METHODS OF PURE CULTURE STUDY 121 



Provisional method. It is recommended that the following 

 method proposed by Rothberg (1917) be put in provisional use 

 until experience shows its value. It is designed to distinguish 

 ''true liquefiers" (organisms producing ecto-enzymes) from the 

 organisms that produce endo-enzymes of proteolytic action that 

 are released from the cell after death and cause liquefaction of 

 the gelatin if incubated for the long period mentioned above. 

 The method is to give the organism a preliminary cultivation 

 for eighteen to forty-eight hours (according to its rapidity of 

 growth) in a 1 per cent solution of gelatin at 25° or 37° according 

 to its temperature relations; then inoculate on surface of gelatin 

 in test tube and incubate 15 days at 20°. 



Relation to free oxygen. Provisional method. Determine by 

 noting the presence or absence of growth in open and closed arm, 

 respectively, of fermentation tubes containing glucose broth. 

 Care must be taken to use fermentation tubes from which the 

 dissolved oxygen has been recently driven off by heating. In 

 case of gas production, this test is of comparatively little value, 

 because bubbles of gas may carry the sediment up with them; 

 hence if an organism produces gas from glucose, the test should, 

 if possible, be made in the presence of some other sugar which 

 it attacks (acidifies) without gas-formation. It must be remem- 

 bered, however, that even anaerobes do not grow in the absence 

 of free oxygen except in the presence of a chemical substance 

 (such as carbohydrate) which they are able to reduce and use 

 as a source of oxygen. 



F ermentatio7i of sugars and glycerin. This is normally to be 

 studied in fermentation tubes. Ordinarily use beef-extract 

 broth containing 1 per cent of the substance investigated; but 

 if the organism does not grow well in such broth and some me- 

 dium is known in which it does grow well, the latter may be 

 used. Generally speaking, organisms of series I and II should 

 be studied in broth, organisms of series III and IV in some other 

 medium. Incubate organisms of series I and III at 37°, organ- 

 isms of series II and IV at 25°. Test ordinarily on 1st, 3rd, and 

 7th days, although the best days for testing will depend upon the 

 rapidity of growth of the culture. Inoculations should always 

 be made at least in triplicate. 



