86 ANAEROBIC ORGANISMS : ISOLATION AND CULTURE 



by passing it lightly two or three times through the flame, and 

 loosening the cotton-wool plug of the test-tube so as to admit 

 of its passage, the pipette is inserted into the inoculated medium 

 and its contents gently aspirated into the tube, which, however, it 

 is well not to fill to more than within from i| to 2 inches from the 

 top. Holding the tube in the right hand, with the upper aperture 

 closed by pressure of the forefinger, elevate the point slightly, and 

 seal it off in a small flame. 



Connect the tube with the tubing of the combined vacuum 

 pump and hydrogen apparatus, and after exhausting, wash some 

 four or five times in hydrogen {see p. 83). Exhaust again, and in 

 order to ensure the expulsion of any oxygen held in suspension in 

 the liquid, it is well at this stage to provoke ebullition in vacuo by 

 raising the temperature to 30° or 35° C. The simplest method of 

 doing this is by the gentle application to the exterior of the tube 

 of the small flame of a spirit or other lamp. Great care, however, 

 must be exercised to avoid such application of the flame as will 

 cause violent ebullition, as in this case the liquid will have a tendency 

 to pass into the exhaust tube. To prevent this, commence always 

 by applying the heat first to the higher part of the tube, just above 

 the level of the liquid. After ebullition has ceased, wash again 

 with hydrogen, re-exhaust, and either seal off at the lower con- 

 striction, strengthening the point of closure by the application of a 

 little Golatz wax, or screw down tightly the screw pinchcock upon 

 a short length of pressure tubing fitted as shown on Fig. 19. This 

 latter, which must of course be fitted previous to exhaustion of the 

 air within the tube, has the advantage of permitting the examination 

 of the culture at intervals. It is only necessary, if such examination 

 is desired, to attach the tube to the hydrogen apparatus, fill the 

 vacuum with the gas, snip off" the extreme point, allow a drop of 

 the culture to fall on a slide, and seal up afresh in the flame. 



3. Cultures in nitrog-en (pyrograllate of potash method)— 



Although not so rigorous as many of the methods to be here- 

 after described, this form of culture is a most valuable accessory, 

 owing to its extreme simplicity and the ready way in which it 

 lends itself to anaerobic cultivations both in liquid and upon solid 

 media. It depends upon the avidity with which oxygen is taken 

 up by a solution of pyrogallate of potash, and is applicable both 

 to test-tube and plate cultivations. 



Test-tube cultures. — In the lower portion of a Buchner's tube, 



