IMPORTANT PATHOGENIC BACTERIA 135 



consuming the oxygen by burning paper, or by sub- 

 stituting hydrogen gas for air. The following methods 

 are most commonly in use. 



1. Park'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) Solidify 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. 



a) 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. 



