12 



A. P. H. A. MILK ANALYSIS 



It is important that the plating shall 

 be completed as rapidly as . possible. 

 The work should be so planned that 

 no more than 15 minutes shall elapse 

 after the dilution of the milk and 

 before the agar is poured into the 

 petri dishes; and in no case shall the in- 

 terval be allowed to exceed 20 minutes. 



After the agar has been thoroughly 

 hardened, place the petri dishes in an 

 incubator. The danger of spreaders 

 may be reduced either by the use of 

 clay tops or by inverting the plates as 

 preferred. 



Incubation. 



Only one period of incubation, and 

 one temperature is regarded as stand- 

 ard, 48 hours at 37.5° C. Piles of 

 plates should not be packed too closely 

 together and in crowded incubators 

 ventilation should be provided. 

 Counting plates. 



If among the different dilutions 

 there are plates containing from 30 to 

 300 colonies these should be counted^^, 

 and the number, multiplied by the 

 dilution, be reported as the final count. 

 All colonies on such plates should be 

 counted, and the numbers from the 

 different plates averaged. If there are 

 no plates within these limits, the one 

 that comes the nearest to 300 is to be 

 counted. No plate that contains less 

 than 20 colonies shall be counted, 

 unless it happens that there are no 

 other plates. If the number of col- 

 onies on the plates to be counted are in 

 excess of 300 per plate, a part of the 

 plate may be counted and the total 

 number estimated; but such plates 

 are admittedly overcrowded and the 

 counts are less than they should be. 



Counting shall be done with a lens, 

 and all recognizable colonies included. 

 A lens magnifying lyi diameters (or 

 what the opticians call a 3>^ x lens) is 



recommended for general use. In case 

 any particles visible by this method 

 are of doubtful nature they should be 

 examined with a compound microscope 

 to determine whether they are colonies 

 or dirt specks. 

 Common sources of error in counts. 



Agar plate "counts" per cc. are to be 

 regarded as estimates of numbers rather 

 than as exact counts, since only a 

 portion of a cubic centimeter is used 

 in preparing the plates. As such they 

 are (like all estimates) subject to 

 certain well known and recognized 

 errors whose size can be largely con- 

 trolled by the care taken in the analysis. 

 Among these errors are: (a) Failure of 

 some of the bacteria to grow because 

 the incubation temperature, or the 

 composition or reaction of the medium, 

 is not suitable, (b) Inaccuracies in 

 measurement of the quantities used. 



(c) Mistakes in counting, recording 

 data, computing results and the like. 



(d) Incomplete sterilization or con- 

 tamination of the plates, dilution 

 waters, etc. The possible errors caused 

 by these things makes it highly im- 

 portant for all routine laboratories to 

 follow carefully a standard procedure. 



Recent investigations^* make it clear 

 that these largely controllable errors, 

 are not so likely to cause serious mis- 

 conceptions of the accuracy of results 

 as are the errors due to the fact that 

 bacteria in milk usually cling together 

 in groups of from two to many hundreds 

 of individuals. These groups are only 

 partially broken apart by the shaking 

 given in preparing the dilutions so that 

 at best the counts from the agar 

 plates represent the number of isolated 

 individuals and groups of two or more 

 bacteria that exist in the final dilution 

 water. Thus the colony counts from 

 the plates are always much smaller 

 than the total number of bacteria 



