ANAEROBIC BACTERIA 167 



developing preferably at the different temperatures, as in the case of 

 thermophilic bacteria. 



5. Use of high dilutions for the separation of organisms before 

 plating. 25 



6. Isolation of the individual colony. This can be accomplished 

 either (a) by the picking of surface colonies from agar or gelatin plates 

 or slants in large tubes, kept under anaerobic conditions; (6) by picking 

 colonies from deep agar tubes, 26 the last procedure being the easiest and 

 most reliable in the process of separation of pure cultures of anaerobic 

 bacteria from all accompanying forms. 



7. Finally the isolation of single cells either by the India ink method, 27 

 by the method of Barber, or by one of the microscopic methods. 28 



A detailed study of the various methods used for the isolation from 

 surface colonies is given elsewhere. 29,30 



In general, plates or large agar slants containing the proper culture 

 media are streaked out and placed either in vacuo, in hydrogen, carbon 

 dioxide, or in an atmosphere from which the oxygen is removed by means 

 of sodium pyrogallate. 31 To produce discreet colonies, the agar plates 

 or slants must be dried before inoculating, but too much drying of the 

 medium is injurious. The slants or plates are streaked out with a loop- 

 ful of the material taken from the enriched culture or using a dilution 

 of it. The plates are immediately placed in the atmosphere of the neu- 

 tral gas; the agar may also be placed into the upper part of a Petri dish, 

 which is then covered directly with the sterile inverted lower half of 

 the dish and the whole covered with a larger Petri dish. 32 



26 Stoddard, J. L. Points in the technic of separating anaerobes. Jour. Am. 

 Med. Assn. 79: 906. 1918. 



26 Burri, R. Zur Isolierung der Anaeroben. Centrbl. Bakt. II, 8: 533-537. 

 1902. 



27 Burri, 1909 (p. 55); also in Krause-TJhlenhut's Handbuch der mikrobiolo- 

 gischer Technik. 2: 329. 1923. 



28 Barber, 1911-1920 (p. 56). Kendall, A. I., Cook, M., and Ryan, M. Methods 

 of isolation and cultivation of anaerobic bacteria. Jour. Inf. Dis. 29: 227-234. 

 1921. Holker, J. Micro- and Macro-methods of cultivating anaerobic organ- 

 isms. Jour. Path. Bact. 22: 28. 1919; 23: 192-195. 1920. 



29 von Hibler, E. Untersuchungen liber die pathogenen Anaeroben. Jena. 

 1908. 



30 Besson, A. Practical bacteriology, microbiology and serum therapy. Lon- 

 don, 1913. 



31 Lentz, O. In Friedberger und Pfeiffer's Lehrbuch der Mikrobiologie. Jena, 

 1919, p. 370. 



32 Marino, F. Mcthode pour isoler les anaerobes. Ann. Inst. Past. 21: 

 1005. 1907; also Ogata, M., and Takenouchi, M. Einfache Plattenkultur- 

 methode der anaeroben Bakterien. Centrbl. Bakt. I, 73: 75-77. 1914. 



