THE STUDY OF OBLIGATELY ANAEROBIC BACTERIA ni^.-is 



a dilution sufficient that well isolated colonies will appear.) If 

 difficulty is encountered in obtaining discrete colonies, reduce the 

 agar concentration in the plating medium to 0.75 to 1.0%. 



Another method is available for colony isolation which may be 

 preferred, particularly if the special apparatus needed for some of 

 the plating methods is not at hand. This method involves the inocu- 

 lation of a column of medium as mentioned in the opening pages 

 of this Leaflet 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 trans- 

 fer it to a suitable liquid medium. (This method is preferred if 

 the tube shows aerobic contamination 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 the new tube of medium, for it is this portion of the 

 medium which will stay reduced the longest. Although it is possible 

 to initiate growth from a small number of cells, in routine studies 

 use a more adequate inoculum. To facilitate the placing of the 

 inoculum in the bottom of the tube with liquid and semisolid media 

 substitute a Wright or Pasteur pipette (used with small rubber 

 bulbs) for the inoculation needle. By this means transfer a small 

 drop (0.1 or 0.2 ml.) of the culture to the new tube. Use pipette 

 also in the isolation of subsurface colonies particularly from media 

 in which the concentration of agar is reduced. Prepare these pipettes 

 from 6 to 8 inch lengths of sterile 8-9 mm. soft glass tubing (with 

 cotton plug in each end) by applying heat to the center of the glass 

 and pulling to form two capillary pipettes. 



In general use a culture from 16-20 hours old. With the jjatho- 

 genic types this time may be extended a few hours with no harm. 



