NUMERICAL DKTKRMIXATIOX OF HACTKRIA. 145 



during tliis interval. They should be kept below 40 F., but care should 

 be taken that the}' are not frozen. 



DILUTIONS. 



Ordinary potable water, sterili/.ed, may be used for dilutions. Occa- 

 sionally spore forms are found in such water which resist ordinary auto- 

 clave sterilization ; in such cases distilled water may be used or the 

 autoclave pressure increased. With dilution water in eight-ounce bottles 

 calibrated for ninety-nine cubic centimeters all the necessary dilutions 

 can be made. 



Short, wide-mouthed ' ' Blakes ' ' or wide-mouthed French square bottles 

 are more easily handled and more economical of space than other forms 

 of bottles or flasks ( 11 ). 



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

 water only about half fills them, leaving room for shaking. I v ong-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 cc. and 9 cc. marks to allow r for loss 

 during sterilization (14). 



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 cc. of the milk to a test tube containing 9 

 cc. of sterile w r ater. 



The 1-100 dilution is prepared in the same way, except that a bottle 

 with 99 cc. 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 cc. of the dilution to a test 

 tube containing 9 cc. of sterile water. 



It is recommended that that dilution be used which will produce 

 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 esti- 

 mated before dilutions are made by direct microscopic examination of a 

 properly prepared sediment. Otherwise it is necessary to make a range 

 of dilutions, thereafter selecting for record the count obtained on that 

 plate which yields between 40 and 400 colonies. 



Plating whole milk is unreliable (15), whatever qualities be used, 

 since the bacteria are not so well separated as in the dilutions, and often, 

 ow T ing to the crowded conditions, only a portion of the bacteria present 

 will develop into visible colonies (6). Moreover, if a cubic centimeter 

 of the milk is used, the turbidity of the jelly, due to the presence of the 

 milk, hides the colonies present from the eye. 



