90 CULTIVATION OF ANAEROBIC MICRO-ORGANISMS 



4. By the use of a vacuum. 



The use of apparatus by means of which a vacuum can be produced 

 simplifies the methods of cultivating anaerobes and at the same time 

 renders them more exact ; and moreover, as both a mercury pump and 

 water pump are in everyday use in the laboratory, the essentials are 

 ready to hand. The use of a vacuum is generally supplemented by 

 washing with an inert gas ; by the combination of the two methods 

 it should be possible to remove all trace of oxygen from the culture 

 vessels. 



In many laboratories the further precaution is taken of adding some oxygen- 

 absorbing solution, generally pyrogallol and potash, to absorb any traces of oxygen 

 which might still remain. 



The reason for washing with an inert gas lies in the physical fact that two gases, 

 which do not enter into chemical combination, rapidly diffuse when brought in 

 contact and form an uniform and constant mixture. The rate of diffusion varies 

 directly as the differences in density of the gases ; the greater the difference the 

 more rapid the diffusion. 



In practice it is impossible to obtain a perfect vacuum, so that after exhausting 

 a vessel full of air a residuum of air remains. Now if the vessel be filled with 

 hydrogen and exhausted again the residuum will consist of a mixture of air and 

 hydrogen ; by repeating the process several times, the amount of air ultimately 

 present will be infinitesimal in amount. 



Suppose that after exhausting a vessel of 2 litres' capacity there remains 1 c.c. 

 of air measured at atmospheric temperature and pressure ; fill the vessel with 

 hydrogen, and the 1 c.c. of air will be diluted 1 in 2000 ; exhaust again until only 

 1 c.c. remains, and the residual gas will contain o J o (T c.c. of air and $$$ c.c. of 

 hydrogen ; after a second washing with hydrogen the volume of air will not exceed 



4.000,000 c ' c> 



A. Mercury pump. With this apparatus an almost perfect vacuum can 

 be obtained, but it is expensive and being delicate is liable to be easily 

 damaged ; moreover time and skill are required to use it to the best advantage. 

 Its use is limited in practice to very delicate investigations and to vessels of 

 small capacity. Without going into the details of the working of the pump 

 the following points of importance in connexion with its use may be 

 noted. 



1. The pump must always be tested to see that it is working properly 

 and that the taps fit well. Any taps not fitting tightly must be lubri- 

 cated. 



2. Connect the vessel containing the culture to the pump, and exhaust 

 until there is a wide difference between the levels of the mercury in the two 

 limbs of the manometer. 



3. Then open the tap connected to the hydrogen supply just a little, and 

 let the hydrogen pass slowly into the receiver until the mercury has reached 

 its original position. 



4. Turn off the supply of hydrogen. Exhaust again, and repeat the 

 process two or three times. 



5. Seal the neck of the culture vessel in the flame in vacuo. 



B. Water pump. On account of its moderate price and of the ease with 

 which it is worked, a water pump is much more often used for producing 

 a vacuum than a mercury pump. The vacuum is only approximate, and 

 exhaustion with a water pump must therefore be combined with washing 

 with an inert gas. 



The pump, which is best made of metal (d'Alvergniat's pattern), should 



