ROUTINE TESTS FOR THE IDENTIFICATION OF BACTERIA 151 



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- 

 hke bodies are present whose exact nature is uncertain, one of the spore 

 stains recommended in Chap. II should be employed. 



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

 duces them. All rather large rods, however (0.8 y, 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 10 

 min ; if still alive, endospores may be regarded as probably present. One 

 should also make repeated transfers of the culture onto agar and examine 

 at various ages. A culture of a large rod should not be recorded as a 

 nonsporeformer unless all these tests are negative. 



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

 ing if an organism is ''acid fast." They are all essentially modifications 

 of the same general procedure and are similar to the spore stains of 

 Moeller (1891) and Foth (1892). The committee is as yet unprepared to 

 recommend any one of them in particular. Several are listed in Chap. II. 



Capsules. An organism should not be recorded as having capsules 

 unless they have been actually stained by one of the methods of capsule 

 staining described in bacteriological textbooks. Four of the common 

 methods of capsule staining, namely those of Anthonj^, of Hiss, of 

 Huntoon, and of Churchman, are given in Chap. II. The committee 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 significance 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, mth sketches of all the shapes that occur. 



Gram stain. The gram stain was until recently an entirely empirical 

 procedure for distinguishing between two groups of organisms, the actual 

 significance of which was not understood. Since 1940, however, the work 

 of Henry and Stacey (1943), Bartholomew and Umbreit (1944), and others 

 has shown that a positive reaction is dependent upon the presence of 

 ribonucleic acid in the outer layers of the cells, which can be removed by 

 treatment with ribonuclease and replated on them by treatment with 



