IMPORTANT PATHOGENIC BACTERIA 13 S 



consuming the oxygen by burning paper, or by sub- 

 stituting hydrogen gas for air. The following methods 

 are most commonly in use. 



1. Pafk's method. 



a) Boil three tubes of dextrose agar vigorously for 5 

 minutes, to drive out the dissolved oxygen. 



b) Cool to 43° C. and inoculate from laboratory 

 cultures of B. tetani, B. edematis, and B. welchii. 



c) SoUdify rapidly by immersion in cold water, 



d) Cover the medium with a thin layer of liquid 

 paraffin or sterilized mineral oil. 



e) Incubate at 37° C. 



Note. — ^The layer of paraffin or oil excludes atmospheric 

 oxygen which is inhibitory to the growth of anaerobes. The 

 oxygen necessary for their multiplication is derived from carbo- 

 hydrates in the medium. 



2. Wright's modification of Buchner's method. 



o) Liquefy, as before, six dextrose agar tubes, 

 plugged with absorbent cotton. Cool three to 43° C. 

 and inoculate while fluid. Let the other three become 

 solid, and make stab cultures. 



b) Sterilize the cotton stoppers in a flame, and with 

 the forceps, sterilized in a flame, push the stoppers into 

 the test tubes for the distance of about i inch (2-3 cm.). 



c) Pour into the tubes (upon the cotton stoppers^ 

 2 c.c. of a saturated solution of pyrogallic acid in water, 

 followed by 2 c.c. of a 2 per cent solution of NaOH. 



d) Cork the tubes immediately with rubber stop- 

 pers, and keep upside down. 



e) Incubate at the required temperature. Pyro- 

 gallic acid in alkaline solution absorbs oxygen, leaving 

 the cultures in an atmosphere of nitrogen. 



