No Formalin, No Mercury, New Fixatives: (Part 1)
The picture above is from a spherical cluster of cells, not identifiable as any organism that I know, with a nucleus of animal characteristics and amoeboid cytoplasm, found in a preparation of material fixed with GALA 60. A very small drop of “brilliant blue” gives them this color. Click on the image for a bigger version of the picture.
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.
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.
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 60 ml
The previous ciliate image,
left, and the
right are all pictures of materials fixed in
GALA 20 by Christian Colin who kindly allows
their inclusion here.
Acetic acid 5%.................... 6 ml
Water................................ 93 ml
Copper sulfate……….......... 5 g
Egg of daphnid, in the brood pouch of a Cladocera. Nuclei of adjacent tissues are seen.
Click to increase.
|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.
|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.
Click to increase
Click to increase
The images of
were composited with
0,2 gram………. copper
0,3 gram ……... copper dichloride
1 gram……… acetic acid
100 ml……….… water
A daphnid with its brood poach full of
eggs and mounted in PVA-G.
Contrary to what I advise, this sample was preserved (as a test) in the same LC for more than two months before making the slide. It is in good condition, but I think it is good not to try the devil.
This final image is of
fixed in LC with its semi-contracted stalk and its
I needed to amalgamate 3 pictures to appropriately show the flexibility of the stalk.
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