168 BIOLOGY AND TECHNIQUE 



In bacteriological work 4 c.c. of the culture fluid is poured into a 

 clean test tube, and to it are gradually added 2 c.c. of the mixed test 

 solutions. A pink color indicates the presence of nitrites, the intensity 

 of the color being proportionate to the amount of nitrite present. 



The reducing powers of bacteria may also be shown by their ability 

 to decolorize litmus, methylene-blue, and some other anilin dyes, 

 which on abstraction of oxygen form colorless leukobases. 



Enzyme Action. — The action of the enzymes produced by bacteria 

 may be demonstrated by bringing the bacteria, or their isolated fer- 

 ments, into contact with the proper substances and observing both the 

 physical and chemical changes produced. In obtaining enzymes free 

 from living bacteria, it is convenient to kill the cultures by the addition 

 either of toluol or of chloroform. Both of these substances will 

 destroy the bacteria without injuring the enzymes. Enzymes may also 

 be obtained separate from the bodies of the bacteria by filtration. 



Proteolytic Enzymes. — The most common evidences of proteolytic 

 enzyme action observed in bacteriology are the liquefaction of gelatin, 

 fibrin or coagulated blood-serum, and the peptonization of milk. This 

 may be observed both by allowing the proper bacteria to grow upon 

 these media, or by mixing sterilized cultures with small quantities of 

 these substances.^ The products of such a reaction may be separated 

 from the bacteria by filtration and then tested for pepton by the biuret 

 reaction. 



Proteolytic ^ enzymes may also be determined by growing the bac- 

 teria upon fluid media containing albumin solutions, blood serum, or 

 milk serum, then precipitating the proteids by the addition of ammomum 

 sulphate (about 30 grams to 20 c.c. of the culture fluid) and warming 

 between 50 to 60° C. for thirty minutes. The- precipitate is then filtered 

 off, the filtrate made strongly alkaline with NaOH, and a few drops 

 of copper sulphate solution added. A violet color indicates the pres- 

 ence of pepton — proving proteolysis of the original albumin. 



(Acetic acid of 1.04 prepared by diluting 400 c.c. of cone, of Sp. Gr. 1.75 with 70C 

 c.c. of water.) 



II. A-Naphthylamin. — Dissolve 0.1 g. in 20 c.c. of water, boil, filter (if necessary), 

 and to clear filtrate add 180 c.c. of acetic acid, Sp. Gr. 1.04. 



The solutions are kept separate and mixed in equal parts just before use. 



In carrying out the test, put 2 c.c. of each reagent in a test tube and add substance 

 to be tested. (In ordinary water analysis use 100 c.c.) Cover tube with watch 

 glass and set in warm water for 20 minutes. Observe presence or absence of pink 

 color promptly. Always run a blank on the distilled water used for rinsing to avoid 

 errors due to nitrites in the water, or in the air of the laboratory. 



1 Bitter, Archiv f . Hyg., v. 1886. 



^ Hankin and Wesbrook, Ann. Past., vi., 1892. 



