III43-4 MANUAL OF METHODS FOR PURE CULTURE STUDY 



or in case of doubt, the following technic will usually suffice to 

 characterize an anaerobic strain and to differentiate if from the 

 aerobes: Inoculate, while the agar is molten, several deep tubes 

 (8-9 cm. columns of medium) of a suitable nutrient agar medium 

 (see Leaflet II) containing 1.0% glucose; allow these to solidify 

 in an upright position, and incubate the tubes at several tempera- 

 tures or at the optimum temperature for the organism in question; 

 adjust the seeding so that relatively few (e.g., 25-50) colonies per 

 tube will result. With an obligate anaerobe, all of the colonies 

 should be localized in the bottom of the tube and none should appear 

 on the surface or in the upper 1 cm. layer. Likewise, with pathogenic 

 organisms cultured in fluid thiogly collate medium, the growth should 

 be confined to the lower section of the medium and no growth 

 should result in the upper layer wherein the methylene blue is 

 recolorized. If growth does occur in the upper layer of either 

 medium, the culture is either not an obligate anaerobe or is con- 

 taminated with an aerobic or a facultative species. 



ANAEROBIC CULTURE METHODS AND EQUIPMENT 



All of the procedures which have been devised for the cultivation 

 of anaerobic bacteria have the single purpose of excluding atmospheric 

 oxygen from the environment in which the growth is to take place. 

 With certain tubed media the oxygen potential may be reduced 

 sufficiently by constituents of the medium to permit anaerobic 

 growth (Hewitt, 1937; Knight, 1931; and Reed and Orr, 1943). 

 Since, however, this is rarely possible for surface cultures on a solid 

 medium, usually plate and slant cultures are incubated within a 

 closed container from which the oxygen is removed by one or another 

 means. A study of the various methods shows that no single 

 procedure may be proposed as the best technic but that the method 

 of choice will depend upon the prevailing circumstances. A pro- 

 cedure which is ideal for one situation may be impractical or im- 

 possible to apply with 'other conditions. Each of the technics out- 

 lined below is recommended within the limits proposed in the dis- 

 cussions. 



Use of Methylene Blue as Indicator of Anaerobiosis. For all types of anaerobic jars 

 and containers, except individual plating or tube culture systems, it is convenient 

 to include an indicator tube which will serve as a check on the development of anaero- 

 biosis. The most commonly used system utilizes the change of methylene blue from 

 the colored (oxidized state) to the leuco form (reduced state) Using the solution 

 prepared as given below, any system which gives sufficient degree of removal of 

 oxygen from the atmosphere for anaerobic growth to develop will cause the blue color 

 of the solution to disappear or will maintain the colorless condition if the solution is 



