METHODS USED IN SYSTEMATIC BACTERIOLOGY 367 



culturing into a favourable medium. This is, of course, merely a rough 

 dififerential test. For accurate purposes, the column of fluid culture should 

 be enclosed in a capillary tube thin enough to ensure rapid heating of the 

 organisms to the temperature of the water in the bath. 



Metabolic Properties. 



Oxygen Pressure required for Growth. — Aerobic, facultatively anaerobic, obligatory 



anaerobic, microaerophilic. 

 Increased Carbon Dioxide pressure required for Growth. — The growth of many 



organisms is favoured by a partial pressure of CO 2 higher than that — 0-03 per 



cent.- — in the atmosphere, and some entirely fail to grow in the absence of a 



raised pressure. 

 Effect of Temperature on Growth. — Limits between which growth occurs ; optimal 



temperature for growth. 



Pigment Formation. — Tested usually on an agar slope incubated at 22° C, or left 

 at room- temperature in the light after preliminary incubation at 37° C. 



Effect of Modifying the Constitution of the Medium. — Effect on growth of adding 

 to the medium blood, serum, ascitic fluid, glucose, glycerine, potassium 

 nitrate, bile salts, or other substances. 



Biochemical Reactions. 



Fermentation of Sugars. — Tested in 1 per cent, peptone water containing 1 per 

 cent, of the sugar and Andrade's indicator. A Durham's tube is included. 

 For certain groups of organisms, which do not grow well in this medium, 

 5 per cent, of serum is added. Horse serum may be used as a rule, but in 

 testing the fermentation of maltose it is better replaced by human or rabbit 

 serum, since it contains an enzyme, maltase, which may lead to a false 

 reaction (Hendry 1938). Acid, or acid and gas production, is noted. 



Litmus Milk. — No change, acid or alkali ; clot ; clot disrupted by gas ; pep- 

 tonization ; saponification. The term " clot " is unfortunately used for 

 both an acid clot and a rennet clot. An acid clot results from the precipita- 

 tion of the caseinogen ; it is soft, gelatinous, does not retract, and can be 

 completely dissolved in alkali. A rennet clot is due to the coagulation of 

 the caseinogen under the influence of bacterial enzymes. A few hours after 

 its formation it retracts with the expression of a clear greyish-coloured fluid 

 called whey ; the clot itself is firm and cannot be dissolved by alkali. 

 Calcium caseinogenate is soluble in water. When acid is produced from 

 the lactose of the milk, the calcium combines with it, and the caseinogen, 

 which is insoluble, is precipitated. This is the mechanism of formation 

 of the acid clot. In coagulation by rennet the soluble calcium caseinogenate 

 is converted into insoluble calcium caseinate, which forms the curd. 



Indole. — Tested in 1 per cent, peptone water after 5 days' growth, using Bohme's 

 reagents. One ml. of ether is added to the culture, which is shaken thor- 

 oughly, and then allowed to stand till the ether collects on the surface. 

 1 ml. Solution A is run down the side of the tube ; if no colour appears 

 within a minute, 1 ml. of Solution B is added. A positive reaction is 

 characterized by a colour varying from a faint pink to a deep magenta. 

 According to Happold and Hoyle (1934) xylene is better than ether. 



