ELEMENTARY BACTERIOLOGY LABORATORY EXERCISES 23 
SECTION 8: PHYSIOLOGICAL REACTIONS 
OF BACTERIA 
EXERCISE 37 
GAS FORMATION 
Using the culture of Bacterium coli furnished, inoculate: 
(1) A Smith fermentation tube containing glucose broth. 
(2) A Durham fermentation tube containing glucose broth. 
(3) A tube of glucose broth covered with a paraffine jelly seal. 
(4) A glucose agar “shake culture”. 
Melt the tube of glucose agar in boiling water. When melted, cool to a tem- 
perature of 42° to 45° C. and inoculate. Thoroughly mix the inoculum with the 
medium, being careful to introduce as little air as possible. Cause the agar to 
solidify quickly by placing the tube in cold water. 
Incubate at 37° C. At the next period notice the collection of gas. 
The gas produced by most gas-forming bacteria consists chiefly of carbon 
dioxide and hydrogen. Analyze the gas formed in the Smith fermentation tube 
for carbon dioxide in the following manner. 
Measure on the arm of the fermentation tube the amount of gas formed. Fill 
the bulb with a 2% solution of NaOH and close the mouth with the thumb, tak-., 
ing care not to leave any air between the thumb and the liquid. 
Tilt the gas back and forth slowly from the closed arm to the bulb and back 
to the closed arm five or six times and finally allow the gas to collect again in the 
closed arm. The NaOH combines with the carbon dioxide and, consequently, on 
releasing the thumb, the volume of gas will become smaller in proportion -to the 
amount of carbon dioxide absorbed. 
Measure the amount of gas again and note the relation of carbon dioxide to 
the total gas formed. 
Test the remaining gas for hydrogen by tilting it back into the bulb, pre- 
viously covered with the thumb, and then holding the mouth of the tube to a 
flame and releasing the thumb. A slight explosion takes place from the combina- 
tion of the hydrogen with the oxygen of the atmosphere. 
Give the ratio of hydrogen to carbon dioxide found according to your results. 
The ratio of these two gases formed varies with different organisms. The results 
obtained from the quantitative analysis of the gas in a Smith fermentation tube 
are Not accurate since a large amount escapes from the open end of the tube. The 
proportion of carbon dioxide to hydrogen as revealed by this test is not accurate 
as the carbon dioxide is soluble and, therefore, a larger proportion of it is lost 
from the open end of the tube than of the insoluble hydrogen. However, the test- 
ing of the gas produced in a Smith fermentation tube is of value for qualitative 
purposes. 
