Dissolve this amount of dry powder in 25 cc. of glycerine at 60°C. Then add 25 cc. of methyl alcohol at the same temperature. Allow the glycerine-methyl alcohol solution to stand overnight and then filter. This is the stock stain. To use: Dilute 1 cc. with 10 to 15 cc. of distilled water. If 1 to 1000 potassium carbonate solution is used instead of water it stains more deeply. These same dyes, mixed with methylene violet, are now obtainable commercially as a powder ready for solution in methyl alcohol.
The alkaline diluent is used to obtain the coarse stippling in malignant tertian (Maurer’s clefts). Having fixed the smear with methyl alcohol for one to five minutes, pour on the diluted stain, and after fifteen to thirty minutes wash off and continue washing with distilled water until the film has a slight pink tinge. For Treponema pertenue stain from one to twelve hours.
Haematoxylin Staining.—While the Romanowsky methods are more satisfactory for differential counts and for the demonstration of the malarial parasites, and especially for differentiating species, yet by reason of the liability to deterioration in the tropics of methylene blue the haematoxylin methods may be preferable. Many workers in blood-work and cytodiagnosis prefer the haematoxylin.
1. Fix the film either by heat, with methyl alcohol for two minutes or with Whitney’s fixative. Heat is to be preferred.
2. Stain with Meyer’s hemalum or Delafield’s haematoxylin for from five to fifteen minutes according to the stain. Frequently three minutes will be found sufficient. To make the hemalum, dissolve 0.5 gram of haematin in 25 cc. of 95% alcohol. Next dissolve 25 grams of ammonia alum in 500 cc. of distilled water. Mix the two solutions and allow to ripen for a few days. The stain should be satisfactory in two or three days.
3. Wash for two to five minutes in tap water to develop the haematoxylin color.
4. Stain either with a 1 to 1000 aqueous solution of eosin or with a one-half of 1% eosin solution in 70% alcohol. The eosin staining only requires fifteen to thirty seconds.
5. Wash and examine.
Differential Count
In making a differential count I would recommend the following from the directions of Schilling-Torgau. It will be remembered that considerable interest was raised a few years ago in what was termed the Arneth index. In this the more normal, more mature, better resisting polymorphonuclears were considered to have 3 or 4 lobes to the nuclear structure, even occasionally 5. The immature cells had only one or at most two lobes to the nucleus. The index was obtained by adding the percentages of cells showing 1 and 2 lobes to ½ the percentage of those with 3 lobes. As will be understood a high percentage of these immature cells was unfavorable in prognosis. These cells are graded from left to right, I, II, III, IV, V, as to separate masses in the nucleus, so that when the percentage is shoved or displaced to the left it indicates an increase in the immature cells.