QUANTITATIVE EXAMINATION OF MILK 49 



3. A liberal supply of test tubes marked on the outside, with 

 a diamond or fine file dipped in turpentine, at the level of 9 c.c. of 

 water. 



4. A liberal supply of small conical flasks marked at the level 

 of 49 ex. of water. 



5. A sufficient number of sterile Petri dishes. 



The pipettes should be carefully cleaned and dried, lightly 

 plugged with cotton-wool at the upper end, placed either in a cylin- 

 drical box made of copper or thin sheet iron (or a glass cylinder 

 of suitable length cut from stout tubing of about i^ in. diameter 

 and plugged at each end with cotton-wool), and sterilised in the 

 hot air steriliser for one hour at 150° C. To avoid contamina- 

 tion of the exterior they should not be removed from the box or 

 cylinder until required for use. 



Into the test tubes and conical flasks, previously plugged with 

 cotton-wool and sterilised in the hot air steriliser for one hour 

 at 150' C, are run 9 c.c. and 49 c.c. of water respectively. It 

 is better to allow a slight margin over the exact quantity in 

 order to avoid possible loss during sterilisation. Any excess is 

 easily withdrawn subsequently by means of a sterile Pasteur 

 pipette^ passed between the stopper and the neck. The tubes 

 and flasks are then either sterilised in the autoclave at 115° 

 C. for thirty minutes, or in the Koch for half an hour on three 

 successive days. After sterilisation they should be provided with 

 an india-rubber cap to prevent evaporation, and placed on one side 

 until required for use. 



Procedure. Dilution to 500 times. — i. By means of a sterilised 

 I c.c. pipette remove from the original sample i cubic centimetre 

 of the milk under examination, and place it in a test tube of 

 9 c.c. of sterile water. Aspirate the mixed milk and water into the 

 pipette at least 3 times in order to well wash down the interior 

 walls, and, replacing the cotton-wool plug of the tube, but keeping 

 the pipette within it, roll the tube well in the hands to ensure an 

 even distribution of the organisms in the original milk. The tube 

 now contains 10 c.c. of liquid containing i c.c. of the original milk. 

 This is termed \h& primary dilution — i in 10. 



2. Transfer, by means of the same pipette, i cc. of this primary 

 dilution to a flask containing 49 cc. of sterile water. Aspirate 3 



^ For the method of making these pipettes, which will be found of the 

 greatest possible service in almost every branch of bacteriological work, see 

 Appendix A, p. 547. 



D 



