2. Qualitative Examination.--

_Collection of Sample._--The water sample required for the routine examination, which it will be convenient to consider first, amounts to about 110 c.c. It is collected in the manner previously described (_vide_ page 416); similar bottles are used, and if four are filled the combined contents, amounting to about 240 c.c., will provide ample material for both the qualitative and quant.i.tative examinations. Unless the examination is to be commenced at once, the ice-box must be employed, otherwise water bacteria and other saprophytes will probably multiply at the expense of the microbes indicative of pollution, and so increase the difficulties of the investigation.

In the routine examination of water supplies it is customary to limit the qualitative examination to a search for

A. B. coli and its near allies.

B. Streptococci,

organisms which are frequently spoken of as microbes of indication, as their presence is held to be evidence of pollution of the water by material derived from the mammalian alimentary ca.n.a.l, and so to const.i.tute a danger signal.

C. Some observers still attach importance to the presence of B.

enteritidis sporogenes, but as the search for this bacterium, (relatively scarce in water) necessitates the collection of a fairly large quant.i.ty of water it is not usually included in the routine examination.

In the case of water samples examined during the progress of an epidemic, of new supplies and of unknown waters the search is extended to embrace other members of the coli-typhoid group; and on occasion the question of the presence or absence of Vibrio cholerae or (more rarely) such bacteria as B. anthracis or B. tetani, may need investigation.

When pathogenic or excremental bacteria are present in water, their numbers are relatively few, owing to the dilution they have undergone, and it is usual in commencing the examination, to adopt one or other of the following methods:

A. _Enrichment_, in which the harmless non-pathogenic bacteria may be destroyed or their growth inhibited, whilst the growth of the parasitic bacteria is encouraged.

This is attained by so arranging the environment, (i. e., Media, incubation temperature, and atmosphere) as to favor the growth of the pathogenic organisms at the expense of the harmless saprophytes.

B. _Concentration_, whereby all the bacteria present in the sample of water, pathogenic or otherwise, are concentrated in a small bulk of fluid.

This is usually effected by filtration of the water sample through a porcelain filter candle, and the subsequent emulsion of the bacterial residue remaining on the walls of the candle with a small measured quant.i.ty of sterile bouillon.

A. ~Enrichment Method.~

(Dealing with the demonstration of bacteria of intestinal origin.)

_Apparatus Required_ (_Preliminary Stage_):

Incubator running at 42 C.

Case of sterile pipettes, 1 c.c. graduated in tenths.

Case of sterile pipettes, 10 c.c. graduated in c.c.

Case of sterile pipettes, graduated to deliver 25 c.c.

Tubes of bile salt broth (_vide_ page 180).

Flask of double strength bile salt broth (_vide_ page 199).

Tubes of litmus silk.

Sterile flasks, 250 c.c. capacity.

Buchner's tubes.

Tabloids pyrogallic acid.

Tabloids sodium hydrate.

Bunsen burner.

Grease pencil.

(_Later stage_):

Incubator running at 37 C.

Surface plates of nutrose agar (see page 232).

Aluminium spreader.

Tubes of various media, including carbohydrate media.

Agglutinating sera, etc.

METHOD.--

1. Number a set of bile salt broth, tubes 1-5, and a duplicate set 1a-5a.

2. Number one flask 7 and another 8.

3. To Tubes No. 1 and 1a add 0.1 c.c. water sample.

To Tubes No. 2 and 2a add 1 c.c. water sample.

To Tubes No. 3 and 3a add 2 c.c. water sample.

To Tubes No. 4 and 4a add 5 c.c. water sample.

To Tubes No. 5 and 5a add 10 c.c. water sample.

4. Put up all the tubes in Buchner's tubes and incubate anaerobically at 42C.

NOTE.--The bile salt medium is particularly suitable for the cultivation of bacteria of intestinal origin, and at the same time inhibits the growth of bacteria derived from other sources.

The anaerobic conditions likewise favor the multiplication of intestinal bacteria, and also their fermentative activity. The temperature 42 C.

destroys ordinary water bacteria and inhibits the growth of many ordinary mesophilic bacteria.

5. Pipette 25 c.c. of double strength bile salt broth into flask 6, and 50 c.c. double strength bile salt broth into flask 7.

6. Pipette 25 c.c. water sample into flask 6, and 50 c.c. water sample into flask 7.

7. Incubate the two flasks aerobically at 42C.

8. After twenty-four hours incubation note in each culture:

a. The presence or absence of visible growth.

b. The reaction of the medium as indicated by the colour change, if any, the litmus has undergone.

c. The presence or absence of gas formation, as indicated by a froth on the surface of the medium, and the collection of gas in the inner "gas" tube.

9. Replace those tubes which show no signs of growth in the incubator.

Examine after another period of twenty-four hours (total forty-eight hours incubation) with reference to the same points.

10. Remove culture tubes which show visible growth from the Buchner's tubes, whether acid production and gas formation are present or not.

11. Examine all tubes which show growth by hanging-drop preparations.

Note such as show the presence of chains of cocci.