24 



MANUAL OF MICROBIOLOGICAL METHODS 



Dried films are stained 10 sec in a mixture of 2 parts of solution 1 and 1 part of 

 solution 2. Wash. Stain 10 sec in solution 3. Wash briefly in water, or not at all. 

 Blot dry. 



Pondefs Diphtheria Stain 

 Ponder {1912); Kinyoun (1915) 



Toluidine blue 



Azure I 



Methylene blue 



Glacial acetic acid 



Ethyl alcohol (see below) 

 Distilled water 



Dissolve the dyes in the alcohol; add the water, then the acid; and let stand 24 hr 

 before using. Do not filter. After prolonged standing, action may be intensified by 

 adding 1 or 2 drops of glacial acetic acid. 



According to Kinyoun, smears are fixed with heat, allowed to cool, and stained 2-7 

 min. 



In the source of the original formula above cited, absolute alcohol is specified; Kin- 

 youn calls for 95 per cent alcohol. On theoretical grounds, indeed, absolute alcohol 

 is not indicated, and the 95 per cent strength may well be substituted even in the 

 original formula. Although the committee has had no personal experience with 

 either formula, information is at hand indicating the superiority of the Kinyoun 

 modification. 



FLAGELLA STAINING— RECOMMENDED PROCEDURES 



Flagella staining is a difficult technic, and there have been numerous 

 methods proposed for the purpose. It has long been realized that flagella 

 are actually below the visual limit in size, but of recent years the electron 

 microscope has given a definite idea how small they really are — around 

 0.02-0.03 fjL in diameter. Electron micrographs, in fact, often show many 

 more flagella than do stained preparations. Until the electron micro- 

 scope, however, has become a routine laboratory instrument, one must 

 have resort to the principle introduced by Loeffler of mordanting the 

 preparations before staining to increase the apparent size of the flagella. 



A second difficulty in staining flagella is the ease with which bacteria 

 shed these delicate appendages unless the cultures are properly handled. 

 To prevent this one ordinarily employs specially cleaned slides and 

 specially prepared smears on the slides. 



Methods for preparing slides. Ordinary cleaning of glassware is not 

 sufficient for the purpose. Various methods have been proposed, but the 

 following directions seem to give as good results as any: 



