THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 125 



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 solidification 

 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 Chap. Ill) ; 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 together 

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

 provide an airtight 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 incu- 

 bator, incubate for 18 hr 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 

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

 method in almost any laboratory at any time. The technic is extremely simple and 

 can be set up by inexperienced individuals. Since each set of plates is an individual 

 unit, observation of the growth of each anaerobe may be carried out without destroy- 

 ing anaerobic conditions for other cultures. Disadvantages. The method is somewhat 

 time-consuming when large numbers of cultures are to be made and is therefore not 

 suitable in laboratories where routine platings of a number of cultures is not an unusual 

 event. Anaerobiosis may not be attained promptly enough to prevent death of the 

 inoculum of nonsporeforming species of vegetative cells of anaerobic sporeformers. 

 Negative results on isolation attempts with this procedure are hence not reliable. 



Chemical Methods for Oxygen Removal 



Many of the methods proposed for removal of oxygen from the environ- 

 ment for anaerobic culture involve the initiation of a chemical reaction in 

 which oxygen is consumed. Of the various systems which have been sug- 

 gested, those which are recommenxled have been tested and used suffi- 

 ciently to show their utility, and they do not require elaborate apparatus. 



Phosphorus Jar 



Materials. (1) Sticks of yellow (or white) phosphorus (which must he 

 kept under water in tightly stoppered, wide-mouth bottle; the small sticks, 

 %6-in.-diameter, are the most useful); (2) Pyrex cylinder, or any con- 

 venient jar or container w^hich may be sealed airtight; (3) pair of long 

 forceps or chemical tongs; (4) plasticene; (5) small amount of tap water. 



