104 CULTIVATION OF ANAEROBIC MICRO-ORGANISMS 



contamination. To overcome these disadvantages, the method has been 

 modified by Veillon and as modified by him is now [one of] the best 

 methods of isolating anaerobic organisms. 



1. Fill a number of large test-tubes (22 cm. x 15 mm.) to a depth of 10 to 

 15 cm. with some quite transparent agar containing 1*5 per cent, of glucose. 

 Sterilize in the ordinary way but do not allow the temperature to exceed 

 120 C. 



2. When ready to sow the tubes, heat five to ten of them to 100 C. in a 

 water bath, and boil them for 20 minutes or so to liquefy the agar and drive 

 off the air dissolved in the medium. Then transfer the tubes to a water bath 

 at 40 C. to keep the agar liquid until sown. 



3. Add one drop of the material to be sown to the first tube, and disseminate 

 it by rolling the tube between the hands. 



4. Sow the second tube with a few drops from the first, the third from the 

 second and so on, as previously described. 



5. Immediately the tubes are sown, cool them rapidly in the upright 

 position. Incubate. 



Aerobic organisms grow in the upper part of the medium which contains 

 a certain amount of air in solution, while the anaerobes multiply in the 

 deeper layer. 



6. When carefully examined it will be found that growth soon makes its 

 appearance, the number of colonies depending upon the extent to which 

 the material with which they were sown was diluted. Examine the 

 different colonies with the naked eye and with a lens and select the 

 tubes containing the smallest number of colonies for the purposes of sub- 

 cultivation. 



To sub-cultivate, take a Pasteur pipette with a fine point, break off the end, 

 and holding the culture-tube horizontally remove the wool plug and pass 

 the fine end of the pipette into the agar towards the colony to be removed : 

 as the pipette is passed through the colony some of the growth is forced 

 into it ; withdraw the pipette and sow the colony in a fresh tube of 

 medium. 



It facilitates the process of sub-cultivation to put, as Guillemot advises, an india- 

 rubber teat on the plugged end of the pipette ; the colony can then be more easily 

 drawn into the pipette by aspiration, and forced out into the new medium by 

 compression of the teat. 



Great care must be taken that the pipette does not touch any colony other 

 than that to be sub -cultivated ; the only way of avoiding such an accident 

 is to work with cultures in which the colonies are few in number and suffi- 

 ciently well isolated one from another. 



Note. It is often an advantage to use a medium containing serum, since many anaerobic 

 bacteria grow better in albuminous media. Prepare the agar as above, melt the contents 

 of the tubes, and cool to 40 C. in a water bath, then to each tube for two parts of agar 

 add one part of sterile liquid serum also heated to 40 C. : mix the agar and serum 

 thoroughly, keeping the medium at 40 C. to prevent the contents solidifying, and sow 

 with the material. 



SECTION IV. VACUUM INCUBATORS. 



Anaerobic organisms can be cultivated in ordinary culture vessels, provided 

 that these are incubated in a special form of incubator which can be exhausted 

 and the vacuum maintained. A little water, or, better, solution of potassium 

 pyrogallate which absorbs oxygen, should always be placed in these 

 incubators to prevent desiccation of the medium. 



In discussing isolation of anaerobic organisms by the plate method, Roux's 



