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PRINCIPLES OF SOIL MICROBIOLOGY 



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However, the deep colony procedure, used first by Liborius for the 

 isolation of anaerobic bacteria, has been preferred by a number of 

 workers. The selection of a suitable medium for this purpose is essen- 

 tial; the medium should be clear and transparent and enough dilution 

 tubes should be used. Some actively growing anaerobes will grow 

 through the agar as if it was a broth; this "permeat- 

 ing growth" will contaminate the other colonies. 

 The deep tubes of sterile agar are placed in boiling 

 water till the agar is melted, tubes are shaken to remove 

 air, and agar cooled down to 45°. Long boiling is 

 inadvisable, since the cotton becomes saturated with 

 moisture. Three tubes are employed for ordinary pur- 

 poses of dilution, but for new material or for weakly 

 growing organisms among rapidly growing forms, more 

 tubes may be used. Tube 1 is inoculated with one 

 loopful of the enriched culture or soil suspension. The 

 tube is then shaken, and transfer is made by means of a 

 sterile pipette (a Pasteur pipette may be used), pre- 

 viously flamed, into tube 2. The inoculum is placed 

 throughout the length of the agar, while withdrawing 

 the pipette, taking care not to blow air into the agar in 

 the tube, the latter being then shaken. The pipette is 

 flamed and, by means of it, some of the agar from tube 

 2 is transferred to tube 3, which is also shaken. The 

 tubes are plugged with cotton, as ordinary aerobic 

 tubes, and incubated aerobically at 25° to 28°. For 

 actively growing species, 12 to 24 hours' incubation are 

 sufficient; for slow growing forms, such as Bac. amylo- 

 bacter, 4 to 8 days may be required. The 

 colonies are examined, by means of a hand 

 lens, for permeating growth and aerobic 

 organisms. Final isolation is made from 

 the colonies of the mixed culture. The 

 tube and colonies to be transferred are 

 selected. A plain glass or metal rod, steri- 

 lized in the flame and cooled, may be used 

 to pierce the agar to the bottom of the tube, so as to admit air and allow 

 the expulsion of the unbroken agar from the tube upon a sterile half of a 

 Petri dish. The agar tube may also be placed for a second or two into 

 warm water so as to separate the agar from the walls of the tube. The 



Fig. 9. Buchner tube for 

 the anaerobic cultivation of 

 bacteria: p, the alkaline 

 pyrogallol solution; inner 

 tube contains culture of or- 

 ganism (after Omeliansky). 



