J. Embryol. exp. Morph., Vol. 15, 2, pp. 131-132, April 1966
Printed in Great Britain
131
A simple technique for the preservation of vital
dyes in fixed and sectioned embryos
By J. PERTUSA 1
From the Institute Biologico de Sarrid, Barcelona
In embryological work using vital dyes it is highly desirable to be able to study
the distribution of the dyes in fixed material, whether examined in toto or after
embedding in paraffin and serial sectioning. However, both fixation and dehydration present problems for the preservation of colour in vitally stained cells.
Some fixatives preserve some dyes but, so far as I am aware, none will preserve
all the vital dyes in common use. On the other hand, ethyl alcohol destroys or
dissolves all vital dyes and its use in dehydration is thus undesirable. Among the
fixatives that have been proposed are those of Golowin (1902), Mitamura
(1923), Parat & Painleve (1925), and Tcheou Tai Chuin (1930) for neutral
red; that of Izquierdo (1955) for toluidine blue; that of Gerard (1925) for
Trypan blue; that of Turchini (1919) for methylene blue; that of Lehmann
(1929) for Nile blue.
I have found that the use of isopropanol saturated with mercuric chloride
both fixes and dehydrates dyed material without loss of colour. It is not, however, a good fixative from the cytological point of view.
Since isopropanol mixes with paraffin, material fixed in it can be passed
directly to paraffin without an intermediate agent. Thus the two methods suggested are as follows:
(a) For sections. Fix in isopropanol saturated with mercuric chloride and
change the fluid several times to ensure complete dehydration, pass to paraffin,
routine cutting and mounting of sections, dewaxing and clearing using xylene
and Canada balsam (Mayer's albumen does not affect the dye). When embryos
or very delicate eggs are to be fixed, they must be hardened by placing them in
distilled water saturated with mercuric chloride before they are transferred to
isopropanol. If cytological detail is required, the material can be fixed in some
other fixative and then dehydrated in isopropanol and mercuric chloride.
(b) For whole mounts. Material fixed and dehydrated as above is passed to
an essential oil and then to xylene before mounting in Canada balsam.
The stains that I have used have been obtained from Messrs Gurr of London.
1
Author's address: Instituto Biologico de Sarria, Calle Dr Amigant 31, Barcelona 17,
Spain.
9-2
132
J. PERTUSA
The stains tested were: Neutral red, Neutral red iodide (vital), Vital new red,
Brilliant vital red, Trypan red (vital), Toluidine blue, Trypan blue, Nile blue,
Methylene blue. The tests I made with these stains were only to see if the fixative
described was able to fix them. No account was taken of the fact that some are
more or less toxic to the embryo.
SUMMARY
The use of a saturated solution of mercuric chloride in isopropanol for the
fixation and dehydration of embryonic material stained intra vitam preserves
the colour of the dye while permitting permanent whole mounts or histological
sections to be prepared.
RESUME
Une technique simple pour preserver les teints vitaux dans les
embryons fixes et sectionnes
L'emploi d'une solution saturee de chlorure mercurique dans l'isopropanol
pour fixer et deshydrater les materiaux embryonnaires teints intra vitam preserve
la couleur de la teinture et, en meme temps, rend possible la preparation des
specimens entiers et des sections histologiques.
REFERENCES
P. (1925). Recherches morphologiques et experimentales sur la vesicule ombilicale
des Rongeurs a feuillets inverses. Archs. BioL, Paris, 35, 269-93.
GOLOWIN, E. (1902). Sur lefixagedu Neutralrot. Z. wiss. Mikrosk. 19, 176-85.
IZQUIERDO, L. (1955). Fixation des oeufs de rat colores vitalement par le bleu de toluidine.
Technique et observations cytologiques. Archs. BioL, Paris, 66, 403-36.
LEHMANN, F. E. (1929). Die Entwicklung des Anlagenmusters im Ektoderm der Tritongastrula. Wilhelm Roux Arch. EntwMech. Org. 117, 312-83.
MITAMURA, T. (1923). t)ber eine neue Fixierungsmethode farbstoffhaltiger Organe. Zentbl.
allg. Path. path. Anat. 33, 593-9.
PARAT, M. & J. PAINLEVS (1925). Techniques relatives a la demonstration du vacuome et a
sa comparaison avec l'appareil de Golgi. C. r. Seanc. Soc. BioL 93, 315-17.
TCH£OU TAI CHUIN (1930). Procede technique pour conserver en preparations durables la
coloration vitale au rouge neutre. C. r. Seanc. Soc. BioL 103, 871.
TURCHINI, J. (1919). Coloration vitale du chondriome des cellules secretrices du rein au cours
de l'elimination du bleu de methylene. C. r. Seanc. Soc. BioL 82, 1134-5.
GERARD,
(Manuscript received 13 September 1965)