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bacteria can now germinate and if gelatin is used there will soon appear the large 

 liquefying colonies of the so remarkable Bacillus septicus, together with the non- 

 liquefying putrefiers, for the greater part recognisable by the flocculent structure of 

 their colonies, a character related to their sensitiveness to extension and contraction of 

 the substratum where in they grow, quite as by B. Zopfii. For the microscopical 

 examination this method undoubtedly affords good material, but it is hardly possible 

 to reach the single anaerobic colonies without touching others. To this end it is 

 necessary to remove the culture gelatin from the by heating it in the flame so that 

 only the outer side of the gelatin melts and the contents may be thrown out as a whole. 

 One may also with a file make an incision in the glass wall in the neighbourhood of 

 favourably situated colonies. But it is clear that there is much chance that thereby 

 also different colonies intermix so that of a pure culture of anaerobes in the usual 

 sense of the word there is no question in such experiments. For examination with 

 the microscope and for studying the appearance of the colonies the method is useful, 

 but for the culture of pure species it is worthless. 



Every good method for pure culture of aerobes and still more of anaerobes should 

 answer the following requirements ; the colonies must be situated quite free and 

 at due distances from each other on the surface of the solid plates, they must further- 

 more be readily attainable with the platinum wire. These requirements can only be 

 satisfied by cultivation in ordinary glass boxes or P e t r i dishes, which may take place 

 in the laboratory by means of the exsiccator method of N o v y (see Mace, 1. c.). 



After this method, the best of the chemical ones, ordinary culture boxes are 

 placed in an exsiccator filled with pure hydrogen and moreover containing some 

 oxygen-removing substance, such as ferro-ferrocyan or alkaline pyrogallol. But this 

 method also has its drawbacks. It is namely impossible quite to prevent the deposition 

 of vapour at the glass covers, so that drops of water falling down come on the plates ; 

 this makes the colonies intermix and spoils the experiment. It is, besides, hardly 

 possible distinctly to see the state of development of the colonies in the closed ex- 

 siccator, which may lead to it being opened too early and oblige the experimenter to 

 begin anew. This is very troublesome considering the complication of the experiment. 



The Oidiitm method has none of these disadvantages, and if well-managed, pro- 

 duces colonies of the anaerobes situated quite free on the surface of the plates and 

 easily reached with the wire. 



The principle of the method is the placing one over the other of two culture 

 plates, separated by a relatively small space of air. One of the plates contains the 

 aerobic microbe which is to absorb the oxygen, while on the surface of the other the 

 anaerobe is to grow. Here, also, I select a definite example for illustration, namely 

 the strictly anaerobic bacilli of the butyric-acid and the butyl-alcoholic fermentations; 

 they have corresponding nutrition conditions and may be isolated in the same way. 

 They are spore-producers, thriving best in malt infusion where they cause strong fer- 

 mentations accompanied with production of hydrogen and carbonic acid. A crude 

 butyric-acid fermentation is prepared as follows. Wheat- or rye-flour, or better a pap 

 of potatoes infected with soil, is mixed in a glass beaker with water to which is 

 added some calcium-carbonate, then heated for a few seconds to 90 or 100 C. Kept 

 at 30 to 40 C. there usually results after two days a strong butyric-acid fermen- 

 tation in which occur various butyric-acid bacteria which are then to be isolated. 



18* 



