92 CULTIVATION OF ANAEROBIC MICRO-ORGANISMS 



which will act in the same way as the bottle and prevent a rush of water 

 into the culture. The only ground of objection to this piece of apparatus is 

 that of cost. 



Tests for oxygen. 



It is often necessary to know whether a gas the hydrogen used in washing 

 anaerobic cultures, for instance is free from oxygen. This may be deter- 

 mined by passing the gas through a solution of indigo white, a substance 

 which turns blue in presence of small quantities of oxygen. 



Indigo white is prepared by treating indigotine (pure indigo) with concentrated 

 sulphuric acid. This solution when neutralized with sodium carbonate gives 

 sodium sulphindigotate, which in presence of an excess of alkali is easily decolourized 

 by reducing agents. Sodium sulphindigotate is generally reduced with sodium 

 hydrosulphite, obtained by adding to powdered zinc a concentrated solution of 

 sodium bisulphite saturated with sulphurous anhydride. Sodium hydrosulphite 

 is a powerful reducing agent and decolourizes the indigo, combining with the 

 oxygen of the atmosphere to form bisulphite. 



The gas may therefore be tested for oxygen by bubbling it, away from air, 

 through a solution of indigo white. 



To make sure that a culture medium contains no free oxygen, a few drops 

 of a O2 per cent, solution of sulphindigotate of sodium may be added until 

 the colour is distinctly blue, then 1 per cent, by weight of a normal soda 

 solution and 1 per cent, of glucose. When all the free oxygen has been 

 removed the blue colour disappears, the glucose reducing the indigo under 

 these conditions. 



If a culture medium tinted with a few drops of a solution of sodium 

 sulphindigotate be sown with an anaerobic organism and freed from oxygen, 

 the blue colour will be destroyed as growth of the organism proceeds, 

 decolourization commencing in the immediate neighbourhood of the colonies. 

 The micro-organism takes the oxygen necessary for its growth from the 

 substances around it, and acts therefore as a reducing agent. 



SECTION II. THE CULTIVATION OF ANAEROBIC ORGANISMS. 

 1. Liquid media. * 



A. Pasteur's method. This is the method originally employed in growing 

 anaerobic organisms. It is now only of historical interest. 



A large round flask A (fig. 79) with two tubulures 

 is filled with broth : the tubulure B dips into a porce- 

 lain dish three-parts filled with the same liquid. The 

 tap R being closed, the flask and porcelain dish are 

 simultaneously heated to boiling for half an hour. 

 The dissolved air is thus driven off. The apparatus 

 is allowed to cool in situ, and then the end of the 

 tube B is transferred to a vessel full of mercury. The 

 funnel E is filled with carbonic acid gas, and then 

 (away from air) with .the fluid to be sown. The tap 

 R is next opened and the fluid runs into the flask, 



_ care being taken that a little remains in the funnel 



FIG. 79. Pasteur's original method to prevent access of air to the flask. The culture is 

 for the cultivation of anaerobic or- then incubated. 



B. Roux's pipette. Method recommended. 



1. Make a constriction in a sterile Pasteur pipette in a small flame of the 

 blow-pipe just below the cotton-wool plug (fig. 67 a, p. 75). 



2. After flaming the point of the pipette, break it off, dip it into a tube of 

 broth already sown with the organism to be cultivated and aspirate the 

 broth into the pipette until the latter is three-parts full. 



