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Fixing specimens for making permanent slides



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.
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10 Comments

  • denis fitt says:

    as a novice it was very interesting thank you,

  • Dave Bolick says:

    Thank you for the information. The aspect that I am most important to me is the effect of fixation on tissue shrinkage.

    Which histology fixatives have the least shrinkage?

    • Oliver says:

      Hi, I think that it is important to always match the type of fixative with the specimen. I would guess that water-based fixatives containing formalin or formaldehyde (careful) do not cause much shrinkage, as these fixatives do not withdraw water from the specimen. The problem is the dehydration process afterwards, which then may cause shrinkage. This problem can be overcome by slowly dehydrating the specimen.
      Oliver

  • Herbdhall says:

    How can a specimen be made permanent on a slide ?

    • Oliver says:

      >How can a specimen be made permanent on a slide?

      This depends on the specimen. Must be fixed, microtomed, dehydrated and mounted. Alternatively it is possible to use a water-based mounting medium in which dehydration is not necessary. This can not be explained in a few words, because the procedure depends very much on the type of specimen investigated. Different specimens have different specific procedures.
      Oliver.

  • Raul says:

    Have you ever heat-fix bacteria for further visualization with fluorescent antibodies using an epifluorescent microscope?

  • how can reference slides of different flora of pollen prepared?

  • azma says:

    hi. do you have any idea how to make a permanent slide for microalgae?

  • Nouman Khalid says:

    Hi! have you any idea about preparation of permanent slides of bacteria?

    • Oliver says:

      Hello,
      1. air-dry the bacterial suspension
      2. heat fix them to prevent them from becoming detached
      3. Possibly stain them.
      4. Allow to air dry completely
      3. Apply mounting medium and cover glass.

      Oliver

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