CHAPTER III 

 STAINING METHODS 



IN order to study a bacterial or blood specimen the first essential is 

 a properly prepared film; the matter of staining is of less importance. 



The slide or cover-glass, after cleaning with soap and water or by special solutions, 

 should be polished with a piece of old linen. If a glass surface is free of grease a 

 loopful of water will smear out evenly and over the entire surface. The only quick 

 practical way to make the slide or cover-glass grease free is to burn the surface for a 

 moment in a Bunsen or alcohol flame. The cover-glass must not be warped. To 

 make a preparation, apply a small loopful of distilled water on the slide or cover-glass 

 and, touching a colony with a platinum needle, stir the transferred culture into the 

 loopful (not drop) of water. The mistake is almost invariably made of taking up 

 too much bacterial growth. Fluid cultures do not need dilution. Smearing the 

 mixture over a large part of the cover-glass or over an equal area of a slide, it is 

 allowed to dry. If very little water is used, the preparation dries readily. Other- 

 wise it can be dried in the fingers high over a flame. As soon as dry, the cover-glass 

 should be passed three times through the flame, film side up, to fix the preparation. 

 Slides may be fixed by passing them five times through the flame, but the method by 

 burning alcohol recommended for fixing blood-films gives more satisfactory bacterial 

 fixation. For routine work the stain recommended is a dilute carbol fuchsin. Drop 

 about 5 to 10 drops of water on the cover-glass, then add i drop of carbol fuchsin. 

 Allow the dilute stain to act from one to two minutes, then wash in water, dry be- 

 tween small squares of filter-paper (4X4 inches), and mount in balsam or the oil 

 used for the K 2-inch immersion objective. LofBer's methylene blue is equally good 

 as a stain. 



By far the best mounting medium is liquid petrolatum. This not only has the 

 advantage of always being of proper consistence for mounts, as opposed to Canada 

 balsam, which must frequently be made thinner with xylol, but it is less sticky and 

 does not develop the acidity which causes balsam mounts of Romanowsky stains 

 to fade. Furthermore, it has superior optical qualities. It is also applicable for 

 mounting small insects and sporangia of moulds. For permanent preparations the 

 border of the cover-glass should be sealed with gold size or some other cement. 

 Some prefer to mount directly in water without preliminary drying. It is good 

 practice to make a rule to always keep the smeared side of the preparations up 

 never allowing it to be reversed. By this simple rule, preparations can be carried 

 through the most complicated staining methods without the necessity of scratching 

 the cover-glass, etc., to see which is the film side. In grasping a cover-glass with a 

 Cornet or Stewart forceps, be sure that the tips are well by the margin of the glass, 

 otherwise the stain will drain off. In staining with slides, the grease pencil and the 



38 



