BIOCHEMICAL REACTIONS 863 



centration of COj of the order of 2-10 per cent. (Rockwell 1924, Dack et al. 1927, 

 Aitken et al. 1936). 



On ordinary media growth of the anaerobes is poor compared with that of the 

 aerobic spore-bearers. Some strains grow better than others — Gl. welchii, CI. 

 bifernientans, CI. hotulmum ; some give poorer growths — CI. chauvoei, CI. cochlearium. 



Glucose fa^vours the saccharolytic species ; blood or serum improves the growth 

 of all. The optimum H-ion concentration for growth is about pH 7-0 to 7-4 (Reddish 

 and Rettger 1924). 



On media containing bile salts, such as MacConkey's medium, growth of CI. 

 sporogenes, CI. botuUnum, CI. histolyticum, CI. welchii, CI. tetani, and CI. septicum 

 is accompanied by a greenish fluorescence. In our experience, CI. chauvoei and 

 CI. cedematiens have failed to grow on this medium. 



Most of the members with which we are dealing here grow best at about 37° C, 

 though many of them are capable of growing at temperatures of 20° C. and even 

 lower. There is a group of thermophilic Clostridia which have an optimum tempera- 

 ture about 50°-60° C, and which sometimes do not grow at all below 30° C. 



HEMOLYSIN Production. — Apart from their action on blood agar plates, many 

 of the anaerobes, such as CI. tetani, CI. welchii, CI. septicum, CI. cedematiens, and 

 CI. chauvoei, produce filtrable hsemolysins capable of dissolving sheep's red blood 

 corpuscles. Kerrin (1930) states that atoxic strains of CI. tetani produce as powerful 

 a hsemolysin as do toxic strains, and that normal rabbit, horse, and human serum 

 have a very strong antihsemolytic effect. For the detection of hsemolysins, care 

 must be taken to buffer the hsemolytic systems at the pH of optimum activity 

 (Walbum 1938). Fibrinolysins are formed by some species (Carlen 1939, Reed, 

 Orr and Brown 1943) and leucocidins by others. 



Biochemical Reactions. — The action on sugars is of some value in differentiating 

 the anaerobes, and constitutes one basis of classification. Great care must be 

 exercised in carrying out the tests, since even with known stock strains the results 

 are often irregular and must be repeated two or three times before they can be 

 relied on. Some Clostridia decolorize indicators irreversibly, so that the formation 

 of acid in a fermentation-tube should always be tested by the addition of fresh 

 indicator to a sample of the culture. 



Reed (1942) points out that both indole formation from tryptophan, and the 

 reduction of nitrates to nitrites, depend on the relative rates of breakdown of the 

 original substrates, and of substances formed from them. Thus, most Clostridia 

 reduce both nitrates and nitrites, and only if the reduction of nitrates is the quicker 

 of the two processes will a positive test for nitrites be obtained. 



One of the striking features of the anaerobic bacteria is the large amount of 

 gas that they are able to produce even in media free from fermentable carbohydrates. 

 Thus Wolf and Harris (1917) found that CI. welchii in casein water produced 90 ml. 

 of gas per litre of medium, and in peptone water 186 ml. CI. sporogenes formed 

 1,044 ml. of gas per litre of casein water in 157 hours, and in peptone water 360 ml. 

 in 24 hours. The gas consists of COj and Hj in different proportions according 

 to the species of anaerobe. The addition of a fermentable carbohydrate to the 

 medium increases the gas production. Acids are formed as the result of the fer- 

 mentation ; with CI. welchii rather more than 50 per cent, are volatile — mostly 

 butyric acid. Ammonia appears to be formed in large quantities by the proteolytic, 

 and in much smaller quantities by the saccharolytic anaerobes. 



An attempt has been made (Anderson 1924) to classify the anaerobes on the 



