498 QUANTITATIVE ESTIMATION OF BACTERIA IN MILK 



1 in 10(30, 1 in 10,000, and 1 in 100,000) into the lower portions of 

 Petri dishes. 1 Then add the melted agar; mix well with the 1 c.c. 

 of diluted milk in the Petri dish by properly moving and shaking. 

 This should be done carefully so that the medium does not run over. 



5. As soon as the agar has again become solid the Petri dishes are 

 inverted, the now upper portion with the solid medium in it is lifted 

 away from the lower portion. Into the latter a piece of filter paper 

 with a drop of glycerin on it is placed. This arrangement insures 

 against moisture collecting on the agar and spoiling the count by 

 spreading the colonies in a diffuse manner. The Petri dish is now 

 placed in an inverted position (culture medium above, plate with 

 filter paper below) into the incubator, and is kept there at 37 C. for 

 forty-eight hours. Another method recommended to prevent the 

 collection of moisture on the agar is to use a porous earthenware 

 cover for the Petri dish. The former method, however, is preferable 

 to the use of a non-transparent cover. 



6. After forty-eight hours the colonies on and in the depth of the 

 agar are counted in the manner described in Chapter XIV, p. 174. It 

 is well, however, to count all the colonies which have developed and not 

 merely a number in a portion of the agar. A so-called blank control 

 should be made for each set of specimens. This is done in the follow- 

 ing manner: One c.c. of the sterile water to which no milk whatever 

 has been added is poured into a Petri dish and then the melted agar 

 is added. The plate is incubated with the others and examined after 

 forty-eight hours. It should be entirely sterile, or it may perhaps 

 have developed one or two colonies on the surface, which might 

 possibly be due to air contamination during manipulation. The 

 count is made on those plates which have developed between 200 

 and 400 colonies. This is considered to be the dilution which gives 

 the most trustworthy count from which to calculate the bacterial 

 contents of the milk. 



Heinemann and Glenn have made some experiments to determine 

 whether it is preferable, in order to get an exact count, to incubate 

 at 20 C. or at 37 C. They found that in dextrose-litmus-agar 2 

 the number of colonies after one day is larger at 37 C.; after two 

 days the number is higher at 20 C., and after three days the number 

 of colonies at 20 C. is about double that at 37 C. In lactose agar 

 the conditions are practically the same. There are no acid colonies 

 in either dextrose or lactose agar after twenty-four hours at 20 C. 

 After two days the number of acid colonies in both dextrose and lactose 

 agar is considerably larger at 37 C. than at 20 C., but after three 

 days the proportion is reversed, as the acid colonies develop more 

 rapidly after two days at 20 than at 37 C. The proportionate rate of 



1 Two Petri dishes should be prepared from each dilution. 



1 The culture media used in these tests were prepared as follows : A sterile litmus solution 

 of Merck's pure extract of litmus was poured into a Petri dish, then the dilute milk was added, 

 and finally the melted dextrose or lactose agar was poured into the dish. 



