No Formalin, No Mercury, New Fixatives: (Part 1)
	WALTER  DIONI                       Durango (Dgo) México

A few years ago, began the tendency to highlight the dangers of many substances normally used in microscopy for more than one century. Successively they were denounced: formalin, glutaraldehyde, mercuric dichloride, picric acid, chloral hydrate, phenol, thymol, gentian violet, Bengal Rose, xylol, benzol, toluol, (xylene, benzene, toluene) etc. etc...

The short preceding list includes the ingredients for almost all the traditional formulas for microscopical reagents including fixatives, dyes, clearing agents and mountants.
 
Even in a world where poisons and dangerous substances invade the streets and houses (see this list of dangerous household substances ), and for those of us who must live in the cities where we are surrounded by the smog and the pollutant gases emitted by cars (whose principal components are the famous triad of BTX (benzene, toluene, and xylene), it is certainly understandable to try to defend the health of the laboratory technicians, who daily spend 8 hours or more, subjected to the action of vapors from all these dangerous substances.

As a measure to limit the use of dangerous reagents, the industrial producers sell only to wholesalers, and the latter only to large distributors, which in turn sell only to professional institutions. Minimum quantities to be ordered (excessive for an amateur) and the high prices, dissuade enthusiasts from buying.

It is obvious that, in parallel, these rigorous preventive measures have other objectives.
 
When I was 15 years old and I made my first microscopic observations of micro-invertebrates, I had gone to the pharmacy on the corner, two blocks from my address, and very naively I had requested 100 milliliters of Rousselet's Liquor, according to the formula provided by Langeron in his "Précis de Microscopie", in those times my Bible.

This formula, which my pharmacist provided without the least objection, includes 1 g of cocaine hydrochloride! Moreover its price was not excessive!

For my smears of protozoa my favorite formula was Schaudinn’s fixative, 2/3 of which consisted of a saturated solution of mercuric dichloride; and I fixed the samples of trematodes which I extracted from frogs in a park pond, with Bouin’s, made with picric acid and formalin, always according to the indications of my Bible.

To make them more transparent and to better see their anatomy I employed chloralphenol and carbolxylol, both with phenol. I employed canada balsam dissolved in xylol for the preparation.
 
Only one minute of thought reminds us that mercury is really terribly dangerous (remember Minamata in Japan). Even the thermometers are filled today with colored alcohol. Picric acid, and picrates and to a lesser degree, chloral hydrate, and phenol, are all usable to prepare homemade explosives, and cocaine is a substance causing a terrible dependence. Solvents with hydrocarbons also cause dependence. They are favorites among poor young people.

The protection of the histologists is thus related to the design of a wider social protection.

Consequently, by legitimate or other reasons, we are subjected to limitations which require that by our spirit of invention we search for new solutions. Which could be made without prohibited substances, and be accessible to no matter whom, amateur microscopist or student, in suitable small quantities.

Personally I only know one English commercial supplier which supports the point of view of the non-professional microscopists, but, although it declares that it can send its products everywhere, in many countries legal mail rules, taxes, and even the economic situation, prevent us from benefiting from this help. (See on the main site index the link to reach Brunel.)
 
I began a series of articles, already published or which will be published in Micscape Magazine, which examines the possibilities of amateurs for homemade preparations.

1) of mounting media, (Micscape Magazine, December 2002 , January 2003 , March 2003 , April 2003 , May 2003)
2) of fixatives for various uses,
3) of nuclear and cytoplasmic dyes.

This article will present the formulas and the characteristics for use of my first two experimental formulas (one published: GALA , and another new). In my opinion, this, with other already known alternative solutions, which I will examine in other articles, will provide the amateur with tools to better develop his vocation.
 

One moment of thought. Nothing can equal the beauty of our critters when they are alive. Photomicrography and drawing are the tools of choice to document their characteristics. One resorts to fixing (and staining also) only to seek details which are not visible in living organisms. Many protist nucleii have a characteristic form, even specific some times (e.g. Euplotes , Ciliates, Hypotricha) and it is very difficult to detect them in the transparent and mobile animal. Undulipodia (the modern name for cilia and flagella) have too much rapid movement for one to appreciate in detail their disposition on the living organism. Moreover, many organs (of the rotifers for example) change much of their form in the living organisms. If in your collections there are some of the smaller larger invertebrates, like entomostracans or acarina, and many other micro-arthropods, by fixing them you can make a collection of samples to be examined a little later, when you have the occasion and the possibility to do it. And certainly, for those who have the chance to have a microtome at their disposal, even if it is borrowed from a school or an academic friend, fixing opens the possibility to investigate the histological structure of the animals.

GALA. (Glycerin, Acetic acid, Lactic acid and Alcohol)

This fixative was published in the first part of my article series on mounting media. So I present here only a summary and some additional notes. The basic illustrations are also included in the previous article. I include here only three pictures captured by Christian Collin, after the use of GALA.

GALA is a gentle fixative which I employ in two versions. The GALA 20 is useful for the smallest protists of ponds on which I work at the laboratory. Most of the time it retains a protist's normal aspect, and returns to non-living cytoplasm part of its transparency.

In a small capsule I add 1-2 drops of GALA to 9-8 drops of sample, let the material fall to the bottom and distribute them on my slides with a pipette. Usually as soon as excess water has evaporated, I seal the coverslide with nail varnish. Naturally this gives me a very long time to study my preparations.

The fixative highlights the macronucleus of the protozoa by a difference in the index of refraction. Cilia and flagella are visible if you play a little with the illumination. For plants (filamentous algae, desmids, diatoms) fixed when they are alive, the green chloroplasts are preserved for several days.
 
The nematodes, the loricate rotifers, the gastrotrichs, are acceptably fixed, although, without anesthesia the gastrotrichs can roll up and their anatomy can be difficult to decipher. As with all the other fixatives which I know, the bdelloid rotifers contract to an uninterpretable ovoid mass. Study them alive. They do not respond even to anesthetics!

To make a complete study of the majority of these groups you must play with the anesthetics, dyes, and clearing agents. This will be the topic of future articles.
 
I do not keep for a long time my samples in GALA 20. They help me understand the details of the anatomy of the creatures that I study alive beforehand, and that I record with my webcam, first alive, then fixed.

To preserve my samples for a longer time I employ GALA 60. I concentrate the sample in the least water quantity that I can, and I add GALA with a hypodermic syringe to have 20% of fixative in the final volume. e.g. typical volumes are as follows:
 

sample, ml	4	8	12	16	20
GALA 60 ml	1	2	3	4	5

Do not leave the samples for more than 24 hours in GALA 60. As is usual with all the traditional fixatives, replace it with 50% alcohol (for 3 or 4 hours) and preserve the sample in 70% alcohol, with some 2 to 5% glycerin added to prevent the occasional evaporation of alcohol, and to improve the transparency of protoplasm. If your material is very fragile, you can first use one 30% alcohol bath. In my experience GALA 60 is not suitable to kill and preserve the Copepods, or the Branchiopods, (both better known to the amateurs under the name of "water fleas"): it is not penetrating enough. It is possible that I did not employ the adequate proportion of the fixative. There are grounds for studies in this area. Ross Ferris, one amateur from New Zealand, and several amateurs from the MicrOscOpies French group tried out GALA 20 with very good results, for the observation of protozoa and rotifers.

Formulas for the GALA fixatives and preparation method can be obtained from my previous article .

The previous ciliate image, the Bosmina at left, and the Volvox at right are all pictures of materials fixed in GALA 20 by Christian Colin who kindly allows their inclusion here.

Mounting materials fixed with GALA

Many materials can be mounted in solidifiable media, like fructose, nail varnish, gum arabic media, etc.

But a special case is to mount microscopic invertebrates, mixed with much detritus, which because of their very small size cannot be handled individually, or be classified into uniform groups, and must be preferably mounted in some liquid medium.
 
A method to mount the required slides in glycerin by carefully increasing the proportion of glycerin is detailed in the section on DIFFICULT MATERIALS section 3) of the article on liquid mountants , first one of the series .

The same method for glycerin mounting is useful for materials fixed with Lactocupric, or with hot water, both of which will be studied thereafter. Master it, because it is really useful.

Just as glycerin you can also use fructose syrup, or polyvinyl alcohol media, that are all miscible with water. Fructose and two formulas of PVA (PVA-L with lactic acid and PVA-G with glycerin) were presented in my series on the mounting media . All need the careful substitution method described for glycerin.

LACTOCUPRIC FIXATIVE

I also needed a fixative which preserved the tissues of microscopic animals and plants. Mercury containing reagents and formalin are prohibited, and I thought of alcohol, the acids, and other heavy metals, which all precipitate proteins, which is the base of fixing. (Although the aldehydes do not precipitate, they make connections between the protein chains (cross-linking), but they are forbidden.)

Zinc was proposed, tested, and found useful, as one substitutive of mercury in histological formulas with formalin. I cannot easily find zinc soluble salts in Durango. Thus I resorted to a metal which caused the aggressiveness and hilarity of some histologists which answered on Histonet, when it was employed by a technician in an urgent pathological sample: copper.

I think that its use as a fixative in microscopic botany and zoology is well established and should not cause hilarity.

The result of my studies is Lactocupric fixative :

Lactic Acid...........................1 ml
Acetic acid 5%.................... 6 ml
Water................................ 93 ml
Copper sulfate……….......... 5 g

Unlike GALA it kills Copepods and Cladocera in almost the same time as 70% alcohol (which, by the way, is the fixative of choice for entomostracans), preserves well the internal anatomy, the chromatin in cell nuclei is well fixed, maintains the general aspect, including ovisacs not collapsed, and does this in less than 24 hours, even while being a very acid fixer. One can distinguish the cells of the ovary, and of testicles and muscles.

Most of the illustrations presented here have been used in the articles on mounting media without mention of the used fixative.

Egg of daphnid, in the brood pouch of a Cladocera. Nuclei of adjacent tissues are seen. Click to increase.

Moreover it fixed the remainder of the microfauna in the samples almost like GALA. All algae in the samples, and for samples of leaf epithelia which I tested, it preserved the green color of chloroplasts.

To study fixed mixed materials one can apply a protocol similar to which was specified for GALA.

I must remark that washing in water is indispensable after fixing with Lactocupric to prevent the appearance of crystals that can spoil the preparations. After this you can pass to alcohol 30, 50 and 70% to store your specimens.

Above there are a small cluster of algae (diatoms, at top right: cyanobacteria, below left and chlorophyta in the center, one month after fixing with the LC and mounted in pure glycerin.	Above is the epithelium of the underside of a leaf, (at 1000X) fixed with LC and mounted in fructose; one sees the chloroplasts of the guard cells of a stomata and the nucleus of an epithelial  cell (at left). Chlorophyll can still be recognized after one month.

I do not take risks and for the small crustaceans and other calcified materials I transfer the major part of the samples to 70% alcohol after more than 24 hours. (But see also the legend for the daphnid picture at the end of this article.) This naturally eliminates the green. But I also file some preparations mounted in glycerin or fructose, after being fixed with Lactocupric and washed in water. The green color lasts for a few weeks at least. I have some that were mounted more than two months ago which have retained their color.
 

Above is a photo taken with homogeneous immersion of the objective (x1000) showing the edge of the egg bag of a copepod. The subjects of the picture are certainly the very small colonial ciliates fixed on eggs by a rigid stalk.	Above one sees the cells of tissues under the carapace of the copepod. Most probably they are ovocytes developing in the ovary.

My experience with LC is very limited however, and I did not perform tests with bulkier animals or plants, nor have I tried it as a histological fixative. It is really a task for a larger team than only one amateur. I think on the lists of amateur microscopists groups, always so active and interested, a suggestion to make extensive experiments would provide results in a short enough time.

Peter Gray, in his old but always useful "The microscopist’s formulary and guide” presents more than 20 formulas with copper salts (nitrate and chloride) but mixed with formalin. A long time ago Blaydes (and others following him) modified AFA (alcohol-formalin-acetic acid) by adding copper sulfate to preserve chlorophyll in fixed algae. Although Gray claims an important role of copper in the fixing of proteins, copper has not had a chance. It is relegated today to the role of a fixative for chlorophyll.

Brachionus bidentata . Click to increase	Cephallodela  sp.	Platyas quadricornis Click to increase
The images of Brachionus and Platyas were composited with CombineZ software.

Jean-Marie Cavanihac communicates to me a formula, which Deflandre recommends in his beautiful and too small book (my first book of microscopy) "Microscopie Pratique" , to fix the microscopic algae. It is also a perfectly safe fixative. I cannot find these salts at Durango (for the prices that I can pay).

It is up to you to try it:   

0,2 gram………. copper nitrate
0,3 gram ……... copper dichloride
1 gram……… acetic acid
100 ml……….… water