STEPS IN QUANTITATIVE BACTERIAL ANALYSIS OF MILK 497 



thoroughly shaking the original bottle the sample from certified 

 milk can best be procured at the laboratory by perforating the card- 

 board cover with a knife sterilized over a flame of a Bunsen burner 

 and inserting the sterile pipette through the hole. In this way all 

 danger of increasing the bacterial contents of the milk during manipu- 

 lation is entirely avoided. Samples should be taken in quantities of 

 not less than 10 c.c. because of the need for duplicates, controls, etc. 

 The sterile pipettes used in the collection of samples outside of the 

 laboratory should be carried in metal boxes or in sterilized glass tubes. 

 To use a single pipette for collecting a number of samples and steril- 

 izing it between samples by dipping into sulphuric acid and then 

 into sterile water is not a good practice. 



2. Milk contains too many bacteria to allow it to be mixed in 

 quantities of 1 c.c. with the culture media employed, hence it must be 

 diluted with sterile water. The water can be kept in ordinary glass 

 bottles. If volumetric flasks are used considerable space should be 

 left over the 10 c.c. or 100 c.c. mark, so that the dilute fluid can be 

 well shaken. The dilutions used are 1 in 10, 1 1 in 100, 1 in 1000, 

 1 in 10,000, 1 in 100,000, and 1 in 1,000,000. For the examination 

 of market milk the dilutions are generally 1 in 1000, 1 in 10,000, and 

 1 in 100,000. Sterilize 9 c.c. and 99 c.c. of ordinary clean tap water 

 in bottles closed with cotton plugs. As a rule, if 100 c.c. are placed in 

 bottles and sterilized in the autoclave for a sufficient time the water 

 will be reduced by evaporation to about 99 c.c. Preliminary tests, 

 however, must determine how much water should be taken to be 

 exactly reduced to 9 c.c. after the sterilization. The dilutions are 

 made in the following manner: Add 1 c.c. of the milk which has 

 been well shaken, with a sterile graduated pipette to the bottle con- 

 taining 9 c.c. and also 1 c.c. to the bottle containing 99 c.c. of sterile 

 water; shake well for several minutes, taking care that the fluid does 

 not come in contact with the cotton plug. This gives the dilutions 

 of 1 in 10 and 1 in 100. From these the dilutions of 1 in 1000 and 1 in 

 10,000 can be prepared and from these again the dilutions of 1 in 

 100,000 and 1 in 1,000,000. 



3. Before the dilutions are made the culture media must be pre- 

 pared. The medium generally used is 10 c.c. of an agar medium 

 containing 1 per cent, of agar, with a reaction of 1.5 per cent, acid to 

 phenolphthalein. The medium should be melted in a water bath, 

 then cooled down to 45 C. To control the temperature exactly a 

 thermometer should be placed in one of the agar tubes in the 

 water bath. When it registers 45 it indicates that this is also the 

 temperature of the agar in the other tubes. The tube containing the 

 thermometer is, of course, not used for pouring plates. 



4. When the media are melted and of the proper temperature, 

 place with sterile pipettes 1 c.c. of the dilutions (for example, those of 



1 Only applicable in the very best forms of certified milk. 

 32 



