158 MANUAL OF MICROBIOLOGICAL METHODS 



For a '* quick '* method, Morse and Weaver (1947) recommend using 

 small tubes containing 0.8-ml portions of 2 per cent thiopentone solution 

 preheated to 37°C in a water bath. These are inoculated from a 6-hr 

 agar slant culture using sufficient quantity to give a turbidity indicating 

 2,100 millions per liter. A strip of paper saturated with lead acetate 

 solution is inserted in the tubes, and they are reincubated for 30-45 min. 

 Clarke (1953) proposes a slightly different test; she calls attention to the 

 fact that very few of the cultures which she tested, using a micromethod, 

 failed to produce H2S from cysteine. Thiosulfate appeared to be a more 

 useful substrate than cysteine, since not all the cultures studied were able 

 to reduce the compound to H2S. When micromethods are used, the 

 importance of recording the conditions of the test cannot be over- 

 emphasized. 



LIQUEFACTION OF GELATIN 



The conventional method of determining liquefaction, which has been 

 given with but slight modification in all the reports on methods, is as 

 follows : 



Make a gelatin stab (plain 12 per cent gelatin), and incubate 6 weeks at 

 20°C, provided the organism under investigation will grow at that tem- 

 perature. Care must be taken to observe whether the organisms produce 

 rapid and progressive liquefaction or merely slow fiquef action not extend- 

 ing far from the point of inoculation. In the latter case the liquefaction 

 may be due merely to endo-enzymes that are released from the cell after 

 death and may not be what is generally called "true liquefaction" (that 

 is, the process resulting from the action of enzymes diffusing out of 

 actively growing cells) . Some slow liquefiers are true liquefiers, however, 

 and the distinction between slow and rapid liquefaction must be regarded 

 as very artificial. 



In early editions of the manual the Frazier (1926) method was given, but it was 

 omitted from later editions as not proving practicable. A recent modification of it 

 by Smith (1946), however, proves useful and has two advantages over the gelatin 

 stab method: (1) It does not require low temperature incubation; (2) it is more sensi- 

 tive in the case of weak liquefiers. The procedure is as follows: Streak culture on a 

 plate of nutrient agar containing 0.4 per cent of gelatin. Incubate at 28°C for 2-14 

 days according to rate of growth. Cover plate with 8-10 ml of a solution of 15 g 

 of HgCh in 100 ml of distilled water and 20 ml of concentrated HCl. This reagent 

 forms a white opaque precipitate with the unchanged gelatin, but a liquefier is sur- 

 rounded by a clear zone. 



There is another method recommended for organisms that do not grow at 20°C. By 

 this technic an inoculated tube of gelatin is incubated at 37°C, or whatever tempera- 

 ture may be the optimum, and then after incubation the tubes are placed in a cold- 

 water bath or in a refrigerator to determine whether or not the gelatin is still capable 



