ANAEROBIC BACTERIA 1G5 



medium is simplified. An enriched culture is first prepared either by 

 adding some soil to a specific medium kept under specific conditions, 

 or the specific substance is added to the soil itself. An attempt is 

 then made to obtain a culture of the specific bacterium free from accom- 

 panying non-spore-forming and spore-forming aerobic and anaerobic 

 organisms. 



For the separation of spore-forming organisms from non-spore- 

 formers, whether aerobes or anaerobes, the soil is heated at 75° to 80°C, 

 by placing 2 grams of soil in 10 cc. of sterile water and keeping in a water 

 bath for 10 minutes. This leads to the destruction of all the vegetative 

 cells, while bacterial spores are not injured. The soil is then inoculated 

 into a proper medium, favorable for the development of the specific 

 organism, which will develop under proper cultural conditions; the cul- 

 ture is then transferred repeatedly upon the selective medium and grown 

 under strict anaerobic conditions. To purify anaerobes from aerobes, 

 the method of Dorner 17 can be used. The deep agar tube, inoculated 

 with the organisms, is allowed to cool and the agar to solidify. Two 

 cubic centimeters of melted agar containing 0.2 per cent of mercury 

 bichloride is then poured on the surface of the cooled agar and the 

 tubes are closed with rubber stoppers. The aerobes are thus completely 

 eliminated. However, neither of these methods will separate the facul- 

 tative anaerobes from the obligate anaerobes. 



To separate anaerobes from spore-forming aerobes, use is made of 

 three procedures: (1) Strict anaerobic methods of cultivation. (2) 

 The inhibitive action of gentian- violet on aerobic growth; 18,19 a 1 : 100,000 

 to 1:400,000 dilution of the dye in the agar medium is sufficient to 

 render cultures of anaerobic bacteria free from spore-forming aerobes. 

 (3) Anaerobic organisms are less sensitive than aerobes to pyrocate- 

 chin, chinon, sodium formate, and sodium sulphindigotate. 20,21 



The most difficult process, often involving a complicated technic, is 



17 Dorner, W. Beobachtungen liber das Verhalten der Sporen und vegetativen 

 Formen von Bac. amylobacter A. M. et Bredemann bei Nachweis- und Reinzucht- 

 versuchen. Landw. Jahrb. Schweiz. 1924, 1-28. 



18 Churchman. The selective bactericidal action of gentian violet. Jour. 

 Exper. Med. 16: 2, 221, 1912. 



19 Hall, I. C. Practical methods in the purification of obligate anaerobes. 

 Jour. Inf. Dis. 27: 576-590. 1920. 



20 Kitasato, S., and Weyl, Th. Zur Kenntniss der Anaeroben. Zeitschr. Hyg. 

 8: 41, 404. 1890. 



21 Rivas, D. Ein Beitrag zur Anaerobenzllchtung. Centrbl. Bakt. 32: 831- 

 842. 1902. 



