854 THE BACTERIOLOGICAL EXAMINATION OF WATER 



No account is taken of the presence of anaerobic organisms in stating the 

 number present in a water : the determination is always made under aerobic 

 conditions and this is always tacitly understood. By employing anaerobic 

 methods of isolation the number of anaerobic organisms could of course be 

 determined, but in practice this is never done, and it presents difficulties other 

 than those of technique such for instance as the occurrence of facultative 

 anaerobes, many of which, as a matter of fact, grow on the aerobic plates. 



Vincent investigated the occurrence of anaerobic organisms in water and found 

 that the number of strictly anaerobic organisms was very small. He used VignaFs 

 tubes and glucose-gelatin, containing sulphindigotate of sodium (p. 92). The 

 medium was sown by the dilution method, and for the detection of pathogenic species 

 the colonies so isolated were sub-cultivated anaerobically in broth and then inoculated 

 into animals (p. 858). 



In determining the number of organisms in an unit volume of water it is 

 usual to work with a fraction of a cubic centimetre because the number of 

 organisms present in a cubic centimetre is generally so large as to make an 

 enumeration impossible. 



A. Dilution method. 1. Chip away the wax with which the cork was 

 sealed and flame the top of the cork in a Bunsen : raise the cork with a 



flamed corkscrew sufficiently to allow of it 

 being removed from the bottle with the 

 fingers. 1 



2. Have ready on the bench : 



A 10 c.c. pipette graduated in 1 c.c. 

 A 2 c.c. pipette graduated in 0*5 c.c. 

 A drop pipette (20 drops to 1 c.c.). 

 (All of which must, of course, have been 

 plugged with wool at the upper end and steri- 

 lized.) 



A sterile glass vessel covered with paper. 

 A tube of sterile water. 

 Several tubes of sterile gelatin in a 

 water bath at a temperature high 

 enough to keep the medium fluid. 



' ?o 8 r-wa?e? i eJimffion y n ' s) Several conical flasks plugged with wool 



and sterilized (fig. 408). 



3. Into the sterile glass vessel measure 9 c.c. of sterile water adopting all 

 precautions to avoid contamination : add 1 c.c. of the water under examina- 

 tion and mix thoroughly. This gives a dilution of 1 in 10. 



4. Flame the mouth of one of the conical flasks, remove the wool plug 

 and with the drop pipette introduce 2 drops of the 1-10 dilution of the water 

 ( = 0-01c.c.). 



5. Flame the mouth of one of the tubes of liquefied gelatin (the tempera- 

 ture of which should be such that the tube can be held quite comfortably 

 in the hand), remove the plug and pour the contents quickly into the conical 

 flask (par. 4). Replace the wool plug in the mouth of the flask, mix the 

 gelatin and water thoroughly by rotating the flask and then stand the latter 

 on some cold and horizontal surface so that the gelatin may set as quicklv 

 as possible. 



In effect, plate O'Ol c.c. of the water on gelatin. 



[6. Incubate at 20-22 C.] 



7. Colonies will soon appear in the gelatin, each colony representing one 



i Before uncorking the bottle shake it well to mix thoroughly the contents and to dis- 

 tribute any deposit there may be uniformly throughout the whole. 



