ROUTINE TESTS FOR THE DESCRIPTIVE CHART V49-I5 



action of enzymes diffusing out of actively growing cells). Some 

 slow liquefiers are true liquefiers, however; and the distinction be- 

 tween slow and rapid liquefaction must be regarded as very artificial. 



In early editions of this Leaflet the Frazier (192()) method was given, but it was 

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

 by Smith (19-i(i), 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% 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 HgCl2 

 in 100 ml. distilled water and 20 ml. concentrated HCl. This reagent forms a white 

 opaque precipitate with the unchanged gelatin, but a liquefier is surrounded bj- 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 

 of solidifying. Suitable uninoculated controls must always be run in parallel, especi- 

 ally if the optimum growth conditions for the organism necessitate prolonged ex- 

 posure of the gelatin to hydrolysis by mild acid, alkali or heat. In addition, pre- 

 cautions should always be taken to prevent evaporation of moisture which might 

 conceivably tend to obscure a slow liquefaction. This method has the advantage of 

 rarely giving positive results except in case of "true liquefaction". On the otlier 

 hand, it may well fail to detect cases of real liquefaction that have proceeded so slowly 

 that the gelatin can still set even after several weeks's incubation. The significance 

 of this test can be increased by using weaker than normal gelatin, — 4% gelatin, for ex- 

 ample, or even less. 



Other methods designed to give more technical information on the 

 subject are given in Leaflet VI. 



Cleavage of Sugars, Alcohols, and Glucosides 



Fermentable substance to employ. Quite a wide range of pure alcohols 

 and carbohydrates is available for use in fermentation tests. In 

 routine work the choice is often limited to the more common and 

 less expensive substances; but in special research work economy 

 is of less importance. The three sugars, glucose, sucrose, and lactose, 

 and the alcohols, glycerol and mannitol, are most widely employed 

 because they are readily available. Whether these compounds give 

 valuable information depends upon the group of organisms being 

 studied. If the group, like the colon group, is capable of fermenting 

 nearly all these substances, these readily fermented sugars and 

 alcohols may have very little value in separating the species one from 

 another; one must then employ one or more of the rarer compounds 

 In other words the selection is based upon the group of bacteria 

 under investigation. 



