Laboratory Outline for General Bacteriology 



laminated. With the first pipette used transfer another cubic 

 centimeter of milk to the dilution bottle. This doubles the amount 

 of milk and decreases the dilution one-half, thus giving the 100 

 dilution. Discard the pipette. Shake sample and make transfers 

 from it to the remaining two plates, using the same pipette as was 

 used for the first plates but first rinsing out any of the first dilu- 

 tion remaining in it by drawing up some of the second dilution 

 and letting it out. Transfers are always made using 1 cc. quanti- 

 ties in dilution work. Melted agar is poured into the plates and 

 mixed with the dilution. Plates are inverted after hardening and 

 incubated at 37 C. for two days. 



Now if the sample of milk is older or of poorer quality and 

 may contain 500,000 bacteria, allowing 100 bacteria per plate 

 would make the required dilution 5000. This is too large a quan- 

 tity of water to be handled in one container. The same result is 

 secured by using two bottles in succession having 50 cc. and 100 cc. 

 of water respectively. Multiplying amounts of water in the bottles 

 gives the dilution. When bottles of different sizes are used always 

 use the smaller first. In this case 1 cc. is taken from milk sample 

 to 50 cc. bottle and pipette discarded. This 50 cc. dilution is used 

 exactly the same as the milk sample was in the preceding case. 

 Laboratory dilution bottles may be had marked for 10 cc., 20 cc., 

 40 cc., 50., 60., 100., 200., 400, and 500 cc. 



After incubation counts are made from the plate cultures. To 

 find the number of bacteria per cc. of milk, multiply the plate 

 counts by their respective dilutions, add together and divide by 

 four. If some plates are covered entirely by spreaders or other- 

 wise spoiled they are left out of the average. 



When it is desired to find the number of acid producing bac- 

 teria and liquefying bacteria in milk, litmus lactose gelatin plates 

 are made in the same manner as the agar plates except that 2 cc. 

 of litmus solution is added to the gelatin just before pouring plates. 

 The plates are incubated right side up at 20 C. for seven days 

 unless liquefying bacteria make it necessary to count earlier. Acid 

 bacteria turn blue litmus red. 



The interpretation of the results must depend upon the his- 

 tory of the milk. 



1. When analysis is made immediately after milking con- 

 clusions may be drawn as to the cleanliness and care in the dairy, 

 the thoroness in cleaning and sterilizing of the utensils, and some- 

 times as to the presence of cows with infected udders. With prop- 

 erly cleaned and sterilized milk vessels and care in milking the 

 numbers of bacteria should not exceed 10,000, and may easily be 

 brought lower. Larger numbers indicate unclean methods, un- 

 sterile utensils or infected udders. 



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