CR Scientific: In introduction to some Fixatives used in Microscopy

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Some Fixatives & Preservatives
for Microscopy
by Christian Thorsten

Fixatives can be thought of as preservatives for tissues or cells that are going to be mounted permanently (or semi-permanently). Without fixation, these cells or tissues would undergo morphologic and chemical changes that could ruin them for study. In some cases there would also be growths of bacteria, yeasts, or fungi that would consume the specimens.

One of the chief functions of any fixative is to halt certain enzymatic reactions that occur when a cell dies; these are what we might call "autolysis", reactions by which the cell's own enzymes destroy its structural integrity. Ideally, a fixative not only halts these reactions but also hardens structural elements so cells can withstand sectioning and permanent mounting without turning to mush.

Because the chemistry of a cell is so complex and heterogeneous, there is no universal fixative. A specific agent may be used to preserve chromatin, for example; another may be preferable for fixing mitochondria. Even when a certain agent is useful for multiple specimen types, there can be marked procedural differences.

It is probably not a coincidence that the better fixatives are poisonous; fixatives are, by definition, agents that irreversibly halt biochemical processes by reacting with cell structures. However, not all poisons are useful as fixatives; furthermore, a few of the most common fixatives-- vinegar, ethanol, lactic acid-- aren't really poisons by the everyday definition (although: sola dosis facit venenum). It is important to understand that many fixatives, while toxic, aren't necessarily the deadliest or most intense poisons known; the histologist inundates cells with fixative in a way that, at least to the cells, is like hitting them with a tidal wave of the stuff.

It remains unknown to this writer just why fixatives tend to make cells adhere better to glass slides, but this is an important (if sometimes overlooked) benefit of their use. It seems possible that macromolecule-rich liquids find their way into the ultra-microscopic pores and valleys in the surface of the glass, and fixing causes these liquids to become unable to run back out again. Such would be analogous to the drying of glue or perhaps gelatin dessert. One may notice a similar habit when certain foods have been cooked or left to stand for a long time in their containers: something is indeed different about the really stubborn patches of residue. Denaturation almost certainly plays a role here.

Now, let's go onward to a discussion of some fixatives. This article cannot pretend to be a complete reference; such would take up one or more volumes. Following is just a summary:

Acetone (CH₃COCH₃ or H₃CC(=O)CH₃): as with ethanol, acetone is used in the biochemistry lab to bring proteins down from solution. It therefore makes sense that acetone could be used as a fixative and a dehydrating agent in the same manner as alcohols. Like alcohol, acetone's mode of action is more a matter of physical chemistry than anything else: it interferes with the hydration cloud that keeps charged protein molecules in aqueous solution. For that matter, it is able to precipitate many other, normally water-soluble materials; a number of metal salts, for example, are very soluble in water but nearly insoluble in acetone.
Acetone is also a good solvent for lipids; like alcohol, acetone can remove exposed lipid components that are not tightly bound.

It is worth mentioning that cold acetone (0 °C and below) has long been used to precipitate enzymes without destroying their activity (Green and Hughes, 1955). Organic solvents in general lower the dielectric constant of an aqueous medium. As an experimenter quickly learns, however, acetone at 0° C may yield an enzyme precipitate that is still active, while acetone at 30 °C may give a completely inactive one. Unless kept very cold, organic solvents such as acetone and alcohol can unravel the tertiary structure of proteins; this should come as no surprise, since much of that tertiary structure depends on hydrogen bonding.

Acetic Acid (CH₃COOH or "HOAc"): a 5% solution is useful as a non-toxic fixative, but it has some pronounced limitations. Acetic acid will partially hydrolyze proteins in the specimen, especially with prolonged exposure. This can lead to unwanted structural changes; the classic "rubber wishbone" experiment is illustrative. However, limited hydrolysis of proteins may be acceptable, depending on what is being studied.

Acetic acid is not good for blood smears if one wants to view erythrocytes (RBC's), since the acid dissolves them away. White blood cells are a bit more robust. (In one specimen this writer prepared, there were quite a few WBC's but not a single RBC in sight. Guess which fixative was used!)

Alcohol is not a very efficient fixative by itself. However, alcohol does have precipitant / coagulant effects similar to those of acetone (see above). Alcohols in general are used more as solvents for other fixatives than as fixing agents of their own.

Glycerol is an alcohol as well; actually it's a tri-alcohol, having three -OH functional groups. Like other alcohols, its mode of action is to draw the water out of cells. If this is done too abruptly, the cells shrivel and are ruined for microscopic observation. If it is done gradually-- by steps of increasing glycerol concentration-- it can be useful for preserving cells.

Alcohol fixation relies more on physical chemistry (hydrogen bonding, hydration shells, etc) than on reactivity. However, alcohol that has been made free or almost free of water is an effective protein denaturant and lipid solvent; this can work either for or against the investigator, depending on what's being studied.

Converting ethanol to acetaldehyde by chemical means, possibly by careful oxidation with a metal catalyst, would turn it into a better fixative (though it wouldn't be ethanol anymore!) by introducing some reactivity to the picture. Converting methanol to formaldehyde gives one of the best fixatives of all (see below), though the best way to get that is just to buy the formaldehyde ready-made. However, it seems likely that methanol, when introduced to the still-active enzyme systems of a cell, should yield some HCHO. Mammalian cells, especially human ones, seem to have well-developed systems to deal with the products of ethanol metabolism; methanol, on the other hand, is toxic even in small amounts. Thus, while methanol is not as good a protein coagulant as ethanol (Green and Hughes, 1955), it seems possible that methanol could be the better fixative for cell preparations.

Other alcohols, being organic solvents, have some fixative effect; isopropanol is an inexpensive and readily-available one. Some time ago we bought some butanol and pentanol for a planned rash of paper chromatography experiments; it has occurred to this writer that these might also work as fixatives, though longer-chain alcohols do become progressively less miscible with water and therefore have less dehydrating action. However, this might work to one's advantage if a non-aqueous mounting medium were used.

Alcohol as a fixative should in theory work better if used warm instead of cold, although its destructive effects on certain cell components might be too rapid; for example, warm alcohol dissolves lipids more readily. Some time ago the author had kludged together a slide heater (fig. 1) for one of our newsletters. The heater limits the slide temperature to 100°C, thus addressing the problem of unwanted specimen charring or alcohol ignition.

Figure 1. A slide heater fabricated from common items. The funnel is clamped in place over a beaker of water which is boiled on a hot plate. A slide containing the specimen is then laid on the copper platform. The heat never exceeds 100°C.

The copper platform was soldered to the copper tube sections; the whole copper portion was joined to the funnel by some high-temperature, aluminum-filled epoxy (Hardman 04002 worked well).

Cetylpyridinum chloride has been used to fix mucopolysaccharides (now known as glycosaminoglycans, though this writer will probably never stop using the old term). Certain brands of mouthwash contain cetylpyridinum chloride; it is not inconceivable that these could act as fixatives for some applications.

Chromium Trioxide (CrO₃): a circa 1 to 3% aqueous solution is used as a fixing agent, sometimes with acetic acid. However, even more dilute solutions (0.2 to 0.5%) have worked acceptably in this author's own experiments.

Alcohol should not be brought in contact with solid CrO₃, as spontaneous combustion can result. In such cases where the reagents are diluted safely before mixing, CrO₃ and alcohol together might cause undesirable effects, such as insoluble precipitates of Cr₂O₃. Since alcohol is commonly used in the dehydration process and may be present in the mountant, unbound CrO₃ residue is removed with water first.

Hexavalent chromium is of course poisonous, and it may be a carcinogen as well. Though hazardous, it still much safer to handle than osmium tetroxide or mercuric chloride and is a reasonable substitute in many cases. Just remember the spontaneous combustion hazard, mentioned above; keep CrO₃, whether solid or in solution, away from any type of combustible organic material.

Chromium trioxide is a very important fixing agent. Its cousin, dichromate ion (Cr₂O₇^2-), is also important but may have a slightly different mode of action.

CrO₃, like a few other compounds, is not only a fixative but also can act as a mordant (Fig. 2).

Figure 2. A cluster of white blood cells from a salivary gland infection. Fixation with 0.2% chromium trioxide in 5% HOAc for a duration of ca. 15 minutes had a pronounced effect on the outcome of the staining. Magnification 400x. A full write-up on this experiment is now available at this link.

After being flooded with CrO₃, there wasn't much left that could autolyze the specimen. Indeed, it is still fully intact after more than a year.

Copper Acetate (Cu(CH₃COO)₂): a fungicide known since ancient times, copper acetate also sees some use in fixative recipes. Other heavy metal salts are also used as fixatives- lead, cobalt, chromium, silver, mercury, sometimes even uranium. These metal ions can act as precipitants for proteins and amino acids, which would at least partially explain their fixative action. The ones that don't cause outright coagulation can still inactivate enzymes via complex formation.

The author recently did an experiment in the preparation of copper acetate by electrolysis. The membrane used was made of protein (actually, sausage casing). During the course of the experiment there was a good illustration of the "fixative" action of copper, especially in warm acetic acid. What happened was that hydrolysis by acetic acid caused some of the membrane protein to go into solution; gentle warming in the presence of the dissolved Cu⁺⁺ ions caused the proteins to coagulate rapidly. The extent was greater than if acetic acid had been used alone. The combination of copper ions and acetic acid was (and is) effective for "fixing" or denaturing proteins and enzymes.

Cupric salts have both fixative and mordant properties.

Iodoform (CHI₃) is a good preservative for microscopy specimens. Like formalin, iodoform kills microbes and other cells but is not a protein coagulant. Iodoform's chemical mode of disinfection is not currently known to this writer, though I₂ and/or HI are probably involved somehow. Iodoform would probably be a useful fixative for protozoa.

This writer has used iodoform to preserve aqueous stain preparations for long-term storage. A vial of aqueous Eosin Y stain treated with it has shown no fungal growth for more than two years; surprisingly, a similar vial treated with thymol developed huge mold colonies after only a few months. What's poisonous to one species can be a nutrient for another, especially in the world of microbes.

Formaldehyde (written HCHO, HC(=O)H, or CH₂O) is actually a gas, but it is typically kept as a 37% aqueous solution (formalin, sometimes called "formol"). Indispensible for the histology lab, formaldehyde is fairly reactive toward the various functional groups in a cell. It can cross-link different macromolecules or different parts of the same macromolecule. In biochemical terms, a covalent adduct or cross-link is irreversible; while it may come apart with some reagent under hours of reflux at 90°C (as organic chemists are accustomed to doing), it generally won't come apart under physiological conditions unless there is an enzyme specific to it. It is this chemical property that makes formaldehyde such an effective fixing agent. For example, alcohol groups on two different molecules can react with formaldehyde to form an acetal cross-link; acetal formation is taught in undergrad organic chemistry courses. Imines, amines, peptides (i.e., amides), thiols, carboxyl groups, and a couple of other functional groups can also react with formaldehyde to give cross-links; this has been mentioned throughout the literature, but see for example Barka and Anderson (1963).

Formalin is also used to kill protozoal infections in fish ponds, by which it undoubtedly has a similar mechanism (in other words, it "fixes" the protozoans).

Formalin is one of the most important of all fixatives. Every professional lab has it, and in this writer's opinion it's still appropriate for use by the teacher or advanced amateur if safety precautions are excercised. Wear gloves and goggles and work in a well-ventilated area. Don't smoke, eat, or drink in the lab (you shouldn't anyway). Wash your hands with soap a couple of times after you're done working.

Lactic Acid is a mild organic acid produced by human muscle tissue during anaerobic exercise. It is used in some fixative and mountant recipes, sometimes with glycerol, and often with phenol as well ("lactophenol"). One benefit of lactic acid is that it is essentially non-toxic (though, like acetic acid, it's corrosive in concentrated form). Pure lactic acid becomes a solid at some point near room temperature, so it's usually sold and kept in aqueous solution; 80-85% is typical.

In the future we may do a separate write-up on preparation of lactic acid from yogurt.

Lead Acetate or other lead salts have been used in fixative formulae. Lead salts, like those of other heavy metals (Cr, Os, Hg, etc.) form complexes with proteins, causing them to become denatured or otherwise biologically inactive. Like these other heavy metals, lead tends to "poison" enzymes irreversibly, halting their catalytic action. Soluble lead salts are quite poisonous, though mercuric salts are considerably more so.

One useful property of lead compounds is that lead ions can be rendered highly insoluble by treatment with H₂S (also a poison). The result is the mineral galena, PbS; this has one of the lowest solubility product constants (K_sp) in water of all the heavy metal compounds. An excess of H₂S therefore precipitates lead very thoroughly from aqueous solutions, although there are other, somewhat safer ways to introduce sulfide ion (e.g., Na₂S).

Mercuric Chloride ("corrosive sublimate") - an intense poison, HgCl₂ is also very useful as a fixative. Mercuric chloride mustn't be confused with the far less-toxic, mostly insoluble Hg₂Cl₂ (mercurous chloride; "calomel").
Many fixative recipes call for mercuric chloride. It has become more difficult to obtain; whether it's an instance of chemophobia is debatable, since HgCl₂ is in fact pretty nasty. As in the case of other poisons, however, there's no reason to lose one's composure at the mere mention of it . It must simply be treated with respect and handled appropriately. Like all hazardous reagents, it is inanimate and follows a set of physical laws; thus, there are ways to handle it safely.

Osmium Tetroxide - this may be the most dangerous of the fixatives to handle, yet it is also one of the most effective. OsO₄ is highly reactive; however, unlike most other heavy metal compounds, it readily emits vapors at room temperature. These vapors can denature living tissues on contact, including the cornea of the eye. OsO₄ must be handled with extreme caution and its vapors avoided by use of a working fume hood.

Phenol is a powerful disinfectant; it kills bacteria and cells rapidly. Because of this, it is also a powerful fixing agent and anti-microbial additive for mounting media. Phenol is often used in formulations containing lactic acid ("lactophenol" formulas).

Phenol is also used in conjunction with formalin (or sometimes formic acid); however, concentrated or solid phenol tends to undergo violently exothermic polymerization in contact with formalin. The reagents must therefore be pre-diluted for use in any formulae that call for phenol-formaldehyde. It makes the most sense to apply these two fixatives in different stages so they react with cell components rather than directly attacking each other.

Phenol is also used in the DNA lab, usually in conjunction with chloroform, to denature and remove proteins from DNA extracts. The pure compound has a high affinity for water and therefore also has some dehydrating action.

It is wise to keep some PEG-300 (polyethylene glycol with a molecular weight around 300) handy in case of accidental skin contact with phenol. The PEG-300 is used to get phenol molecules off the skin; it evidently has a higher affinity for phenol than does skin. PEG-300 and 400 are, by the way, useful in certain embedding and mounting protocols.

There is no reason to be irrationally afraid of phenol; just treat it with respect. It is far too useful in the laboratory to start substituting it for some inferior compound. (To put things in perspective, consider that it's nowhere near as toxic as HgCl₂, for example; recall that phenol is an ingredient in mouth rinses and canker sore medicines.)

Figure 3. The author treated some dinoflagellates with phenol and dilute methylene blue. Under just the right conditions, the normally golden-brown organisms became grass-green or even deep emerald-green.

Predictably, PEG-400 interferes with the reaction. On standing, it even seems to leach the green color out of phenol-treated dinoflagellates.

Picric Acid is an old standby that's still used often (for example, in Bouin's Fixative, a formulation containing picric acid, acetic acid, and formalin). Although picric acid is an explosive and can detonate if struck or overheated, the quantities used in microscopy are small, and the compound is used in solution.
Picric acid is toxic and can be absorbed through the skin. It must be stored wet to mitigate the explosion hazard. Metal picrates are very sensitive to detonation; picric acid must therefore be kept away from compounds of lead, copper, etc.
Despite these hazards, one shouldn't fall into catalepsy at the mere mention of "picric acid". It is, after all, a useful laboratory reagent, provided that it's treated with respect.

While professionals still use picric acid in the lab, it is probably best for the teacher or amateur scientist not to keep it around. A preparation of Bouin's Fixative, on the other hand, shouldn't be of much hazard other than for its fixative (poison) properties. However, some people may get dangerously wrong ideas as soon as they hear the name 'picric acid', regardless of how it's being used.

Picric acid, though not a metal compound, has both fixative and mordant properties.

Silver Nitrate (AgNO₃) is a metal salt with peculiar properties: it is strongly caustic without being strongly acidic or basic. That is because AgNO₃ readily forms a nearly-irreversible complex with proteins, denaturing them. That includes the proteins that make up living tissue. In this respect, silver nitrate is like osmium tetroxide but without the fuming. The author has at least once gotten aqueous AgNO₃ on the skin; it immediately produced a purplish coloration that would not come off until the dead skin grew away (which means... wear those safety goggles!)

The half-reaction Ag⁺ + e^- ----> Ag⁰ has a highly positive standard reduction potential. Silver nitrate has a tendency to be reduced spontaneously to extremely fine particles of metallic silver on contact with organic material (on the macro scale this can be a fire hazard). Although one might expect reduction to happen immediately with every attempt to use AgNO₃ as a fixative, the interaction of Ag salts with cell proteins is a bit more complex. Although Ag⁺ is indeed reduced to metallic Ag by any readily-oxidizable functional groups it contacts, some Ag⁺ ions bind at non-reducing sites on proteins. These sites release Ag⁺ very slowly, freeing it to react later with any remaining, oxidizable groups that might be nearby. This writer has not experimented enough with AgNO₃ as a fixative to know what kind of long-term qualitative effects this might have on a mounted specimen; however, it probably means slow darkening, just as happens when impure silver salts are left exposed to light.

Uranium compounds (e.g., uranyl nitrate) are fixatives by virtue of uranium's being one of the so-called "heavy metals". One might expect close contact between uranium atoms and cellular components to have some effect due to the radiation. While alpha is normally stopped by a sheet of paper or a few cm of air, the highly destructive alpha particles might act without interference over the nano-distances of a cell. However, evidence points to the toxicity of uranium in living systems as being due to uranium's heavy metal chemistry, not its radioactivity (q.v. Casarett and Doull's Toxicology). U-238, comparatively speaking, is not that radioactive; it's just not a strong enough emitter for the radioactivity to be of more concern than the toxicity.

Updated: the writer had wondered about possible effects that radiation may have had on uranium's fixative action. After thinking more on the subject, it seems radiation effects are probably negligible unless there is a huge amount of uranium.

Given that U-238 has a half-life of some 4.5 billion years, an individual U-238 atom has a very small chance of decay at any particular moment. If a ship has a million cannons pointed at it, but the firing of those cannonballs is spread out over a span longer than the earth is old, the crew has plenty of time to repair each hole before the next impact. This analogy relates to why most of us are able to survive despite having radioactive potassium-40 in our bodies, even inside our cells.

Uranyl nitrate, uranyl acetate, and other compounds should be treated with the same caution as any other heavy metal compound. In small quantities the radiation isn't that great a concern, since most of the alpha particles cannot get through the clothing or the outer layer of dead skin. Nevertheless, don't keep U or Th (or their compounds) near the skin; close, chronic proximity can still in theory be dangerous. They do emit some beta and gamma, both of which are more penetrating than alpha.

References:

Barka, Tibor and Anderson, Paul. Histochemistry: Theory, Practice, and Bibliography. New York: Hoeber Medical Division, 1963.

Green, A.A and Hughes, W.L. "Protein Fractionation on the Basis of Solubility in Aqueous Solutions of Salts and Organic Solvents". Methods in Enzymology 1:67-90 (1955).

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