PROTEOLYTIC ENZYMES OF BACTERIA 99 



Bs. sub tilts, Bact. prodigiosum or Bact. liquefadens and in- 

 oculate for three days at 30 C. 



To the cultures add about .25 c.c. of toluene and a very 

 small amount of barium sulphate powder so as to render the 

 liquid turbid : shake thoroughly. 



Pour some of this culture into a tube containing ordinary 

 nutrient gelatine which has been made slightly alkaline with 

 sodium carbonate. 



Allow the whole to stand and when the insoluble barium 

 sulphate has fallen on the surface of the solid gelatine mark the 

 point on the outside of the tube with a piece of gummed paper. 



Keep the tube at 20 C. 



If the solution contains a proteolytic enzyme, the solid 

 gelatine will be slowly dissolved and the thin layer of barium 

 sulphate will descend in the tube. 



Try the experiment with Bact. coli and Sir. lacticus and note 

 the results. 



Ex. 57. Cultivate one of the bacteria mentioned in the 

 previous Ex. in nutrient broth. 



Add .25 c.c, of toluene, shake thoroughly and then suspend 

 in the solution a small piece of Griibler's " Karmin fibrin." 



If the culture contains a enzymes the fibrin dissolves and the 

 carmine with which it is stained diffuses and colours the solution- 

 Try the experiment with Bact. coli, Bact. acidi lactiri, and 

 Sir. lacticus, and note the results. 



Ex. 58. Melt a tube of nutrient agar in a beaker of boiling 

 water. Pour into it some sterilized skim milk which has been 

 heated to about 50 to 60 C. : the proportion of milk to agar 

 medium should be about one of milk to three of agar. Colo 

 to about 40 C., inoculate with Bs. subtilis, Bact. vulgare, or 

 other organism which possesses proteolytic powers. Pour into 

 a small Petri dish and inoculate at 30 C. 



The casein of the milk is peptonised when the colonies 

 develop, the medium at those points becoming clear. 



Try the experiment with Bact. coli and Str. lacticus. 



