114 STAINING OF BACTERIA 



cipitate on the filter, however, is carefully dried in the incubator or 

 in a drying oven at a low temperature. It is then scraped off the paper 

 filter and from it is prepared a saturated solution in C. P. methylic 

 alcohol (Merck's). Three-tenths of a gram of the dry stain will 

 saturate 100 c.c. of C. P. methylic alcohol. This is the staining fluid, 

 ready for use. It is very permanent in character, provided that it is 

 kept in a tightly glass-stoppered bottle. Care must be taken from the 

 start that the solution does not become diluted with water, hence 

 the bottle in which it is prepared must have been washed out with 

 methylic alcohol (not with water). 



The stain also fixes the preparation, and it is used as follows: 



1. When thoroughly air dry do not draw through flame. Prepare 

 cover-glasses or slides as usual. 



2. Pour undiluted stain from glass-stoppered bottle on the cover- 

 glass or slide and allow it to act for one minute. 



3. Now add with a dropper, drop by drop, distilled water (dis- 

 tilled water must be used, not ordinary tap or well water) until the 

 mixture becomes semitransparent, with a reddish tint visible at its 

 margins and a metallic scum forms on the surface. The amount of 

 water required will vary with the amount of staining fluid on the 

 preparation, but in general eight or ten drops will be sufficient if a 

 seven-eighths inch square cover-glass is used. 



4. Wash in distilled water for about one-half to one minute. This 

 will so differentiate the stain that nuclei and bacteria appear blue, 

 while cell protoplasm appears pinkish. (The stain also differentiates 

 well neutrophilic, eosinophilic, and basophilic granula of the various 

 leukocytes.) 



5. Dry between filter paper and mount in Canada balsam. 

 Giemsa's Stain for Spirochetse and Protozoa. This permanent stain 



is prepared in the following manner: Take 



Azur II, eosin (this is a combination stain) ..-.'.. . . 3 grams 

 Azur II .'. . . _ . . . . ... . ... .) . 0.8 grams 



Dry in a desiccator over sulphuric acid, powder very fine and sift 

 through a very fine meshed sieve. Dissolve in C. P. glycerin (Merck) 

 250 c.c. at 60 C. and shake continually. Add 250 c.c. Kahlbaum's 

 C. P. methyl alcohol which has been warmed to 60 C. Keep for 

 twenty-four hours at room temperature and then filter. It is best to 

 buy this stain ready made as prepared by Gruebler, in Leipzig. 

 Use it as follows: 



1. The very thin cover-glass or slide preparation must be allowed 

 to become air dry. 



2. Fix for fifteen to twenty minutes or longer in absolute alcohol. 



3. Dilute the Giemsa stain as follows: Take distilled water and 

 add to each cubic centimeter a few drops of a y 1 ^ per cent, solution 

 of carbonate of potassium. To each cubic centimeter of this slightly 

 alkaline watery solution add one drop of the Giemsa stain. 



