The Elements of Bacteriological Technique Part 115

Tubes nutrient gelatine or gelatine agar.

Tubes of wort gelatine.

Tubes of nutrient agar.

Water-bath regulated at 42 C.

Bunsen burner.

Grease pencil.

METHOD.--

1. Arrange four sterile capsules in a row; number them I, II, III, and IV.

2. Fill 9 c.c. sterile bouillon into the first, and 9.9 c.c. bouillon into each of the three remaining capsules.

3. Remove 1 c.c. milk from one of the bottles by means of a sterile pipette and add it to the bouillon in capsule I; mix thoroughly by repeatedly filling and emptying the pipette.

4. Remove 0.1 c.c. of the milky bouillon from capsule I, add it to the contents of capsule II, and mix as before.

5. In like manner add 0.1 c.c. of the contents of capsule II to capsule III; and then 0.1 c.c. of the contents of capsule III to capsule IV.

Then 1 c.c. of dilution I contains 0.1 c.c. milk sample.

1 c.c. of dilution II contains 0.001 c.c. milk sample.

1 c.c. of dilution III contains 0.00001 c.c. milk sample.

1 c.c. of dilution IV contains 0.0000001 c.c. milk sample.

6. Melt the gelatine and the agar tubes in boiling water; then transfer to the water-bath and cool them down to 42 C.

7. Number the gelatine tubes consecutively 1 to 12.

8. Inoculate the tubes with varying quant.i.ties of the material as follows:

To tube No. 1 add 1.0 c.c. of the milk sample.

2 add 0.1 c.c. of the milk sample.

{ 3 add 1.0 c.c. from capsule I.

{ 4 add 0.1 c.c. from capsule I.

{ 5 add 1.0 c.c. from capsule II.

{ 6 add 0.1 c.c. from capsule II.

{ 7 add 0.5 c.c. from capsule III.

{ 8 add 0.3 c.c. from capsule III.

{ 9 add 0.2 c.c. from capsule III.

{ 10 add 0.5 c.c. from capsule IV.

{ 11 add 0.3 c.c. from capsule IV.

{ 12 add 0.2 c.c. from capsule IV.

9. Pour plates from the gelatine tubes; label, and incubate at 20 C.

10. Liquefy five wort gelatine tubes and to them add 1.0 c.c. of the milk sample and a similar quant.i.ty of the diluted milk from capsules I, II, and III and IV respectively.

11. Pour plates from the wort gelatine; label, and incubate at 20 C.

12. Inoculate the liquefied agar tubes as follows:

To tube No. 1 add 0.1 c.c. of the milk sample.

2 add 0.1 c.c. from capsule I.

3 add 0.1 c.c. from capsule II.

4 add 0.1 c.c. from capsule III.

5 add 1.0 c.c. from capsule IV. } 6 add 0.1 c.c. from capsule IV. }

13. Pour plates from the agar tubes; label, and incubate at 37 C.

14. After twenty-four hours' incubation "inspect," and after forty-eight hours' incubation, "count" the agar plates and estimate the number of "organisms growing at 37 C." present per cubic centimetre of the sample of milk.

15. After three, four, or five days' incubation, "count" the gelatine plates and estimate therefrom the number of "organisms growing at 20 C." present per cubic centimetre of the sample of milk.

16. After a similar interval "count" the wort gelatine plates and estimate the number of moulds and yeasts present per cubic centimetre of the sample of milk.

NOTE.--Many observers prefer to employ gelatine agar (see page 193) for the quant.i.tative examination. In this case gelatine-agar plates should be poured from tubes containing the quant.i.ties of material indicated in step 8, incubated at 28 C. to 30 C. and after five days the "total number of organisms developing at 28 C." recorded.

~Qualitative.~--The qualitative bacteriological examination of milk is chiefly directed to the detection of the presence of one or more of the following pathogenic bacteria and when present to the estimation of their numerical frequency.

Members of the Coli-typhoid group.

Vibrio cholerae.

Streptococcus pyogenes longus.

Micrococcus melitensis.

Staphylococcus pyogenes aureus.

Bacillus enteritidis sporogenes.

Bacillus diphtheriae.

Bacillus tuberculosis.

Some of these occur as accidental contaminations, either from the water supply to the cow farm, or from the farm employees, whilst others are derived directly from the cow.

In milk, as in water examinations, two methods are available, viz.: Enrichment and Concentration--the former is used for the demonstration of bacteria of intestinal origin, the latter for the isolation of the micro-organisms of diphtheria and tubercle. The first essential in the latter process is the concentration of the bacterial contents of a large volume of the sample into a small compa.s.s; but in the case of milk, thorough centrifugalisation is subst.i.tuted for filtration.

_Apparatus Required_:

A large centrifugal machine. This machine, to be of real service in the bacteriological examination of milk, must conform to the following requirements:

1. The centrifugal machine must be of such size, and should carry tubes or bottles of such capacity, as to enable from 200 to 500 c.c. of milk to be manipulated at one time.

2. The rate of centrifugalisation should be from 2500 to 3000 revolutions per minute.

3. The portion of the machine destined to carry the tubes should be a metal disc, of sufficient weight to ensure good "flank" movement, continuing over a considerable period of time. In other words, the machine should run down very gradually and slowly after the motive power is removed, thus obviating any disturbance of the relative positions of particulate matter in the solution that is being centrifugalised.

4. The machine should preferably be driven by electricity, or by power, but in the case of hand-driven machines--

(a) The gearing should be so arranged that the requisite speed is obtained by not more than forty or fifty revolutions of the crank handle per minute, so that it may be maintained for periods of twenty or thirty minutes without undue exertion.

(b) The handle employed should be provided with a special fastening (e. g., a clutch similar to that employed for the free wheel of a bicycle), or should be readily detachable so that, on ceasing to turn, the handle should not, by its weight and air resistance, act as a brake and stop the machine too suddenly.

One of the few satisfactory machines of this cla.s.s is shown in figure 212.