The best-known modification is one due to Duboscq and Brasil, and often known as Bouin-Duboscq Fluid. Its formula is as follows:—
| Alcohol, 80 per cent. | 150 | c.c. |
| Formalin, 40 per cent. | 60 | " |
| Acetic acid, glacial | 15 | " |
| Picric acid | 1 | grm. |
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 Mann[1283] 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.