96 ANAEROBIC ORGANISMS: ISOLATION AND CULTURE 



the second lateral tube, and repeat with a culture of the second 

 organism. Seal off the point, and connect up with the vacuum 

 pump and hydrogen apparatus. Exhaust, wash well in hydrogen, 

 and seal off in vacuo or hydrogen as desired, by screwing down 

 tightly the screw pinchcock on a short fitting of pressure tubing 

 fitted as shown on Fig. 19 (p. 85). Incubate at 37°, or room tem- 

 perature as desired. When growth has taken place in one or both 

 of the tubes, incline the apparatus and allow a few drops of the one 

 culture to gain entry to the tube containing the other, without 

 disturbing the vacuum. In order to obtain at any time a sufficient 

 quantity of culture for examination, it is only necessary to connect 

 up with the hydrogen apparatus, allow the tube to fill with the gas, 

 snip off the end of one or other of the pipette tubes, and force out 

 by means of the pressure of hydrogen, a few drops of the culture. 

 The point can then be sealed and the tube closed without in any 

 way disturbing the anaerobic conditions. 



Form " B " is simply a modification of form " A," and is useful in 

 the study of one organism only. 



Form " C " can be used either for liquid or solid media, and has 

 been specially used for the latter by Dr Roux at the Pasteur 

 Institute.^ A sufficient quantity of nutrient gelatine or agar is 

 introduced, the two tubes of entry are lightly plugged with cotton- 

 wool, and the whole is then sterilised. Inoculation is made, by 

 means of a finely-drawn Pasteur pipette, through the vertical tube, 

 which is then sealed off in the flame. The lateral tube, which 

 should have in it a double constriction in order to maintain the 

 cotton-wool in place, is connected up with the vacuum pump and 

 hydrogen apparatus, the air exhausted, and the culture well washed 

 in hydrogen while the medium is still liquid. The lateral aperture 

 is then sealed off in the flame. The tube is then inclined and 

 rolled in the hands in order to obtain an even distribution of the 

 organisms introduced, and the medium is then allowed to solidify 

 on the slope. A modification of Petri dish culture is thus obtained 

 under strictly anaerobic conditions. To pick out the colonies after 

 growth it is only necessary to admit air or hydrogen through the 

 cotton-wool filter of the lateral orifice, to make a file mark on any 

 portion of the tube, and to carry the crack round by means of 

 a red-hot wire. 



The methods described above may be said to comprise all those 



^ Roux, " Sur la Culture des Microbes anaerobies " {Ann. de Vlnst. Pasteur^ 

 1 887, p. 49). This article is specially worthy of study by those who wish to 

 obtain a mastery of anaerobic technique. 



