14 



BULLETIN 819, U. S. DEPARTMENT OF AGRICULTURE. 



Protease. — Fifty cubic centimeters of blood serum and milk serum, 

 filtered through a porcelain candle, were prepared, inoculated, and 

 incubated at room temperature for 3 days. For control purposes cul- 

 tures of B. proteus^ which is known to produce a proteolytic enzyme, 

 were also made, and flasks of serum which had not been inoculated 

 were likewise incubated with test cultures for controls. At the end of 

 the incubation period the liquid was made faintly acid with acetic 

 acid, and boiled to precipitate the unaltered proteins. These were 

 filtered out, and the filtrate was tested for polypeptids, peptones, 

 tyrosine, and tryptophane with copper sulphate and sodium hydroxid, 

 Millon's reagent and giyoxylic acid. 



Diastase. — Tube cultures of the pink yeast were prepared in sugar- 

 free broth. As controls, cultures of B. subtilis, which produces dias- 

 tase, and sterile broth tubes were incubated for controls, and all the 

 cultures were incubated at room temperature for 3 days. A thin 

 starch paste with 2 per cent thymol was prepared, and equal parts 

 of the cultures and the paste were mixed. The mixtures were incu- 

 bated for 6 hours at 37° C, and then filtered. The filtrates were 

 tested for sugar with Fehling's solution. 



Invertase. — In testing for the production of invertase, tube cul- 

 tures were made of the pink yeast in sugar-free broth as in testing 

 for diastase. Cultures were also made of the Saccharomyces cere- 

 visice, which inverts saccharose, and uninoculated cultures were in- 

 cubated for controls. A 2 per cent saccharose solution was made, 

 and 2 per cent phenol added. Equal parts of the cane-sugar solution 

 and the cultures were mixed and allowed to stand for several hours. 

 They were then filtered, and the filtrates tested for reducing sugar 

 with Fehling's solution. 



Rennin. — Tube cultures of the pink yeast and of B. prodigiosus 

 were prepared in sugar-free broth, and incubated for 3 days with 

 uninoculated cultures for controls. At the end of the incubation 

 period the cultures were heated to a sufficient temperature to sterilize. 

 By means of a sterile pipette 5 cc. of the culture were introduced 

 into each of 3 litmus milk tubes. The litmus milk tubes were placed 

 in the incubator at 22° C, and examined daily for 10 days. 



Table 4. — Ensyme production. 



Enzyme. 



Organism. 



Result. 



Enzyme. 



Organism. 



Result. 











+ 

 







+ 







Invertase 



Rennin 





+ 

 + 

 





\ B. pToteus 







(Control 



IControl 





( Pink yeast 



( Pink yeast 













+ 





[Control.. 



[control 















