1758 Journal of Applied Microscopy 



atmospheric oxygen reenters the medium and checks the growth of the anaerobic 

 organisms. 



Hammerl (\^QV) tested the efficiency of Trenkmann's NagS, of K2S and of 

 Ammonium sulph-hydrate NH^SH, as reducing agents. Parallel experiments 

 showed that NH^SH was much more efficient than either Na2S or KgS. In the 

 ammonium sulph-hydrate he believes he has found a substance which, without 

 checking the bacterial development, reduces the oxygen in the nutrient medium 

 and thus prepares ideal conditions for anaerobic cultures. Unfortunately the 

 ordinary NH^SH as kept in the laboratory is not fit for this purpose. It is nec- 

 essary that it should be fresh and sterile. To obtain sterile NH^SH the follow- 

 ing procedure is recommended : Fill a glass stoppered bottle of 100 to 150 c. c. 

 capacity completely with sterile water. Replace the glass stopper by a cotton 

 plug and sterilize the apparatus in the steam sterilizer. After sterilization cool 

 it to room temperature and introduce by means of a sterile glass tube, reaching 

 to the bottom of the bottle, a vigorous current of washed HgS for about six 

 minutes. The open end of the bottle is loosely plugged with sterile cotton. Now 

 draw from the sulphurated water accurately measured portions of 10 c. c. into 

 each of 6-8 sterile test tubes ; by means of a pipette drop into the first tube 2 

 drops, into the second 4 drops, etc., etc., of a 1 per cent, solution of ammonium 

 chloride NH^Cl. After vigorous shaking add three drops of a concentrated solu- 

 tion of methylene blue to each tube. The latter operation is done most easily by 

 pouring the 10 c. c. into a tube containing the three drops of methylene blue. 

 Mark the time required for complete decolorization. Generally the optimum 

 amount of ammonium salt lies between 4-8 drops and the minimum time for de- 

 colorization from i^ to 1 minute. Upon finding the optimum amount of NH^Cl 

 a corresponding number of drops of a 1 per cent, solution of NH^Cl is added 

 to the sulphurated water in the bottle. Then add to ten parts of the nutrient 

 medium one part of the thus prepared solution. If about three drops of con- 

 centrated methylene blue are added to this medium, the latter decolorizes rapidly 

 and completely in 'l-o minutes. 



From the above review of the various reducing agents and their comparative 

 efficiency as a means for producing conditions favorable for the development of 

 anaerobic bacteria in the presence of air, it may be seen that none of the agents 

 and substances so far in use are entirely satisfactory. If aerobic species are 

 used for this purpose, it is difficult to obtain the anaerobes in pure cultures with- 

 out the aid of some other method for the cultivation of anaerobic bacteria. If we 

 resort to chemical reducing agents we find that while they may favor anaerobic 

 growth, they may do it at the expense of spore formation, and that they may 

 cause the development of degeneration forms, as in the case of glucose, or the 

 reducing substance may have a poisonous effect on the microorganisms as in the 

 case of Hydroxylaminehydrochloride, or the preparation of the reducing agent 

 in sterile form may be too complicated for practical purposes, as in case of 

 Ammoniumsulph-hydrate, and finally that, no matter how great the reducing 

 power of any one of these chemical reducing agents may be, they are not able 

 to make harmless the atmospheric oxygen, which reenters the medium when the 

 latter is poured into petri dishes. Generally speaking then, in order to use re- 

 ducing agents successfully, they should be used in connection with some other 

 method for cultivating anaerobic bacteria. 

 New York State Veterinary College, Cornell University. Otto F. Hunziker. 



