add 0.01 c.c. liquefied butter.

5. Pour a plate cultivation from each of the gelatine-agar tubes and
incubate at 28 deg. C.

6. "Count" the plates after three days' incubation, and from the figures
thus obtained estimate the number of organisms present per cubic
centimetre of the sample.

~Qualitative.~--

_Apparatus Required_:

Sterile beaker, its mouth plugged with sterile cotton-wool.

Counterpoise for beaker.

Scales and weights.

Sterilised spatula.

Water-bath regulated at 42 deg. C.

Separatory funnel, 250 c.c. capacity, its delivery tube
protected against contamination by passing it through a
cotton-wool plug into the interior of a small Erlenmeyer
flask which serves to support the funnel. This piece of
apparatus is sterilised _en masse_ in the hot-air oven.

Large centrifugal machine.

Sterile tubes (for the centrifuge) closed with solid rubber
stoppers.

Case of sterile pipettes, 10 c.c.

Case of sterile graduated pipettes, 1 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 deg. 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 deg. 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 deg. C., and centrifugalise at once.

6. Pipette off the supernatant fluid and fill the tubes with sterile 1
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 centrifugalise, 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