A. Cultural Examination

Quantitative.—

Apparatus Required:

Sterilised tin opener, (if necessary.)

Erlenmeyer flask (500 c.c. capacity) containing 200 c.c. sterile bouillon and fitted with solid rubber stopper.

Counterpoise.

Scissors and forceps.

Scales and weights.

Water steriliser.

Hypodermic syringe.

Syringe with intragastric tube.

Rat forceps.

Case of sterile capsules.

Filtering apparatus as for water analysis.

Case of sterile plates.

Case of sterile graduated pipettes, 10 c.c. (in tenths of a cubic centimetre).

Case of sterile graduated pipettes, 1 c.c. (in tenths of a cubic centimetre).

Plate-levelling stand.

Tubes of nutrient gelatine.

Tubes of nutrient agar.

Water-bath regulated at 42° C.

Bulloch's apparatus.

Method.—

1. Place the flask containing 200 c.c. sterile broth on one pan of the scales and counterpoise accurately.

2. Mince a portion of the sample by the aid of sterile scissors and forceps, and add the minced sample to the bouillon in the flask to the extent of 20 grammes.

3. Make an extract by standing the flask in the incubator running at 42° C. (or in a water-bath regulated to that temperature) for half an hour, shaking its contents from time to time. Better results are obtained if an electrical shaker is fitted inside the incubator and the flask kept in motion throughout the entire thirty minutes.

Now every centimetre contains the bacteria washed out from 0.1 gramme of the original food.

4. Inoculate tubes of liquefied gelatine as follows:

To tube No. 1 add 1.0 c.c. of the extract.
2 add 0.5 c.c. of the extract.
3 add 0.3 c.c. of the extract.
4 add 0.2 c.c. of the extract.
5 add 0.1 c.c. of the extract.

Pour plates from these tubes and incubate at 20° C.

5. Prepare a precisely similar set of agar plates and incubate at 37° C.

6. Pipette 5 c.c. of the extract into a sterile tube, heat in the differential steriliser at 80° C. for ten minutes.

7. From the heated extract prepare duplicate sets of agar and gelatine plates and incubate anaerobically in Bulloch's apparatus at 37° C. and 20° C. respectively.

8. After three days' incubation examine the agar plates both aerobic and anaerobic and enumerate the colonies developed from spores (7), and from vegetative forms and spores (5), and calculate and record the numbers of each group per gramme of the original food.

9. After seven days' incubation (or earlier if compelled by the growth of liquefying colonies) enumerate the gelatine plates in the same way.

10. Subcultivate from the colonies that make their appearance and identify the various organisms.

11. Continue the investigations with reference to the detection of pathogenic organisms as described under water (page 429 et seq.).

Qualitative.—

I. Cultural.

The micro-organisms sought for during the examination of unsound foods comprise the following:

Members of the Coli-typhoid groups (chiefly those of the Gaertner class).

B. anthracis.

Streptococci

Anaerobic Bacteria:

B. enteritidis sporogenes.
B. botulinus.
B. cadaveris.

The methods by which these organisms if present may be identified and isolated have already been described under the corresponding section of water examination with the exception of those applicable to B. botulinus, and B. cadaveris. These can only be isolated satisfactorily from the bodies of experimentally inoculated animals.

II Experimental.

Tissue.—

1. Feed rats and mice on portions of the sample and observe the result.

2. If any of the animals die, make complete post-mortem examinations and endeavour to isolate the pathogenic organisms.

Extract.—

1. Introduce various quantities of the bouillon extract into the stomachs of several rats, mice and guinea-pigs repeatedly over a period of two or three days by the intragastric method of inoculation (see page 367) and observe the result. Guinea-pigs and mice are very susceptible to infection by B. botulinus by this method; rabbits less so.

2. Inoculate rats, mice, and guinea-pigs subcutaneously into deep pockets, and intraperitoneally with various quantities of the bouillon extract, and observe the result.

3. Filter some of the extract through a Chamberland candle and incubate the filtrate to determine the presence of soluble toxins.

4. If any of the animals succumb to either of these methods of inoculation, make careful post-mortem examinations and endeavour to isolate the pathogenic organisms.