XVIII. BACTERIOLOGICAL ANALYSES.

EACH bacteriological or bacterioscopical analysis of air, earth, sewage, various food-stuffs, etc., includes, as a general rule, two distinct investigations:

I. Quantitative.

2. Qualitative.

The first aims simply at enumerating (approximately) the total number of bacteria present in any given unit of volume irrespective of the nature and character of individual organisms.

The second seeks to classify the bacteria found, and to accurately identify individual organisms. As a subdivision of the qualitative examination, an estimation is often made, and with a fair degree of accuracy, of the numbers of some particular organism (e. g., B. coli communis), present per unit, in the sample under examination.

The general principles underlying the bacteriological analyses of water, sewage, air and dust, soil, milk, ice cream, meat, and other tinned stuffs, as exemplified by the methods used by the author, are indicated in the following pages, together with the methods of testing filters and chemical germicides. It is hoped that the methods given will be found to be capable of expansion and adaptation to any circumstance or set of circumstances which may confront the student.

BACTERIOLOGICAL EXAMINATION OF WATER. 1. Quantitative.—

Collection of the Sample.-As the quantity of water actually used for this examination rarely exceeds 2 c.c., the most suitable vessels for the reception of the sample

are small glass bottles, 25 c.c. capacity, with narrow necks and overhanging glass stoppers (to prevent contamination of the bottle necks by falling dust). These must be carefully sterilised in the hot-air steriliser (vide page 35).

If the sample is obtained from a tap or pipe, turn on the water and allow it to run for a few minutes. Remove the stopper from the bottle and retain it in the hand whilst the water is allowed to run into the bottle and three parts fill it. Replace the stopper and tie it down, but do not seal it.

If the sample is obtained from a stream, tank, or reservoir, fasten a piece of stout wire around the neck of the bottle, remove the stopper, and retain it in the hand. Then, using the wire as a handle, plunge the bottle into the water, mouth downwards, until it is well beneath the surface; then reverse it, allow it to fill, and withdraw it from the water. Pour out a few cubic centimetres of water from the bottle, replace the stopper, and tie it down.

Fig. 159.-Esmarch's collecting bottle for water

samples.

Or, use the apparatus designed by v. Esmarch (Fig. 159), in which the stopper can be removed, the bottle filled, and the stopper replaced, whilst the bottle is below the surface of the water, even at depths previously determined upon. When the apparatus is taken out of the water, the small bottles are filled from it, and packed in the ice-box mentioned below.

To prevent the multiplication of the bacteria contained in the water during transit from the place of collection to the laboratory, enclose the bottles, rolled in cotton-wool, in a double-walled metal box, pack the

space between the walls with pounded ice, close the metal box, and place it in a felt-lined wooden case (Fig. 160). (It has been shown that the majority of bacteria will survive exposure to the temperature of melting ice for some days, while practically none will multiply at this temperature.)

On reaching the laboratory, the method of examination consists in adding measured quantities of the

water sample to several tubes of nutrient media previously liquefied by heat, pouring plate cultivations from each of these tubes, incubating at a suitable temperature, and finally counting the colonies which make their appearance on the plates.

The bacteria present in the water may comprise not only varieties which have their normal habitat in the water and will consequently be developed at 20° C., but also varieties which have been derived from,

or are pathogenic for, the animal body, and which will only develop well at a temperature of 37° C. In order to demonstrate the presence of each of these classes it will be necessary to plant both gelatine and agar plates.

Further, the sample of water may contain moulds, yeasts, or torulæ, and the development of these will be best secured by plating in wort gelatine and incubating at 20° C.

Apparatus Required:
Plate-levelling stand.
Case of sterile plates.

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

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

Case of sterile capsules, 25 c.c. capacity.

Tubes of nutrient gelatine.

Tubes of nutrient agar.

Tubes of wort gelatine.

One 250 c.c. flask of sterile distilled water.

Tall cylinder containing 2 per cent. lysol solution.

1. Arrange the plate-levelling platform with its water compartment filled with water, at 45° C.

2. Number the agar tubes 1, 2, and 3; the gelatine tubes, consecutively, 1 to 6, and the wort tubes, 1, 2, and 3. Flame the plugs and see that they are not adherent to the lips of the tubes.

3. Place the agar tubes in boiling water until the medium is melted, then transfer them to the waterbath regulated at 42° C. Liquefy the nutrient gelatine. and wort gelatine tubes by immersing them in the same water-bath.

4. Remove the bottle containing the water sample from the ice-box, distribute the bacterial contents

evenly throughout the water by shaking, cut the string securing the stopper, and loosen the stopper, but do not take it out.

5. Remove one of the 1 c.c. pipettes from the case, holding it by the plain portion of the tube. Pass the graduated portion twice through the Bunsen flame, raise the bottle containing the water sample from the bench in the other hand, grasp the stopper as if it were a cotton-wool plug, and remove it from the bottle with the hand holding the pipette; flame the mouth of the bottle.

6. Pass the pipette into the mouth of the bottle, holding its point well below the surface of the water. Suck up rather more than I c.c. into the pipette; empty the pipette by blowing. Now draw up exactly 1 c.c. into the pipette. Withdraw the pipette from the bottle, replace the stopper, and put the bottle down.

7. Take the first melted agar tube in the left hand, remove the cotton-wool plug, and add to its contents 0.5 c.c. of the water sample from the pipette; replug the tube and put it down. In a similar manner add 0.3 c.c. water to the contents of the second tube, and 0.2 c.c. to the contents of the third.

8. Drop the pipette into the cylinder containing lysol solution.

9. Mix the water sample with the medium in each tube in the manner described under plate cultivations; pour a plate from each tube. Label each plate with (a) the distinctive name or number of the sample, (b) the quantity of water sample it contains, and (c) the date.

10. Allow the plates to set, and incubate at 37° C. 11. Empty the water chamber of the levelling apparatus and refill it with ice-water.

12. By means of the sterile 10 c.c. pipette deliver 9.9 c.c. sterile distilled water into a sterile capsule. 13. Add 0.1 c.c. of the water sample to the 9.9 c.c.