18 



A. P. H. A. MILK ANALYSIS 



made, it must be recognized that ac- 

 curately carried out microscopic counts 

 of individual bacteria give the truest 

 picture of the actual conditions of raw 

 milk that can be obtained with any 

 technique. 



Where there is reason to fear the 

 presence of large numbers of dead 

 organisms, as for example in pas- 

 teurized milk, it is improper to place 

 reliance upon microscopic counts. Val- 

 uable information may, however, some- 

 times be obtained by making both 

 plate and microscopic counts from 

 samples of pasteurized milk. 

 Reports. 



As only a few ordinances^' have yet 

 been adopted in which both official 

 and microscopic count standards have 

 been given, the form of report used 

 will need to be adapted to the cir- 

 cumstances under which each lab- 

 oratory is working. Specific counts 

 should not be given under normal 

 circumstances, and care should be 

 taken to avoid making finer distinc- 

 tions in grade than are justified by the 

 accuracy of the grading. A series of 

 samples , should be examined in all 

 cases before rendering judgment as 

 to the quality of any milk supply. 



(c) microscopic colony count (peost 

 method) 



Although this technique is not rec- 

 ommended at this time as a standard 

 of official technique, it is described in 

 this report because of the need for 

 more extensive comparative investiga- 

 tions upon which to base judgment as 

 to its real merits. The technique in 

 question has been described by its 

 author as follows: 



"An area of four square centimeters 

 is marked off on an ordinary mi- 

 croscopic slide with a wax pencil. 

 The slide is sterilized in a flame, and 



then 0.05 (1/20) cc. of the milk to be 

 examined is placed on it with an 

 accurately calibrated pipette. An 

 equal amount of sterile nutrient 

 liquefied agar, at 42-45° C, is added 

 and the two drops thoroughly mixed 

 with a sterile loop and carefully spread 

 over the area marked off. The mix- 

 ture is allowed to harden and then a 

 'little plate' cultur.e is formed. 



"The bacteria in the milk are allowed 

 to grow into colonies by keeping the 

 preparation in a moist sterile chamber 

 for a few hours. The period of in- 

 cubation should be long enough to 

 allow the bacteria to grow into distinct 

 colonies although they may not be 

 visible to the naked eye. In practice 

 it seems best to allow eight hours at 

 37.5° C. although good sized colonies 

 are frequently formed in four hours. 

 On the other hand, if more convenient, 

 they may be allowed to grow 16 or 

 more hours before they are counted. 

 In order to count the colonies most 

 readily the plates are thoroughly dried 

 at a little less than 100° C, treated 

 with a 10% solution of glacial acetic 

 acid in 95 per cent alcohol, and stained 

 with a methylene blue or carbol- 

 thionine solution about y^, its usual 

 strength. In this way the colonies 

 are deeply stained while the back- 

 ground is colorless. 



"The colonies are counted under the 

 microscope. Usually this can be done 

 with the 16 mm. (2/3 inch) objective, 

 although where the colonies are small 

 or very numerous the higher powers 

 may be used, e. g. the 4 mm. (1/6 inch) 

 or the 1.9 mm. (1/12 inch) oil im- 

 mersion lens. The factor needed to 

 convert colonies per microscopic field 

 into the number of colonies that 

 would develop per cc. of milk must be 

 determined for each microscope and 

 each combination of lenses of the 



