2. Fixing the Film.--In general, films must be "fixed" before they are stained. Fixation may be accomplished by chemicals or by heat. Those stains which are dissolved in methyl-alcohol combine fixation with the staining process. * * * * * Chemic Fixation.--Soak the film five to fifteen minutes in pure methyl-alcohol, or one-half hour or longer in equal parts of absolute alcohol and ether. One minute in 1 per cent. formalin in alcohol is preferred by some. Chemic fixation may precede eosin-methylene-blue and other simple stains. {171} Heat Fixation.--This may precede any of the methods which do not combine fixation with the staining process; it _must_ be used with Ehrlich's triple stain. The best method is to place the film in an oven, raise the temperature to 150° C., and allow to cool slowly. Without an oven, the proper degree of fixation is difficult to attain. Kowarsky has devised a small plate of two layers of copper (Fig. 77), upon which the films are placed together with a crystal of urea. The plate is heated over a flame until the urea melts, and is then set aside to cool. This is said to give the proper degree of fixation, but in the writer's experience the films have always been underheated. He obtains better results by use of tartaric acid crystals (melting-point, 168°-170° C.). The plate, upon which have been placed the cover-glasses, film side down, and a crystal of the acid, is heated over a low flame until the crystal has completely melted. It should be held sufficiently high above the flame that the heating will require five to seven minutes. The covers are then removed. Freshly made films of normal blood should be allowed to remain upon the plate for a minute or two after heating has ceased. Fresh films require more heat than old ones, and normal blood more than the blood of pernicious anemia and leukemia. [Illustration: FIG. 77.--Kowarsky's plate for fixing blood (Klopstock and Kowarsky).] Fixation by passing the film quickly through a flame about twenty times, as is often done in routine work, is not recommended for beginners. * * * * * 3. Staining the Film.--The anilin dyes, which are extensively used in blood work, are of two general classes: {172} basic dyes, of which methylene-blue is the type; and acid dyes, of which eosin is the type. Nuclei and certain other structures in the blood are stained by the basic dyes, and are hence called _basophilic_. Certain structures take up only acid dyes, and are called _acidophilic_, _oxyphilic_, or _eosinophilic_. Certain other structures are stained only by combinations of the two, and are called _neutrophilic_. Recognition of these staining properties marked the beginning of modern hematology.