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.

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

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).

Method:

1. Make sure that the specimen in completely dry. You may first place the specimen in alcohol to withdraw water, and then let the alcohol evaporate. Note, that this procedure may deform the specimen, however.
2. Stick a piece of the double-sided tape on the slide. The tape should have about the same size of the cover slip, or be slightly smaller.
3. Using the knife (not suitable for children!), cut out a square in the center part of the tape and discard this piece of tape. You should now have a square “frame” of double sided tape on the microscope slide.
4. Place the specimen into the center, it is now surrounded by the tape. The specimen should not be thicker than the thickness of the tape.
5. Place a cover slip on the tape and carefully (!) press the glass against the tape. The tape will hold the cover glass in place. You should not apply pressure to the center part of the glass slide, or it may break. You could roll a round pencil over the cover glass to press it against the tape.
6. Observe using low magnification. The specimen is not embedded in a mounting medium with an appropriate refractive index. The resolution of the image will therefore be lower at higher magnifications.

Suitable objects for dry mounting:

• Wings of insects
• Whole small insects
• Scales of butterfly wings
• Sand or soil particles
• Dust samples
• Dried skin, dandruff
• Different types of paper, etc.

Fructose syrup is a water-based mounting medium, which is suitable for a wide variety of specimens. It is safe to use and it is easy and cheap to make. Spills can be easily washed out with water. One disadvantage is that the color of the specimens may fade and that some stains will loose intensity over time. This is due to the low pH of the medium. Fructose syrup is not suitable for making slides that last for many years, but is should be sufficient for classroom usage, where students would like to re-examine their specimens over and over again over a period of time. The medium will not completely solidify, so it is necessary to seal the cover glass at the side.

Materials: distilled water, fructose, dropper bottle or other container, optionally nail polish / nail varnish.

Method for making fructose syrup:

1. Fill several grams of fructose into the dropper bottle.
2. Using a marker, mark the level of the fructose on the glass bottle.
3. Using the dropper, add distilled water to the fructose. The fructose will dissolve and the volume will decrease. Add more water to maintain the total volume level.
4. Store the bottle for several days in a warm place, or use a warm water bath. It takes this time for all of the fructose to dissolve. At the end, you should have a clear, sticky liquid. It is then ready for use.

Method for using fructose syrup:

1. The specimen to be mounted (eg. a small insect, some plant sections etc.) must be first placed into water. In most cases, fresh material is already stored in water. It could, however, be that due to previous processing or storage the specimens are soaked in alcohol or other organic solvents. This solvent must be removed first. If the specimens were stored in alcohol, then slowly transfer them into distilled water by placing them gradually into more and more dilute alcohol. If you transfer the specimen directly from concentrated alcohol into pure water, then there is the danger that the specimen changes its shape.
2. Place a drop of the mounting medium on the slide, then place the specimen (not wet) into the drop. Place another drop of mounting medium on top of the specimen. The specimen is now surrounded by the medium from top and bottom. Finally, place a cover glass on top of the mounting medium.
3. Store the slide for a few days horizontally. Some water will evaporate, but the syrup will not solidify completely. If you store the slide for a long time (in a dry environment), then the fructose may start to crystallize out. You can then observe the specimen under the microscope.
4. Optional (careful, organic solvents involved!): Seal the corners of the cover glass with some nail polish (nail varnish). This will prevent the syrup from flowing out and will prevent moisture exchange. The slide should be stable for a few months.