CHAPTER IX 



METHODS OF DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA 

 ANIMAL EXPERIMENTATION 



Gas Formation. — Bacteria of many vaiieties produce gas from the 

 proteid and the carbohydrate constituents of their environment. 



Gas formation can be observed in a very simple manner by making 

 stab cultures in gelatin or agar containing the fermentable nutrient 

 substances. In such cultures bubbles of gas will form along the track 

 of the inoculation, or, in the case of such semisolid media as the tube 

 medium of Hiss, will spread throughout the tube. In the case of some 

 anaerobes gas formation in stab cultures will occur to such an extent that 

 the medium will split and break. It should be borne in mind in carrying 

 out such methods that air is readily carried into the medium with 

 the inoculating needle or loop by splitting of the medium, also that 

 media which have been stored in the cold may absorb air. Expansion 

 of the air in such tubes may simulate small amounts of gas formation 

 and lead to error. It is advisable, therefore, whenever making stab 

 inoculations with the above purpose, to heat the media and rapidly 

 cool them before use. 



A more accurate method of gas determination is by the use of fer- 

 mentation tubes, such as those devised by Smith. The gas which is 

 formed collects in the closed arm of the fermentation tube and may be 

 quantitatively estimated. The fermentation, with gas production, of 

 certain substances such as carbohydrates, may be determined by adding 

 these materials in a pure state to the media before inoculation with 

 organisms. 



In the case of carbohydrates this method has proved of great differ- 

 ential value, since the power of splitting specific carbohydrates with gas 

 production is a species characteristic of great constancy for many forms 

 of bacteria. 



Analysis of Gas Formed by Bacteria. — Carbon Dioxide. — For 

 the estimation both qualitatively and roughly quantitatively of carbon 

 dioxide produced by bacteria, cultures are grown in fermentation tubes 

 containing sugar-free broth (see page 125) to which one per cent of pure 

 dextrose, lactose, saccharose, or other sugars has been added. The tubes 

 are incubated until the column of gas formed in the closed arm no longer 



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