In the second case the preparations are placed on a half concentrated watery methylene blue solution* for half an hour, washed in water, dried with filter paper, and counterstained with a 2 per cent, eosin solution in 60 per cent, alcohol for five minutes, washed in water, thoroughly dried, and mounted in xylol Canada balsam. The leukocytes and malarial parasites are stained blue, the red blood corpuscles and the eosinophile granulations, red.
* It is best to have ready at hand a concentrated alcoholic solution of methylene blue, from which the watery solution is prepared fresh when desired. This is readily done by mixing about five drops of the alcoholic mother solution in a watch glass of water.
Instead of methylene blue, hematoxylin may be used.
Thionin has been highly recommended by Marchoux. It is employed in the following solution: Concentrated alcoholic thionin solution (alcohol 60 per cent.), 20 c.c; 2 per cent, watery carbolic acid solution, 100 c.c.
The mixture may be used after fifteen days. It stains in a few seconds. The leukocytes and parasites are stained red violet.
For the staining of hematozoa in tissues Bignami proposes the following: Fix very small cut pieces in 1 per cent, watery solution of bichlorid of mercury to which has been added 0.75 per cent. NaCl and 0.5 per cent, to 1 per cent, acetic acid. The sections should remain in this from one half hour to several hours. They are then transferred to alcohol containing a little iodin (sherry color), and finally hardened in alcohol.
The staining is effected in a saturated watery or alcoholic solution of safranin, methylene blue, vesuvin, or magenta red. The preparation should remain for five minutes, and is then washed in water.
Under special circumstances it may become necessary, for the purpose of diagnosis, to examine the blood of the internal organs. This occurs in cases in which there is strong suspicion of malarial infection, though the examination of the peripheral blood is negative. There are, for instance, rare cases of very severe malarial infection in which the capillaries of certain organs, especially the spleen, liver, and brain, are filled with parasites, while the peripheral vessels show only a few infected red blood corpuscles. In order to assure the diagnosis in such a case puncture of the spleen is indicated. Though, as a rule, this operation is without injurious results, it is not to be undertaken without sufficient reason. We must consider as a decided contraindication to puncture of the spleen any disease in which there is an inclination to hemorrhage, since profuse, even fatal, hemorrhages might result from the wound.
The puncture is to be done by a Pravaz syringe under strict aseptic precautions. During the operation the patient should be told to hold his breath, so that the wound is not enlarged by the upward and downward movement of the spleen in respiration. After the operation an ice bag is applied to the site in order to relieve any pain and produce contraction of the spleen, so as to prevent hemorrhage.
[For diagnosis the practitioner should adopt the Romanowsky method, but examination of unstained fresh preparations should not be neglected, though to the beginner the pitfalls in an unstained specimen are many.-Ed.]