6o 



METHODS OF CULTIVATION OF BACTERIA. 



FlG. 24. Esmarch's 

 roll-tube adapted for cul- 

 ture containing anaerobes. 



ing from the last bottle to the vessel containing the medium 

 ought to be sterilised by passing through a Bunsen flame, and 

 should have a small plug of cotton wool in it to filter the hydro- 

 gen germ-free. 



Separation of Anaerobic Organisms. (a) By Roll-tubes. A 

 i \ inch test-tube has as much gelatin put into it as would be 

 , i used in the Esmarch roll-tube method. It 



is corked with an india-rubber stopper hav- 

 ing two tubes passing through it, as in 

 Fig. 24. The ends of the tubes are partly 

 drawn out as shown, and covered with plugs 

 of cotton wool. Three such test-tubes are 

 prepared, and they are sterilised in the 

 steam steriliser (p. 31). After sterilisation 

 the gelatin is melted and one tube inocu- 

 lated with the mixture containing the 

 anaerobes ; the second is inoculated from 

 the first, and the third from the second, 

 as in making ordinary gelatin plates. 

 After inoculation the gelatin is kept liquid by the lower ends of 

 the tubes being placed in water at about 30 C., and hydrogen is 

 passed in through tube x for twenty minutes. The gas-supply 

 tubes are then completely sealed off at x and z, and each test- 

 tube is rolled as in Esmarch's method till the gelatin solidifies 

 as a thin layer on the internal surface. A little hard paraffin 

 may be run between the rim of the test-tube and the stopper, 

 and round the perforations for the gas-supply tubes, to ensure 

 that the apparatus is air-tight. The gelatin is thus in an atmos- 

 phere of hydrogen in which the colonies may develop. The 

 latter may be examined and isolated in a way which will be 

 presently described. The method is admirably suited for all 

 anaerobes which grow at the ordinary temperature. 



(b)- By Novys and Buchner s Apparatus. The separation of 

 anaerobes may be more readily carried out than in the fore- 

 going methods by making dilutions of the bacteria in agar, 

 glucose agar, or gelatin, and pouring into Petri's dishes and 

 placing them in either a Novy's anaerobic jar (see Fig. 25) 

 or in a modified Buchner apparatus. If one uses the Novy 

 jar, the air is displaced by allowing a good stream of hydrogen 

 gas from a Kipp generator to pass through it for at least 30 



