METHODS FOR MICROSCOPIC STUDY OF BACTERIA 249 
(c) Goidh of Bacteria in 6*e/fl/i».— Gelatin is added to cultural 
media both to confer upon the media the property of solidifying, and 
to enrich the content of nitrogenous substances. 
Pure gelatin does not contain tyrosine and it is relatively rich in 
diamino-acids; according to Hausmann/ nearly 36 per cent of the 
nitrogen in gelatin is diamino-nitrogen— about 63 per cent in the 
form of mono-amino-acids. Chemically, gelatin media are convenient 
for the demonstration of soluble, proteolytic enzymes. In the absence 
of utilizable carbohydrate, several types of bacteria "liquefy" gelatin, 
that is, through the activity of their proteolytic enzymes the gelatin 
molecule is split by hydrolytic cleavage to molecules so simple in 
their state of aggregation that they can no longer produce a "gel." 
The presence of utilizable carbohydrate prevents the liquefaction of 
gelatin by many bacteria.^ 
Formerly the morphology of the liquefied zone in gelatin stab cul- 
tures was regarded as distinctive for individual organisms; thus, the 
napiform liquefaction produced by cholera vibrios was supposed to 
be sufficiently constant to possess diagnostic value. It is now generally 
conceded that this morphological characteristic is of comparatively 
little importance for the identification of the organism. On the other 
hand, the liquefaction of gelatin and of coagulated blood serum and 
casein as well is important from a chemical viewpoint, because it 
indicates the activity of a soluble proteolytic enzyme.' 
2. Growth of Bacteria in Fluid Media, — (o) Plain Broth.— V\aAn 
broth, prepared from meat infusion and peptone in the usual manner, 
and freed from sugar with Bacillus coli,* contains, on the average, 
from 150 to 250 mg. of nitrogen per 100 cc. A small but variable 
amount of the nitrogen exists as free ammonia.^ Less than 10 per 
cent of the total nitrogen, as a rule, exists as amino-nitrogen (deter- 
mined by the method of Van Slyke).^ 
The visible changes in the appearance of broth cultures incidental 
to the development of bacteria are usually not of great importance; 
they consist essentially of turbidity, sediment and occasionally a ring 
or pellicle. The development of a pellicle is of importance in the pro- 
duction of toxin by the diphtheria bacillus, however, because it indi- 
cates the maximum oxygenation of the bacteria. The character of 
the turbidity and sediment— the viscidity and color— may afi'ord 
some information of the character of the organism. Products of 
importance are frequently detected by chemical or physiological 
examination in plain broth cultures of bacteria. Thus, in the absence 
1 Ztschr. f. physiol. Chem., 1899, 27, 95. 
2 Kendall and Walker: Jour. Infec. Dis., 1915, 17, 442. 
' The enzyme may be obtained sterile and in an active state in the filtrates of liquefied 
gelatin, blood serum, casein, and from plain broth cultures as well, if the bacteria are 
removed by filtration through unglazed porcelain. (Auerbach: Arch. f. Hyg., 1897, 
31, 311. Rubner: Arch. f. Hyg., 1904, 48, 260; 1906, 57, 161, 19.3. Bcrghaus: Ibid., 
1908, 64, 1. It is precipitated by azine dyes. Walker: Proc. Soc. Exp. Biol, and Med. 
1927, 24, 839. 
* Theobald Smith: Centralbl. f. Bakteriol., 1897, 22, 45. 
6 Determined by the Folin method, Jour. Biol. Chem., 1912, 11, 523. 
6 Jour. Biol. Chem., 1912, 12, 275; 1913, 16, 121. 
