V49-8 MANUAL OF METHODS FOR PURE CULTURE STUDY 



ing it more adaptable to routine use on bacteria of various types, pub- 

 lished by Fisher and Conn (1942), is also given in Leaflet IV. 



Presence of endospores. Routine examinations should be made on 

 agar slant cultures a week old, employing methylene blue or dilute 

 crystal violet, to stain the vegetative rods and leave spores unstained. 

 If spore-like bodies are present whose exact nature is uncertain, one of 

 the spore stains recommended in Leaflet IV should be employed. 



In most cases there is little trouble in finding spores if the organism 

 produces them. All rather large rods, however, (0.8 micron or more 

 in diameter) should be regarded as possible spore-producers even if 

 microscopic examination does not show spores. Such bacteria 

 should be mixed with sterile broth or physiological saline solution and 

 heated to 85°C. for ten minutes; if still alive, endospores may be re- 

 garded as probably present. One should also make repeated trans- 

 fers of the culture onto agar and examine at various ages. A culture 

 of a large rod should not be recorded as a non-spore-former unless all 

 these tests are negative. 



Acid-fast staining. Various methods have been proposed for de- 

 termining whether an organism is "acid fast." They are all essen- 

 tially modifications of the same general procedure, and are similar 

 to the spore stains of Moeller (1891) and Foth (1892). The Commit- 

 tee is as yet unprepared to recommend any one of them in particular. 

 Several are listed in Leaflet IV. 



Capsules. An organism should not be recorded as having capsules 

 unless they have been actually stained by one of the methods of cap- 

 sule staining described in bacteriological text books. Four of the com- 

 mon methods of capsule staining, namely those of Anthony, of Hiss, 

 of Huntoon, and of Churchman, are given in Leaflet IV. The Com- 

 mittee has obtained good results with Anthony's and Hiss' methods. 

 Capsules do not appear in all media; the medium of choice should be 

 milk serum slants, or exudates from infected animals. 



Irregular forms. Forms that differ from the typical shape for the 

 organism, such as branching forms, clubs, spindles, or filaments should 

 be noted and sketched. Simple observation is enough to show 

 that these irregular forms occur quite uniformly in certain cultures, 

 hence their existence must not be ignored; the interpretation of these 

 forms is at present under dispute and the decision as to their signifi- 

 cance must be awaited. The Committee recommends that the 

 microscopic study of any culture include an examination of the 

 growth on various media and at various ages upon each medium, 

 with sketches of all the shapes that occur. 



