106 STUDY OF BACTERIA 



Tubercle Bacillus Stain 



1. Spread the sputum, pus or culture, over the surface of the cover-slip. 

 Allow the preparation to thoroughly dry. 



2. Fix in flame and cool. 



3. Pour carbol-fuchsin over the slide and heat with steaming for five min- 

 utes. Young bacilli in tubercles and other fluids are very difficult to stain 

 in this way. The preparation containing them should be stood in cold carbol- 

 fuchsin for twenty-four hours. This method stains everything on the slide. 



4. Wash in water. 



5. Decolorize the preparation with a 25 percent solution of sulphuric acid 

 in water until the red color is lost. Repeat this once or twice. 



6. Wash and counterstain with Loffler's methylene blue. 



7. Dry and mount. 



Gabbet's solution, methylene blue 2 grams, H 2 SO4 25 c.c., 

 water 75 c.c., is a very useful, convenient decolorizer and counter- 

 stain for sputum. 



In such a preparation, if tubercle or other acid-fast bacilli are 

 present, the bacilli will be colored a brilliant red, while the pus 

 cells, epithelial cells, and other bacteria will be stained blue. 



The microscope dark field illumination enables one to see 

 flagella and capsules. This illumination is obtained by blocking 

 out the central portion of the Abbe condenser in the substage of 

 the microscope. Light is admitted only from the sides and objects 

 in the field at the point of crossing of the rays reflect these from 

 their sides. India ink may be used as a background for bacteria 

 that stain poorly and have low refractive index. 



Protozoa are stained by Wright's or Giemsa's method in one of 

 its various forms. Spirochetes, particularly that of syphilis, 

 may be stained by Giemsa's methocl but show up more clearly 

 in the following technic of Stern. 



1. Dry film in incubator for several hours. 



2. Immerse in 10 percent aqueous silver nitrate in diffuse 

 daylight, six hours to three days, depending on thickness of smear 

 and need for haste. 



