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.

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

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

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.

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.

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”.

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.

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

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