THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA 135 



of technic. Since some of the anaerobes tend to spread rapidly over the 

 surface of the agar, in many instances it will be found that ''poured" agar 

 plates are to be preferred to plates inoculated by streaking the surface. 

 Two common methods are available for preparing these: (1) Melt tubes 

 of the plating medium, cool, and inoculate before pouring; (2) place a 

 small amount of sterile tap water in the culture dish, inoculate, and pour 

 the agar into the dish immediately. If conditions warrant, use crystal 

 violet in the agar. Place the plates in the anaerobic environment as soon 

 as possible. (The size of inoculum to be used will vary so that some prac- 

 tice may be necessary to give a dilution sufficient so that well-isolated 

 colonies will appear.) If difficulty is encountered in obtaining discrete 

 colonies, decrease the agar concentration in the plating medium to 0.75- 

 1.0 per cent. 



Another method is available for colony isolation which may be pre- 

 ferred, particularly if the special apparatus needed for some of the plating 

 methods is not at hand. This method involves the inoculation of a 

 column of medium as mentioned in the opening pages of this chapter in 

 the discussion of methods useful to determine whether or not a particular 

 strain is an obligate anaerobe. For isolation purposes the fewer the 

 number of colonies appearing in the medium the better. The percentage 

 of fermentable sugar should be reduced to the lowest amount which gives 

 good growth of the organism in order to prevent the production of gas, 

 which may crack the medium. Assuming that we have available a deep 

 tube of agar in which there appear several isolated colonies, two methods 

 of isolation are available: (1) If soft glass tubes are used, cut the glass and 

 break the tube at a short distance below the desired colony. Deposit the 

 agar quickly in a sterile petri dish. Using a hot needle or small blade, cut 

 across the plug of agar near the colony and transfer it to a suitable liquid 

 medium. (This method is preferred if the tube shows aerobic contamina- 

 tion in the upper layers.) (2) If Pyrex tubes are used, eject the plug of 

 agar into the sterile dish by applying a bunsen flame to the bottom end. 

 Before this, heat the sides of the tube and sterilize the mouth of the tube 

 in the flame. During the ejection step of the technic, hold the mouth of 

 the tube so that it points directly into the sterile dish. After the column 

 of agar is deposited in the dish, proceed as discussed above. 



Inoculation Technics 



The following points of culture transfer and other routine technics are 

 sufficiently different from the procedures used with aerobes so that some 

 note is needed : 



Steam or boil most liquid media for a few minutes immediately prior to 

 inoculation in order to drive off oxygen which may have been absorbed 

 following sterilization. Attempt to deliver the inoculum to the bottom of 



