CERTIFIED MILK 503 



66. Samples of milk for plating shall be diluted in the proportion of 1 

 part of milk to 99 parts of sterile water; shake twenty-five times and plate 

 1 c.c. of the dilution. 



The committee on bacterial milk analyses of the American Public Health 

 Association in Part IV of its report presented details with respect to plating 

 apparatus and technic in part as follows: 



Plating Apparatus. For plating it is best to have a water-bath in which 

 to melt the media and a water-jacketed water-bath for keeping it at the 

 required temperature; a wire rack which should fit both the water-baths for 

 holding the media tubes; a thermometer for recording the temperature of the 

 water in the water-jacketed bath, sterile 1 c.c. pipets, sterile Petri dishes, 

 and sterile dilution water in measured quantities. 



Dilutions. Ordinary potable water, sterilized, may be used for dilu- 

 tions. Occasionally spore forms are found in such water which resist ordi- 

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

 autoclave pressure increased. With dilution water in 8-ounce ^bottles cali- 

 brated for 99 c.c. . . . all the necessary dilutions may 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. 



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 weh 1 to use care in select- 

 ing cotton for this purpose, to avoid short-fiber or dusty cotton, which gives 

 a cloud of lint-like particles on shaking. Bottles . . . should be filled a little 

 over the 99 c.c. ... to allow for loss during sterilization. 



Pipets. Straight sides 1 c.c. pipets are more easily handled than those 

 with bulbs; they may be made from ordinary ^-inch glass tubing and should 

 be about 10 inches in length. 



Plating Technic. The agar after melting should be kept in the water- 

 jacketed water-bath between 40 and 45 C. for at least fifteen minutes 

 before using to make sure that the agar itself has reached the temperature 

 of the surrounding water. If used too warm the heat may destroy some of 

 the bacteria or retard their growth. 



Shake the milk sample twenty-five times, then with a sterile pipet trans- 

 fer 1 c.c. to the first dilution water and rinse the pipet by drawing dilution 

 water to the mark and expelling; this gives a dilution 1 to 100. 



. . . Then with a sterile pipet transfer 1 c.c. to the Petri dish, using care 

 to raise the cover only as far as necessary to insert the end of the pipet. 



Take the tube of agar from the water-bath, wipe the water from outside 

 the tube with a piece of cloth, remove the plug, pass the mouth of the tube 

 through a flame, and pour the agar into the plate, using the same care as 

 before to avoid exposure of the plate contents to the air. 



Carefully and thoroughly mix the agar and diluted milk in the Petri dish 

 by a rotary motion, avoiding the formation of air bubbles or slopping the 

 agar, and after allowing the agar to harden for at least fifteen minutes at 

 room temperature, place the dish bottom down in the incubator. 



Plating should always be done in a place free from dust or currents of air. 



In order that colonies may have sufficient food for proper development 

 10 c.c. of agar shall be used for each plate. 



67. Determination of Taste and Odor of Milk. After the plates have been 

 prepared and placed in the incubator, the taste and odor of the milk shall be 

 determined after warming the milk to 100 F. 1 



68. Counts. The total number of colonies on each plate should be 

 counted, and the results expressed in multiples of the dilution factor. Colonies 



1 Should it be deemed desirable and necessary to conduct tests for sedi- 

 ment, the presence of special bacteria, or the number of leukocytes, the 

 methods adopted by the committee of the American Public Health Associa- 

 tion should be followed. 



