162 MANUAL OF MICROBIOLOGICAL METHODS 



that produce a small amount of acid in some tubes but not in others con- 

 taining the same carbon source and in cases where the addition of some 

 carbon source results in a distinctly improved growth without the appear- 

 ance of demonstrable acid or gas. In routine work, accordingly, one should 

 record as positive only those organisms that produce considerable acid 

 or gas from a given compound and as negative only those that con- 

 sistently fail to show any acid or gas or any increase of growth when 

 supplied with the carbon compound under investigation. All others 

 should be regarded as border-line cultures, calling for further investigation. 

 It is especially important to recognize that the question whether or not 

 cleavage of a carbohydrate occurs depends greatly on the cultural charac- 

 teristics. Clarke and Cowan (1952) remark that tests of fermentative 

 ability are often tests of the ease with which the buffer capacity of a 

 medium is overcome. Accordingly some bacteria fail to produce an acid 

 reaction in a beef-extract-peptone medium but will do so from the same 

 carbohydrate in a synthetic or semisynthetic medium. It must accord- 

 ingly be recognized that although tests for fermentative ability in some 

 medium may have diagnostic usefulness, they do not necessarily indicate 

 actual ability to metabolize the carbohydrate therein. 



HYDROLYSIS OF STARCH 



The breaking down of starch is rather more complicated than that of 

 sugars because of the extensive hydrolysis that is necessary before it can 

 be utilized by the bacteria. The first stage of this process is generally 

 known as diastatic action because of the similarity to that brought about 

 by the enzyme diastase. The final end result is usually an increase in 

 acid, so one may obtain good evidence as to the utilization of starch by 

 substituting it for sugar in the above methods (pages 159ff.) and deter- 

 mining acid produced or increase in H-ion concentration. It is often 

 desirable, however, to secure evidence as to the intermediate products 

 and as to whether the starch has been entirely consumed or not, and 

 various methods have been proposed for this purpose. 



This test may be made on raw starch, dissolved by boiling, or on the 

 so-called "soluble starch." The latter is a partly hydrolyzed product, 

 but it is often used as "starch" in this test because its iodine reaction is 

 like that of true starch and different from that given by typical dextrins. 

 If soluble starch is used, its true nature must be taken into account, but 

 at the same time it must be remembered that true starch is partly 

 hydrolyzed when sterilized in culture media, and even cultures growing 

 in such a substratum are not furnished with raw starch as the sole carbo- 

 hydrate. When such media are filtered, possibly "soluble starch" is all 

 that remains. 



