QUANTITATIVE BACTERIAL ANALYSIS 255 



Eight-ounce bottles are the best, as the required amount of 

 dilution water only about half fills them, leaving room for shaking. 

 Long-fiber, non-absorbent cotton should be used for plugs. It is 

 well to use care in selecting cotton for this purpose to avoid short- 

 fiber or " dusty " cotton, which gives a cloud of lint-like particles 

 on shaking. Bottles and tubes should be filled a little over the 99 

 c.c. and 9 c.c. marks to allow for loss during sterilization.* 



The dilutions recommended are 1/10, 1/100,1/1,000, 1/10,000, 

 1/100,000 and 1/1,000,000. 



For certified milk the 1/10 and 1/100 dilutions should be used, 

 while the 1/10,000 will usually be found best for market milk. 



The 1/10 dilution is prepared by shaking the milk sample 

 twenty-five times and then transferring 1 c.c. of the milk to a test 

 tube containing 9 c.c. of sterile water. 



The 1/100 dilution is prepared in the same way,_ except that 

 a bottle with 99 c.c. of sterile water is substituted for the test tube. 



The 1/1,000 dilution is prepared by first making the 1/100 

 dilution, shaking twenty-five times and transferring 1 c.c. of the dil- 

 ution to a test tube containing 9 c.c. of sterile water. 



The 1/10,000, 1/100,000 and 1/1,000,000 dilutions are made 

 in the same manner by dilutions of the 1/100, 1/1,000 and 

 1/10,000 dilutions, 1 c.c. to 99 c.c. of sterile water. 



It is recommended that that dilution be used which will pro- 

 duce about two hundred colonies to a plate, ranging from 40 to 

 400; where a 1/10 dilution exceeds this number the 1/100 dilution 

 is more accurate, etc. The number of bacteria present may if desired 

 be approximately estimated before dilutions are made by direct 

 microscopic examination of a properly prepared sediment. Other- 

 wise it is necessary to make a range of dilutions, thereafter select- 



* If the loss during sterilization has not sufficed to bring the water level down to the 

 marks, sufficient water may be further removed with a sterile pipette. If sterilization 

 has removed a little too much, sufficient sterile water maybe added with a sterile pipette. 

 Ordinarily, however, experience will show how much to overfill before sterilization, so that 

 no change is required afterwards. 



