darkfield image of a tick. Ticks are blood-sucking arthropods. They possess 8 legs and are not insects, but rather are related to the spiders. Ticks are known to transmit various diseases, such as Lyme’s disease and encephalitis.

  For more information on the tick, read the following post: The Tick (Ixodidae).

  ]]> http://www.microbehunter.com/2010/01/13/virtual-microscope-the-tick/feed/ 0 http://www.microbehunter.com/2010/01/12/stains-and-reagents-for-microscopy/ http://www.microbehunter.com/2010/01/12/stains-and-reagents-for-microscopy/#comments Tue, 12 Jan 2010 13:30:32 +0000 Oliver http://www.microbehunter.com/?p=1461 I found an article in Popular Science Magazine (see link below) which gives a general overview of different stains that can be used in microscopy. The article divides the stains into three categories:
  • Common household chemicals: this includes Iodine, for example. They are very readily available.
  • Substances used mostly for microscopy: Methylene blue, Hematoxyline, and Eosine belong to this group.
  • Commercial substances: they are sold by companies specializing in microscopic chemicals.

  The article also provides a step-by-step guide on how to stain a blood sample (don’t do this in schools due to danger of infection).

  Link to the article: Help Your Microscope with Stains and Reagents (Popular Science, March 1937)

  ]]> http://www.microbehunter.com/2010/01/12/stains-and-reagents-for-microscopy/feed/ 0 http://www.microbehunter.com/2010/01/11/virtual-microscope-maple-leaf-skeleton/ http://www.microbehunter.com/2010/01/11/virtual-microscope-maple-leaf-skeleton/#comments Mon, 11 Jan 2010 09:34:58 +0000 Oliver http://www.microbehunter.com/?p=1455
  

  
  This is a scan of maple leaf vascular tissue, done with a normal flat-bed scanner.

  Method: Preparing the leaf was the difficult and time-consuming part. The leaf was boiled for several hours until the cells started to separate. I then carefully lifted the leaf out of the pot and placed it on a plate with water. The soft tissue was then removed with a stiff brush, trying not to damage the delicate veins. The veins were then rinsed in alcohol to remove the remaining chlorophyll, washed in water to remove the alcohol. The alcohol also shrinks the structures, but it will expand again when washed in water. The leaf skeleton was then, pressed and dried. Not all leaves work equally well! The leaves of some plant species are so stiff that the cells do not want to come off when boiled. Don’t waste your time on these leaves.

  A confession: Because the stem of the leaves come off very easily, I had to scan it separately and then integrate it into the picture later using some photo editing. I could not scan the veins and the stem at the same time, because it then would not be flat on the scanner. You will also notice that some parts of the leaf are not in focus. This too is because the leaf was not completely flat on the scanner.

  For teachers and parents: Boil some leaves with your students/children and let them prepare the leaf skeleton. Then observe the leaf skeleton under the stereo microscope.

  ]]> http://www.microbehunter.com/2010/01/11/virtual-microscope-maple-leaf-skeleton/feed/ 2 http://www.microbehunter.com/2010/01/11/virtual-microscope-female-pine-cone-pinius/ http://www.microbehunter.com/2010/01/11/virtual-microscope-female-pine-cone-pinius/#comments Mon, 11 Jan 2010 08:17:40 +0000 Oliver http://www.microbehunter.com/?p=1453
  

  
  For more information on the pine cone, have a look at the following post: Female Pine Cone (Pinus) The specimen size is approximately 20mm from left to right.

  ]]> http://www.microbehunter.com/2010/01/11/virtual-microscope-female-pine-cone-pinius/feed/ 0 http://www.microbehunter.com/2010/01/10/virtual-microscope-aristolochia-sipho/ http://www.microbehunter.com/2010/01/10/virtual-microscope-aristolochia-sipho/#comments Sun, 10 Jan 2010 22:09:58 +0000 Oliver http://www.microbehunter.com/?p=1451
  

  If you can not see anything, then you need to install a flash player. The image shows the cross section of the stem of the Aristolochia sipho plant. The image is an inverted (negative) image, and not a dark-field image. Why did I choose to invert the colors? The reason is surprisingly unscientific: it simply looks better… The diameter of the stem is about 6mm across. The annual rings are also visible.
  

  ]]> http://www.microbehunter.com/2010/01/10/virtual-microscope-aristolochia-sipho/feed/ 0 http://www.microbehunter.com/2010/01/10/digitizing-photographic-slides-with-a-digital-camera/ http://www.microbehunter.com/2010/01/10/digitizing-photographic-slides-with-a-digital-camera/#comments Sun, 10 Jan 2010 18:00:40 +0000 Oliver http://www.microbehunter.com/?p=1430
  
  Slide duplicator attachment: The left duplicator is mounted instead of the camera objective (via T2 adapter ring). The right one is attached to the existing objective (via filter threading)
  
  
  Both systems compared.
  
  
  The slide/film holder is the same in both cases.
  
  
  Digitized slide showing vitamin C.
  
  
  Digitized slide showing vitamin C.
  
  Several years ago, at a time when digital single-lens reflex (SLR) cameras were still financially unobtainable, I used slide film to document my microscopic observations. These slides are now sitting, more or less nicely sorted, in a folder, doing pretty much nothing. I don’t even have a slide projector to look at them. Evidently the slides need to be digitized so that the resulting images can be used more widely.

  There are several ways to digitize the slides:

  • Using a slide or film scanner: This is the method of choice if you want to retain image quality. These devices are connected over USB to a computer. On the down side, scanning takes a long time and a film scanner is also not cheap. Some better slide scanners have a dust removal system.
  • Use a flat-bed scanner: This is possible, if the resolution of the scanner is high and if there is a background lighting. Some flat bed scanners come with an appropriate slide holder. I found this system too time consuming, however.
  • Get the slides scanned by a company: I did this once, it was expensive, but the quality was good. This is probably suitable for a smaller number of slides
  • Photographing slides with a dedicated slide duplicator: This duplicator is directly mounted on the camera, instead of the existing objective. There is a slide/film holder attached. The slide duplicator that I initially tried was designed to reproduce 36mm slides again on 36mm analog systems (or digital cameras with a large sensor – the “full-format” systems). My digital camera’s sensor is smaller than film size. As a consequence it was not possible to fit the whole slide on the image and I always had added magnification. The objective allowed me to zoom in, but not zoom out (what I would have needed.) There are objectives like this that are specifically made for digital SLR cameras with a smaller sensor. So watch out if you get one of these devices.
  • Photographing with a duplicator in front of the objective: This system is mounted in front of the camera’s existing objective. It contains extra lens elements to magnify the slide. This is the system that I used, and it worked well. The adapter is screwed into the filter threading of the camera’s original objective, so be careful that they are compatible (or use an extra adapter ring). One possible problem may be, that there are now many lens elements between the slide and the camera’s sensor. The image quality may suffer because of this. For my purposes, this was perfectly fine. Considering the generally low resolution of microscopic images, the quality loss was negligible. This duplicator also allows me to zoom in. This way I can take overlapping pictures of the slide and assemble them (“stitch” them) using panorama software. This way it is possible to reproduce the slide with an extremely high total resolution – but it’s time consuming (and it’s questionable if the slide / microscopic image has the necessary resolution in the first place.)

  About exposure time

  It’s very important to rest the camera body as well as the objective (whatever system is used) solidly on a stable surface. The objective should not be able to vibrate in relation to the camera body. If both are stable, then the optimum exposure time (to minimize vibrations) should not be too critical. Because I am in no hurry, I set the exposure to about 2 sec. The whole system will have vibrated out (and be steady) for the most part of the exposure. Long exposure times are more important when the camera is mounted on a microscope. In this case the effects of vibrations are much more evident. To minimize vibrations even more, I use the mirror-lock up feature of my camera.

  About white balance

  My camera allows me to adjust a custom white balance. I first take a blank picture of the white screen (the adapter system has a white screen) of the and use this as a reference image. The camera will then automatically adjust the white balance of all images that are taken. If your camera does not allow for the use of a reference image, then you should set the white balance manually based on the actual light source used. It’s not a good idea to use auto-white balance, as there is a color drift. Depending on the algorithm used, the camera may assume that the brightest spot on the image represents white (or a shade of grey, if darker), which may not be the case.

  ]]> http://www.microbehunter.com/2010/01/10/digitizing-photographic-slides-with-a-digital-camera/feed/ 0 http://www.microbehunter.com/2010/01/07/dirty-microscope-objective-its-effect-on-image-quality/ http://www.microbehunter.com/2010/01/07/dirty-microscope-objective-its-effect-on-image-quality/#comments Thu, 07 Jan 2010 21:42:36 +0000 Oliver http://www.microbehunter.com/?p=1435
  
  Macro image of the front lens of a dirty and cracked 40x objective.
  
  
  
  
  A clean 40x objective provides a sharp and crisp image.
  
  
  A dirty objective produces soft, low-contrasty images. The picture was taken with the above 40x objective.
  
  The microscopes in the school where I work have been in operation almost 30 years (!!) now. And the fact that most of them have remained usable says quite something about the quality of these devices. They are soon going to be collectively sent in for maintenance, and this is the last opportunity to do a little quality check.

  Most devices were still in a reasonably good condition, with the biggest problems in the mechanics. A check of the optics revealed that most of them were still quite OK, but the 40x objective of one of the scopes was in a particular desolate condition. A macro image of the front lens can be seen on the right. I suspect highly that one of two things happened to the objective:

  The objective could have been rotated into immersion oil and was subsequently not cleaned. Students sometimes want to use a lower magnification after they used the 100x oil immersion objective.

  A second possibility is, that the “dirt” on the objective is in reality resin for making a permanent slide. Maybe some students attempted to make a permanent slide and used too much resin, and did not wait for the resin to dry out. The front part of the objective was then rotated into the resin.

  The origin of the crack in the lens, remains a mystery. The lens is spring loaded , and retracts when crashed into the specimen.

  The resulting image was not usable at all. I included two pictures of the same area, one with an intact 40x and one with the dirty and cracked objective from above. I think that the two images speak for themselves.

  What do we learn from this? Proper microscope instruction saves money. And be really careful about using immersion oil and resin in the classroom. Don’t even get the students into the position of making such mistakes. In my view, a 100x oil immersion objective is not even necessary for most microscopic work (unless you deliberately want to teach the students different microscopic techniques). Remove the objectives from the microscopes and store them in a safe place.

  ]]> http://www.microbehunter.com/2010/01/07/dirty-microscope-objective-its-effect-on-image-quality/feed/ 0 http://www.microbehunter.com/2010/01/04/what-in-the-world-is-microbe-hunting/ http://www.microbehunter.com/2010/01/04/what-in-the-world-is-microbe-hunting/#comments Mon, 04 Jan 2010 22:18:53 +0000 Oliver http://www.microbehunter.com/?p=1427 A quick Google search of the term “microbe hunting” revealed 2460 hits, the term “microbe hunter” a mere 21300 hits. The combination of these words with “amateur microscopy” returned a total of… 1 and 4 hits respectively. This is not much. The one returned hit is particularly interesting. It is from Popular Science, September 1934, entitled “Microbe hunting with your Microscope”. It gives a nice description on how to prepare bacteria for microscopic observation.

  Microbe hunting – a new term to an old pastime and hobby? The terms seems to be around now for over 70 years, but is still not used widely. Maybe it is time to establish this term a bit more. I have to admit that “Amateur Microscopy?” does sound a bit more “professional” (is this a paradox?), but the sentence “I’m a microbe hunter” flows much easier than “I’m an amateur microscopist”, so maybe this is enough justification to establish that term, even if amateur microscopists observe specimens other than microorganisms as well.

  In any case, I herewith propose that the term “Microbe Hunting” be used interchangeably for amateur microscopy. Comments?

  ]]> http://www.microbehunter.com/2010/01/04/what-in-the-world-is-microbe-hunting/feed/ 1 http://www.microbehunter.com/2009/12/06/mitosis-stages-of-the-lily/ http://www.microbehunter.com/2009/12/06/mitosis-stages-of-the-lily/#comments Sun, 06 Dec 2009 15:33:52 +0000 Oliver http://microscopy.okim.info/?p=1400
  
  Interphase. The nucleus is visible.
  
  
  Prophase. Chromosomes are starting to form.
  
  
  Metaphase. The chromosomes align at the equator of the cell.
  
  
  Metaphase. The chromosomes align at the equator of the cell.
  
  
  Anaphase. The two sister chromatids are separated.
  
  
  
  
  Anaphase. The two sister chromatids are separated.
  
  
  Possibly metaphase or anaphase seen head-on. The chromosomes are possibly pointing towards the viewer.
  
  
  Telophase. The spindle fibers are still visible between the two nuclei. The cytoplasm has not yet divided.
  
  
  Background Information: Mitosis is cell division in eukaryotes. During mitosis the chromosomes are visible. Interphase is not considered part of cell division. The following stages are Prophase, Metaphase, Anaphase and Telophase.
  
  Image Information: Higher resolutions are, unfortunately not available. A magnification of 400x was used to obtain these images. The cells were treated with a dye that has a high affinity for DNA.
  
  ]]> http://www.microbehunter.com/2009/12/06/mitosis-stages-of-the-lily/feed/ 3 http://www.microbehunter.com/2009/11/28/buttercup-ranunculus-repens-root/ http://www.microbehunter.com/2009/11/28/buttercup-ranunculus-repens-root/#comments Sat, 28 Nov 2009 16:56:14 +0000 Oliver http://microscopy.okim.info/?page_id=1397
  
  Vascular tissue of a Buttercup, Ranunculus, root.
  
  
  Image Information: The root was microtomed and stained. The triangular structure on the left is the vascular tissue, used for transporting substances up and down the plant.
  
  Background Information: Ranunculus is a large genus encompassing about 400 different species. They possess bright yellow or white flowers and some have orange or red flowers. All members of the genus are poisonous. The toxin is inactivated when dried, hay used for livestock is therefore safe.
  
  ]]> http://www.microbehunter.com/2009/11/28/buttercup-ranunculus-repens-root/feed/ 0