CULTIVATION OF ANAEROBIC BACTERIA. 185 



illuminating gas was recommended for this purpose by R. WURTY 

 and A. FOUREUR (I.), but, according to the researches of TH. KLA- 

 DAKIS (I.), it must be rejected, since he found it acting as a poison 

 on many bacteria. Nitrogen may be regarded as perfectly inno- 

 cuous, and would long ago have been employed for anaerobic 

 cultures were it not that the method of preparation is too cum- 

 brous and costly, at least for the physiologist. Many methods 

 have been proposed for the expulsion of oxygen by one of the 

 above gases, but only two will now be briefly mentioned here. 

 That of C. FRAENKEL (IV.) is concerned with the treatment of 

 test-tube cultures, ordinary wide test-tubes with two-holed stoppers 

 being employed. One of the glass tubes inserted therein reaches 

 almost to the bottom of the test-tube, whilst the second is cut off 

 close below the stopper. When filled with nutrient gelatin, agar- 

 agar (or bouillon, wort, &c.), the tubes are sterilised in the steamer 

 in the usual way, and inoculated, a current of hydrogen being 

 introduced through the longer tube and passed through. When 

 all the air is expelled the small tubes are hermetically sealed by 

 fusion (Fig. 48) and the stopper smeared with warm paraffin. 

 Esmarch roll cultures can then be prepared. If plate cultures 

 (e.g. in Petri dishes) are to be exposed to an inert gas, they are 

 (according to P. LIBORIUS (I.)) placed under a bell (of copper, &c.) 

 which can be tightly fixed by screw clamps against a caoutchouc 

 plate. The gas (when hydrogen is used) enters through a tube 

 fixed in the crown of the bell, and leaves by way of another tube 

 situated below. 



It is impossible to refrain from mentioning that there is another 

 method capable of taking rank with those described, and fulfilling 

 all the conditions usually prevailing during anaerobic growth in 

 Nature, viz., the simultaneous presence of strongly aerobic organ- 

 isms. It is certain that anaerobic organisms can often be detected 

 in liquids to which air has unrestricted access, and such associa- 

 tions of aerobic and anaerobic organisms are not difficult to bring 

 about by artificial means, this having been successfully attempted 

 by R. PENZO (I.), BEYERINCK (II.), and others. The application 

 of this method is, however, somewhat limited, since, of course, only 

 mixed cultures can be produced by its aid. 



It was remarked by Pasteur that the growth of anaerobic 

 organisms could be promoted by an addition of sugar to the 

 nutrient medium. Now, an alkaline solution of grape-sugar is 

 well known to have a strongly reducing action ; hence these two 

 facts induced KITASATO and WEYL (I.) to ascertain whether other 

 reducing bodies were equally efficient ; and they strongly recom- 

 mended the addition of 0.3-0.5 per cent, of sodium formate, or of 

 o.i per cent, of sodium indigo-sulphate. A solid nutrient medium, 

 qualified and stained blue by the last-named substance, is decolorised 

 as far as the growth of the reducing organism extends. The use 

 of indigo-sulphuric acid as a test for reducing action was first prac- 



