With all thick-film methods it is extremely important to have thorough drying of the smear before dehaemoglobinizing or staining. This ordinarily requires one or two hours in the air or twenty to thirty minutes in the incubator. It is particularly important in working with such smears, although holding for ordinary smears, to protect them from flies, ants, etc., as such insects will eat up the smear in a few minutes if left exposed.
Fixation of Film.—In Wright’s, Leishman’s, and other similar stains the methyl-alcohol solvent causes the fixation. In staining with Giemsa’s stain, or haematoxylin and eosin, separate fixation is necessary. For Giemsa either absolute alcohol (ten to fifteen minutes) or methyl alcohol (two to five minutes) answers well.
For haematoxylin and eosin, heat gives the best results. The best method is to place the films in an oven provided with a thermometer. Raise the temperature of the oven to 135°C. and then remove the burner. After the oven has cooled, take out the fixed slides or slips.
One of the handiest methods is to drop a few drops of 95% alcohol on the slide or cover-glass. Allow this to flow over the entire surface; then get rid of the excess of alcohol by touching the edge to a piece of filter-paper for a second or two. Then light the remaining alcohol film from the flame and allow the burning alcohol to burn itself out.
Staining Blood-films.—As separate staining with eosin and methylene blue rarely gives good preparations and as the modifications of the Romanowsky stain recommended are easy to make and employ, and give much greater information, the separate method of staining is not recommended.
Wright’s Method.—The stain is made by adding 1 gram of methylene blue (Grubler) to 100 cc. of a ½% solution of sodium bicarbonate in water. This mixture is heated for one hour in an Arnold sterilizer. The flask, containing the alkaline methylene-blue solution should be of such size and shape that the depth of the fluid does not exceed 2½ inches. When cool, filter the methylene blue solution, and add 500 cc. of a 1 to 1000 eosin solution (yellow eosin, water soluble). Add the eosin solution slowly, stirring constantly until the blue color is lost and the mixture becomes purple with a yellow metallic lustre on the surface, and there is formed a finely granular black precipitate. Collect this precipitate on a filter-paper and when thoroughly dry (dry in the incubator at 38°C.) dissolve 0.3 gram in 100 cc. of pure methyl alcohol (acetone-free). Wright lately has recommended using 0.1 in 60 cc. methyl alcohol. This constitutes the stock solution. For use filter off 20 cc. and add to the filtrate 5 cc. of methyl alcohol.
A modification by Balch is very satisfactory. In this method instead of polychroming the methylene blue with sodium bicarbonate and heat, the method of Borrel is used. Dissolve 1 gram of methylene blue in 100 cc. of distilled water. Next dissolve 0.5 gram of silver nitrate in 50 cc. of distilled water. To the silver solution add a 2 to 5% caustic soda solution until the silver oxide is completely precipitated. Wash the precipitated silver oxide several times with distilled water. This is best accomplished by pouring the wash-water on the heavy black precipitate in the flask, agitating, then decanting and again pouring on water. After removing all excess of alkali by repeated washings, add the methylene-blue solution to the precipitated silver oxide in the flask. Allow to stand about ten days, occasionally shaking until a purplish color develops. The process may be hastened in an incubator. When polychroming is complete, filter off and add to the filtrate the 1 to 1000 eosin solution and proceed exactly as with Wright’s stain.
In Leishman’s method the polychroming is accomplished by adding 1 gram of methylene blue to 100 cc. of a ½% solution of sodium carbonate. This is kept at 65°C. for twelve hours and allowed to stand at room temperature for ten days before the eosin solution is added. The succeeding steps are as for Wright’s stain.
In all Romanowsky methods distilled water should be used. If not obtainable, the best substitute is rain-water collected in the open and not from a roof.