BUTTER 459 



Case of sterile pipettes, 10 c.c. 



Case of sterile graduated pipettes, i c.c. (in tenths of a cubic 

 centimetre) . 



METHOD. 



1. Weigh out 100 grammes of the sample in a sterile 

 beaker. 



2. Plug the mouth of the beaker with sterile cotton- 

 wool and immerse the beaker in a water-bath at 42 C. 

 until the contents are completely liquefied. 



3 . Fill the liquefied butter into the sterile separatory 

 funnel. 



4. Transfer the funnel to the incubator at 37 C. 

 and allow it to remain there for four days. 



At the end of this time the contents of the funnel 

 will have separated into two distinct strata. 



(a) A superficial oily layer, practically free from 

 bacteria. 



(b) A deep watery layer, turbid and cloudy from 

 the growth of bacteria. 



5. Draw off the subnatant turbid layer into sterile 

 centrifugal tubes, previously warned to about 42 C., 

 and centrifugalise at once. 



6. Pipette off the supernatant fluid and fill the tubes 

 with sterile i per cent, sodium carbonate solution 

 previously warmed slightly; stopper the tubes and 

 shake vigourously for a few minutes. 



7. Centrifugalise again. 



8. Pipette off the supernatant fluid; filling the tubes 

 with warm sterile bouillon, shake well, and again centri- 

 fugalise, to wash the deposit. 



9. Pipette off the supernatant fluid. 



10. Prepare cover-slip preparations, fix and clear 

 as for milk preparations, stain carbolic methylene-blue, 

 Gram's method, Ziehl-Neelsen's method, and examine 

 microscopically with a T V inch oil-immersion lens. 



11. Proceed with the examination of the deposit 

 as in the case of milk deposit (see pages 450 et seq.) . 



