iii43-« MANUAL OF METHODS FOR PURE CULTURE STUDY 



Advantages: The method is inexpensive and employs easily available materials. 

 No special apparatus is required — an advantage in laboratories where anaerobic 

 cultures are not usually prepared. It may be used at any incubation temperature 

 without danger of explosion. It is particularly suitable in problems requiring large 

 numbers of plate cultures. It is recommended especially for cultural and physiologi- 

 cal studies of strains which have been purified by other methods. Disadvantages: 

 Several hours may be needed for anaerobic conditions to become established and there- 

 fore the method is not suitable when the results are required quickly. It is not recom- 

 mended for routine clinical use where speed of isolation of pure culture is an important 

 factor. With certain enrichments it is not suitable for purification of species con- 

 taminated with aerobic spore-forming bacteria due to the quick growth of these forms. 

 In plate culture experiments, as in the isolation of new strains, no one plate may be 

 removed from the cylinder for observation until the end of the incubation period, for 

 to do so would destroy the anaerobic conditions within the cylinder. 



USE OF AEROBE TO ABSORB OXYGEN 



Another biological method for oxygen removal utilizes the growth 

 of an aerobic organism (usually Staphylococcus aureus, Serratia 

 marcescens, or Saccharomyces cerevisiae). A wide variety of applica- 

 tions of this system have appeared in the literature. The technics 

 suggested^ below involve the growth of the aerobic organism in 

 pure culture on a medium separate from that on which the anaerobe 

 is to be cultured. 



Method A 



Materials for method of Snieszko, 1930: (1) Two petri dishes of 

 ordinary size; (2) paper tape, scotch tape, adhesive plaster, or 

 plasticene; (3) culture of Serratia marcescens or other fast growing 

 aerobic organism; (4) tube of nutrient agar. 



Method: Select two petri dishes which have bottoms of exactly 

 the same size and sterilize these in position in their usual top sections. 

 Pour nutrient agar into the bottom half of plate A, and after solidifica- 

 tion, streak the medium heavily (or flood across surface with 0.5 ml. 

 of broth culture) with the aerobic organism. (As an alternate 

 method, seed the agar before pouring.) Pour into plate B, a medium 

 suitable for the anaerobe (see Leaflet 11, 9th Ed.); when hard streak 

 with the sample or culture of the anaerobe (or seed with the latter 

 prior to pouring). 



Remove the two bottoms from their respective tops and fit to- 

 gether at their rims. Use tape or other sealing device around the 

 juncture to provide an air-tight seal. Place plate in the incubator 

 immediately. If thermophilic anaerobic cultures are to be made, 

 replace the »S. marcescens by a thermophilic aerobe, or before placing 

 plates in thermophilic incubator, incubate for 18 hours at 32° C. to 

 allow S. marcescens to grow and to use the oxygen. 



Advantages: No elaborate equipment is needed, since the method uses ordinary 

 peLri plates and other common materials. Thus it is available as an emergency 



'These are similar to the Fortner method and are recommended in place of it. In 

 the Fortner method the aerobe is streaked on one half of the plate and the anaerobe 

 on the olluT lialf of the same dish. 



