Two modifications of Schaudinn’s formula may be found useful. A saturated solution of corrosive sublimate in physiological salt solution may be substituted for the aqueous one, and the addition of a few drops of glacial acetic acid to either of the preceding mixtures may be made.

Some workers prefer to use hot fixatives, raised to a temperature of about 50° C.

Fixation by corrosive sublimate solutions must be followed by thorough removal of the mercury salt by washing repeatedly in 30 per cent. alcohol or with iodine-alcohol.

Bouin’s Fluid, or modifications thereof, is also very useful for wet fixation. Bouin’s picro-formol solution consists of:—

The best-known modification is one due to Duboscq and Brasil, and often known as Bouin-Duboscq Fluid. Its formula is as follows:—

Thorough washing of the smear or cover-slip preparation with 70 per cent. alcohol until the yellow colour disappears is necessary to remove excess of fixative.

Other fixatives, which may be of use, more especially for fixing small pieces of tissue for sectioning, are the solutions of Flemming (chromo-aceto-osmic acids) and of Zenker (sublimate-bichromate-acetic, with sodium sulphate).

Regarding the time of fixation, there is much difference of opinion. Usually, exposure to or contact with the fixative for five minutes is sufficient in the case of films or smears. Material for sections should be cut into small cubic pieces, of a thickness of about 5 mm. ( 1/5 in.). One or two hours should be sufficient time for the fixation of such pieces of tissue, though some, as Langeron, prefer a longer time of fixation. On the other hand, Gustav Mann1283 recommends a short fixation period. The excess of fixative should be thoroughly washed out of the tissue in the manner appropriate to the particular fixative used. If it is desired to keep the tissue for some time before sectioning and staining, it should be transferred to 70 per cent. alcohol.

When fluid fixatives are employed, large quantities of the fixing media are necessary. The volume of the fixative should be at least ten to twenty times that of the object, and the latter should be suspended in the middle of the fixative. The tissue should be fixed as soon as possible after the death of the host.

For sectioning tissue parasitized by Protozoa, embedding in paraffin is generally recommended. Microtome sections should not, if possible, exceed 5 µ in thickness. Details of special procedures must be sought in larger works.

Staining.—Here, as with fixatives, much choice is presented. The various modifications of the Romanowsky stain have aided greatly in the detection of various Protozoa parasitic in the blood. Such stains, however, leave something to be desired in the revealing of finer cytological details. Other stains, more especially the hæmatoxylins, must be employed for cytological purposes.

Formulæ of some of the principal Romanowsky and hæmatoxylin stains may now be given.

The underlying principle of the Romanowsky Stain is the reaction between alkaline methylene blue and eosin, forming the so-called eosinate of methylene blue which stains chromatin purplish-red. A solution of medicinal methylene blue after having been subjected to the action of an alkali, such as sodium carbonate, becomes partly converted into certain derivatives, the chief of which are methylene azure and methylene violet. These substances are also present in matured polychrome methylene blue.

The formula of a slightly modified Romanowsky Stain which gives excellent results is given below:—

Two stock solutions are required—

Keep in a warm incubator for two or three days, until the solution is distinctly purple in colour. It improves with age.

This solution must be kept in the dark, in dark-tinted (amber-coloured) bottles, as unfortunately it is decolorized by light.

Before use each stock solution must be diluted. Thus, make up 5 c.c. of each stock solution to 100 c.c. by adding distilled water. For staining, 1 volume of solution A is added to 2 or 3 volumes of solution B. Mix thoroughly by shaking, pour the mixture over the film, previously fixed in absolute alcohol, and stain for ten to fifteen minutes. Wash carefully in running water, then dry. The cytoplasm of a protozoan parasite will be stained blue, the chromatin purplish-red and vacuoles or very tenuous protoplasm will remain colourless.

The exact proportions of solutions A and B, which must be mixed together, should be determined by experiment. Freshly mixed stain must be used on each occasion.

Leishman’s Stain is the precipitate resulting from the interaction of alkaline methylene blue and eosin. The washed and dried precipitate is collected and dissolved in pure methyl alcohol, which acts as a fixative; 0·015 grm. of Leishman powder may be dissolved in 10 c.c. of methyl alcohol for staining films. The film is covered with the solution for one minute, twice the volume of water is then added and mixed with the stain on the slide. The staining is then continued for five to ten minutes, and the film is finally washed with water.

Giemsa’s Stain.—This should be procured ready made. Azure II is a mixture of methylene azure and methylene blue. (Methylene azure is sometimes known as Giemsa’s Azure I.) The formula given by Giemsa himself in 1912 is:—

The film is first fixed in absolute alcohol. The proportion of stain usually used is one drop of stain to 1 c.c. of water. Stain for about ten minutes and then wash in water.

The details of the application of the Giemsa stain to films fixed wet and to sections must be sought in larger works on technique. These works should also be consulted for information regarding the use of Pappenheim’s Panchrome mixture.

There are numerous formulae of stains containing ripened Hæmatoxylin or its essential principle, Hæmatein. A mordant is necessary, one of the alums being usually employed. The mordant may be included as an ingredient in the staining mixture, or it may be used separately as in the case of the so-called iron-hæmatoxylins, wherein ferric ammonium alum is used separately and is followed by staining with hæmatoxylin or hæmatein. A few of these stains of general application may now be mentioned.

Delafield’s (or Grenachier’s) Hæmatoxylin.

Mix these ingredients, and leave exposed to light and air for three to four days. Filter and add—

Allow the mixture to stand until the colour is sufficiently deep, then filter and place in a stoppered bottle. The solution should be allowed to ripen for at least two months before use. Dilute aqueous solutions of the stain are of service for films and for sections. A trace of acetic acid may be added at the moment of use, for sharp differentiation.

Ehrlich’s acid hæmatoxylin, Mayer’s hæmalum, and Mayer’s glychæmalum are also useful. Their formulæ will be found in larger works.

The chief Iron-Hæmatoxylin Stain is that devised by Heidenhain. Unfortunately the procedure involved is a long one, and various modifications have been made to obviate this disadvantage. Hæmatein may be used instead of ripened hæmatoxylin.

One efficacious modification of Heidenhain’s stain is that of Rosenbusch. The smear or tissue, after fixation, must be graded downwards through the alcohols to water. Mordant for one and a half hours in a 3 1/2 per cent. aqueous solution of ferric ammonium sulphate. Stain for about three minutes in 1 per cent. solution of ripe hæmatoxylin or hæmatein in absolute or 96 per cent. alcohol, to which a drop of saturated aqueous solution of lithium carbonate, sufficient to produce a wine-red colour, has been added. Differentiate under the microscope with a very dilute solution of the ferric ammonium sulphate. Wash, gradually dehydrate, clear and mount in balsam. It must be remarked that iron-hæmatoxylin is a regressive stain, hence great care must be exercised in differentiating with the iron alum.

Gentian Violet.—A 1 per cent. alcoholic solution of gentian violet, or of methyl violet, or of crystal violet, will be found useful for staining spirochætes.

Methyl Green.—This substance is considered to be a chromatin stain, for either fresh or perhaps recently fixed tissues. A concentrated aqueous solution contains about 1 per cent. of the stain. This should be added to a 1 per cent. solution of acetic acid. It may be used for demonstrating the nuclei of ciliates.

In conclusion it is essential to remember that the actual magnification of figures of Protozoa should be given, and not merely the combination of objective and ocular that has been used, for unless the tube-length and distance of the drawing board from the ocular be also given, it is not possible to compute the magnification from such information. Drawings should always be made with the aid of a camera lucida, drawing prism or other form of projection apparatus.