Of all the products
used to prepare microscopic materials for their definitive mounting on
the dyes are the more difficult ones to be substituted. Their selection
has been made over decades by specialized chemists and microscopists who have chosen
with exquisite care the products suitable for each task. There is a
chemical balance between the stain and the organelles or
products that they are intended to color.
than 100 years of experience, any proposed substitute is destined to
be third rate, as opposed to the professional dyes, and many of these
absolutely irreplaceable. For more information search the Stain File (below).
amateurs understand this, and, when really in need, if they can,
they take recourse
to the most common classic colorants as Hematoxylin, Eosin, Carmine,
blue, Methyl green, Fuchsine, and the like. The big problem is
to acquire them
in small quantities and at reasonable prices. The Europeans have at
resources (listed in the
the young person, and sometimes the not so young amateur microscopist, may wish
to highlight nuclei, cytoplasm, cilia, cirri, plastids, and
study them easily but without setting out, of course, to make a
professional result, and to prepare professional formulae.
It is for
them for whom I have written up these notes.
|This is an
impossible picture. It is an illustration mixing picture
enhanced with a little design when needed. It was composed in
PhotoPaint to show the most abundant inhabitants in my
microscopic jungle of Cladophora.
and METHODS USED
Three years ago
before moving to Durango, I wanted to keep some samples from
the very varied microfauna that lived on or between a Cladophora tuff that
inhabited my freshwater aquarium at Cancún. And I took the opportunity to try a pair
of dyes and a probable mounting medium whose possible utility
Cladophora is a chlorophyte with a
thin and profusely branched filamentous thallus
that sometimes invades like a plague the aquariums of the amateurs. My
was not an exhibition aquarium but a source of biological materials, so
alga grew on the understanding that it had the right to balance its
own development with the many
other inhabitants of the aquarium which included a numerous population
guppies. These and their abundant young passed their time exploring the
tuffs of weed
probably to feed on the microfauna that lived between and on it, and also on the new growth of alga.
In order to
obtain the required sample, I introduced underwater a small part of the
a plastic tube (discarded from a photographic 35 mm film) I cut the
I removed the tube.
I filtered the
water, without applying any
type of anesthesia, covering the mouth with a piece of silk
a mesh of 70 microns, and I filled it with hot AFA,
a fixative with alcohol, formalin and acetic acid.
the fixation (and dehydration) squashes the thalli and turns them into
unattractive flattened ribbons but it preserves very well the microfauna.
COLORATION with ALLURA RED
on the dietetic dyes: practically all, including Red 40 have
been accused of being
carcinogenic, although their morbidity incidence is very low. There are
types of dyes, the water soluble ones, which are normally sold to color
ice creams, etc. And the alcohol soluble (called lakes) and which are
sold as a dust, for commercial and industrial use. The ones I used were
simple water-based solutions sold in the food stores for domestic use. In the
text and in the appendix I include the USA and EU
codes for the
out the fixative with 30% alcohol, and finally with demineralized water,
discarding this, always making use of the silk mesh, I filled
the tube with
a solution of Allura Red (a dye which in the U.S.A. has the FD&C
Red #40 code,
and which is known in the European Union under the E129 number). The
is a dye approved for foods and sold in the form of a 2.8% aqueous solution.
commercial solution is extremely concentrated and I used a dilution of
of solution in 30 milliliters of water. I leave the materials in
contact for 3
hours. (Probably one hour could be enough.)
following treatment included the washing of the dye with water (two
and a progressive dehydration with 30%, 50%, 75% alcohol, (half an hour
in each one)
and 96% (of this last two changes, each of one full hour).
It is very
probable that the dehydration protocol has been excessively long, but
behavior of the nail enamel that I thought to use as a mounting media
to me and I preferred to err on the safe side.
MOUNTING IN NAIL
I placed a
heavy drop of nail enamel on a coverslide. I passed a piece of alga to
center of one slide, I extended its
with a pair of needles, and removed with an absorbent paper and very
most of the surrounding alcohol and turning
down the coverslide I lowered it taking care with the material.
I put the
preparation safe from dust and under a 10 g weight (a flat headed screw
of 6 mm diameter by 2.0 cm. long, with two nuts screwed on) for 10
the following day the slides were hard enough to allow safe use even with
the immersion objective. As a precaution I made an additional seal
was extremely successful, and supports my
recommendation for the use of the nail enamel as a synthetic
medium at least at the amateur level.
preparations are now more than 2 years old and as it will be seen from
the enclosed images they
perfectly maintain the morphology of the subjects, and also
retain the color of dye used.
Most of the
protozoa that I found in this first sample were heliozoans of the
eichornii. Finding them was lucky because its morphology is extremely
interesting, as demonstrated by the attached photos. I include the live
organism for a comparison of the information given by both methods.
eichhornii the larger cell, and Actinophrys sol, both alive,
to compare their sizes. The insert at right shows one axopod of A. eichhornii,
with the characteristic beads of cytoplasmic flow over the rigid
eichhornii, stained with Allura Red, which displays its
cellular structures, including the multiple nuclei.
other organisms were not taken then, but I believe that the onion
epithelium presented in my article on the onion skin, which is still
in the same condition, and the ones of Actinosphaerium,
are sufficient to think
that it would behave more or less like the Fast Green, which I discuss
FAST GREEN FCF
three other water soluble dyes; sold in Mexico and the U.S.A. for dietetic aims (other manufacturers
sell similar products in the United Kingdom and the European Union).
I prepared a sample with the
same method using Green
#3. This was a dye very
and used in professional microscopy: the Fast Green FCF (see
note on the probable present composition of the green dye).
I had been lucky
because this time (a month after the first sample), the alga was
luxuriant and the
population very diverse.
following pictures are a small gallery of the organisms found. Of
those the most interesting one is Euplotes (that also prompted
a special article)
which not only shows very good
anatomical details, but even allows differentiation of the macro and micronucleus characteristic of the
species. Pictures of this organism are gathered in a special table at the end
of this section.
species with enormous representation in the sample was Actinophrys sol.
in the case of both heliozoans, the axopodia were fixed in different
retraction. But no professional method can do better than that. There
is a clear
differentiation between ecto and endoplasm, and the nuclei and
vacuoles are clearly visible in both species.
An x 4 obj.
view of some branches of the thallus, showing epiphytes and “neighbors”
One live Actinophrys,
displaying its normal relationship with the algae. This is its hunting
The fixed Actinophrys.
Also seen in the pictures are many epiphytic cyanophyta.
100x objective, the cell anatomy is shown, you also see the epiphytic
cyanophyta, which will be discussed in another article.
|Four steps in the
development of a cyst. Probably a reproductive one.
was also extremely abundant. But the rough treatment applied was not
one to fix them in good conditions of conservation. Nevertheless it is
discover the striated pellicle, as well as the peristomal ring and the
and gullet, although the opacity of the voluminous cytoplasm hides the details of the long beaded nucleus.
cluster of contracted Stentors fixed to a Cladophora thallus.
stentors showing pellicle striation.
crown of membranelles around the peristomal field and the spiral
another stentor individual.
A Carchesium (Ciliatea,
was also abundant. With shame I share a very bad picture of the little
and one contracted individual showing the characteristic horse-shoe shaped
(all the zooids are contracting independently) stopped in its
the hot fixative.
zooid showing the well stained horse-shoe shape nucleus
they were Bdelloids distributed throughout the preparation, but we know
that these rotifers hardly lend themselves to being fixed in extension.
Nevertheless the following figure shows that the internal anatomy,
the germovitellarium, are well preserved and
differentiated, telling us that many Monogononta,
that could respond to anesthesia, could probably be fruitfully colored.
taken through a COL-D3 filter
semi-contracted x 40 Obj.
eggs in many stages of development were adhered to filaments of the
To add a
different example of the uses I found for my green dye, these are
colored in Fast Green FCF
and mounted in PVA-G. Of
course the onion
behaves very well with it.
Ipomoea colored with Fast Green and mounted in PVA-G, Obj. x 100.
Petunia grandiflora, idem.
The new web page of McCormick, the manufacturer of the dyes
that I tried, declares the green one as a mixture of Yellow #5
Blue #1 (Blue brilliant). Apparently the Food & Drug Administration
of the EU decided
finally to prohibit the dietetic use of the Fast Green. I have not
samples and do not know what their behavior is. The sample that I still
use, was declared at the time to be Fast Green FCF.
euristomum. Two pictures selected from 10 focus levels showing the
of the long ribbon nucleus, and the little round micronucleus
the first picture) under the frontal border.
BRILLIANT and TARTRAZINE
two dyes are the blue brilliant
and the tartrazine.
proven in different and smaller samples, fixed with Gala 20, and
mounted in Glycerin, with more or less promising results.
following images are one picture of a gastrotrich and one of paramecium,
showing their structures stained with the blue brilliant. It must be tried
extensively. It is a promising dye.
A gastrotrich, Gala 20, Blue brilliant, mounted in glycerin
from the same slide
The tartrazine is much
less useful because it stains in a diffuse manner,
differentiating the nucleus, or other cytoplasmic structures.
stained with Blue Brilliant, in another similar preparation
stained with tartrazine
ciliate (x 100 obj.) stained with malachite green. But it took more
than 8 hours
to reach that level of staining. I do not recommend it (at least with
think that tartrazine
can be used with advantage to color in diffuse but intense
micro-invertebrates in samples in which they must be counted. Small
especially protozoa, gastrotrichs and rotifers are many times hidden by
sediments in the sample. To color them aids searches and counting. For
function a stain very much used was Rose
Bengal, extremely expensive and now discarded as extremely
dangerous. The tartrazine
must be proven as an alternative. It is
colors cytoplasm fundamentally and not detritus, it is very visible.
all basic features for bulk sample coloring.
dietetic dyes, economical and easily found in any supermarket, can render
services to the amateur. Not all microscopists, especially the young
reach, by their price or its availability, the professional dyes.
microscopists are used to working with methylene blue, which is
sold for aquarium use; eosin,
sold in pharmacies like a disinfectant, (see JMC article for reference)
and gentian violet,
(see my April 2004 article)
also obtainable in
pharmacies like a therapeutic for pathogen yeast infections. Adding to
them the very
well known Lugol’s Iodine
(or the Rhode's
fixative, see my September 2003 article),
and Chinese (or Indian) Ink,
that can be
for example like a
substitute for Nigrosine,
in the so called "negative"
coloration of ciliates (see
reached the number of nine available dyes for beginner microscopists, that
cover an ample range of techniques.
other dietetic dyes and lakes that must be tried, and other sources of
colorants (e.g. the textile dyes). I hope that this article
search for the useful ones, and that those who attain some good
the kindness to share these with other amateur microscopists.
W. Dioni, 'About microscopy and the chemistry of nail polish' Micscape August 2002
Marie Cavanihac, www.microscopies.com
XXX Microscopie Practique. Lechevalier, 1947, 430 pgs.
W. Dioni, 'Drawing your microscopic subjects. Euplotes euristomum ...' Micscape Sept. 2002
W. Dioni, 'No formalin, no mercury fixatives. Part 2' Micscape Sept. 2003
W. Dioni, 'A cheap and precise slicer for teaching botany' Micscape April 2004
sources for professional dyes