112 TEXT-BOOK OF BACTERIOLOGY. 



mains open to the influence of the air and its oxygen, so that they 

 give us a good key to the different amounts of oxygen required, or 

 borne without injury to the various species which develop in them. 

 Colonies which appear only in the upper portion of the medium 

 belong to strictly aerobic (or semi-anaerobic) class, and those, 

 lastly, which are only found in the deeper portions to the strictly 

 anaerobic class. This forms a mark of distinction extremely useful 

 in examining matter containing both strictly anaerobic and semi- 

 anaerobic germs, and which facilitates our labor very considerably. 



Yet this method is not wholly satisfactory for several reasons, 

 and there is abundant room for improvement. The exclusion of 

 oxygen which it affords is neither complete nor enduring. Fur- 

 ther, it is impossible, or next to impossible, to submit the separate 

 colonies to a direct microscopic examination. Lastly, such a cul- 

 ture must be utterty destroyed before we can obtain from it the 

 material for further inoculation. The best way to get such mate- 

 rial is to break the test-tube in a sterilized Petri dish, and then 

 endeavor with sterilized instruments to free the gelatin or agar 

 from the glass with a view to further operations. This will not be 

 accomplished without some difficulty, and the danger of pollution 

 to the culture in the course of such manipulations is always im- 

 minent. 



Another process, somewhat similar to the one already described, 

 allows of direct microscopic examination, but it is equally unsatis- 

 factory as to further inoculations. In the usual manner bring the 

 matter for inoculation into liquid food gelatin which has been pre- 

 viously well boiled, and then spread the liquid over the inner sur- 

 face of a test-tube to harden, according to Esmarch's plan. The 

 interior of the test-tube is then filled with gelatin at about 26 C. 

 so far cooled as to be just short of solidification. This excludes the 

 oxygen, and the anaerobic colonies can thrive over the whole inner 

 surface, with the exception of those parts nearest to the top. The 

 colonies thus formed may be immediately examined under the 

 microscope, yet the removal of portions for further inoculations is 

 more troublesome and difficult than before. 



All the other methods endeavor to accomplish this in another 

 way, by entirely removing the oxygen from the food medium; in 

 which latter case they either establish a vacuum or replace the 

 air by some gas of an indifferent nature, and so create an atmo- 

 sphere without oxygen. 



The first of these processes is that of Buchner. A wide test- 

 tube is closed with an India-rubber cap, arid a pyrogallic solution is 

 shaken up in it. This solution absorbs the oxygen of the air, es- 



