114 CLASSIFICATION AND DESCRIPTION OF BACTERIA 
being studied should be streaked across the surface of the agar. After 
incubation a clear zone will be visible, which, if due to true proteolysis, 
will not be rendered opaque if treated with weak acid. If a clear zone is 
present after incubation, it may be due to either proteolysis or the 
acid formed in the metabolism of the organism. ‘To prove that proteo- 
lytic enzymes produced it, flood the plate with dilute acetic acid and 
if the clear zone remains, the presence of a protease is indicated. 
The Descriptive Chart calls for the determination of the reaction 
on certain days. In the past, this has been determined by titrating 
5 ¢.c. of the culture with N/20 NaOH or HCl. That this method is 
not accurate has been shown by Clark and Lubs. In order to take 
advantage of their work the Committee has advised the following 
procedure: 
Acid production in milk can be detected by adding brom eresol purple to 
the culture and comparing with the color obtained by adding the same propor- 
tionate quantity of the indicator to sterile milk. (Brom thymol blue does not 
give satisfactory results in milk.) Four degrees of acidity that can be recog- 
nized in milk are listed in Table IJ. They correspond closely to those listed in 
Table I, differing only on that brom cresol purple is used instead of brom thymol 
blue to show “neutrality ’’ and that the curdling point (Px=4.7) is used to 
separate between “ moderate’? and “strong” acidity instead of the less 
definite point of maximum red to methyl red. The same method of expression 
used in recording acidity in clear media should be used in recording that of 
milk. 
TABLE VI 
DEGRIEES OF ACIDITY EASILY RECOGNIZED IN MILK 
(After Conn e¢é al., 1918) 
ERR nee 
Acidity. Indicator Reaction, ete. Ap ee alue. 
Neutral....... Same color with brom cresol purple * as sterile milk; | 6.2-6.8 
i.e., blue to gray green. 
Weak.....,.... Color with brom cresol purple lighter than in sterile | 5.2-6.0 
milk; ie., gray green to greenish yellow. 
Moderate...... Yellow with brom cresol purple. Not curdled..... | 4.7-5.0 
Strong........ Curdled. Blue or green to brom phenol blue. ...... | 3.2-4.6 
Very strong....} Yellow to brom phenol blue..................... Under 3.0 
* Use a 0.04 per cent solution. 
