90 ANAEROBIC ORGANISMS: ISOLATION AND CULTURE 



4 



exhausted again, and the cultures left in vacuo or hydrogen as 

 desired, by screwing down tightly the screw pinchcock on the 

 short length of vacuum tubing. The ease with which cultures can 

 be examined and again replaced under anaerobic conditions renders 

 this method at times of great service. 



6. Esmarck roll culture (modified) for isolation of 

 org-anisms— 



Take tube as shown in Fig. 24, plug with cotton-wool, and 

 sterilise in the hot-air steriliser. Take two 

 tubes of liquefied gelatine and inoculate the 

 one with a trace of the culture to be studied, 

 or from which it is desired to isolate any 

 particular organism. Tint the other with sodium 

 sulphindigotate solution, and boil in a beaker 

 of water to get rid of the oxygen in suspension. 

 Cool down rapidly to 36° C, and inoculate 

 with one or two loopfuls of the first dilution. 

 Thoroughly incorporate, and by means of a 

 fine drawn sterile pipette transfer to the Esmarck 

 tube, after removal of the cotton-wool plug, 

 sufficient of this second dilution to give a tJiin 

 coating to the walls of the tube when making 

 the roll culture (from \ to | an inch from the 

 bottom of the tube will be found sufficient). 

 Replace the cotton-wool plug, and slightly con- 

 strict the tube-opening by turning the extremity 

 in the Bunsen flame. Allow the tube to cool, 

 and connect up with the hydrogen apparatus. 

 Exhaust, and wash four or five times with 

 hydrogen in the manner already explained. 

 Exhaust again, and seal off in vacuo at the 

 point of constriction. Liquefy the medium by 

 placing in a beaker of warm water not exceed- 

 ing 36° C, and, holding the tube horizontally, 

 turn and tilt it until the medium is seen to 

 cover the whole of the interior walls of the tube. 

 Solidify by turning under the tap or in a basin 

 of cold water, care being taken to get a perfectly even layer 

 distributed over the tube walls. Incubate at 22° C. When 

 growth has taken place, snip off the sealed point in the heated 

 air above the Bunsen flame (to prevent accidental contamina- 



\J 



Fig. 24. — Esmarck 

 roll culture. 



