Bacteriological Examination of Water

The bacteria present in the water may comprise not only varieties which have their normal habitat in the water and will consequently develop at 20° C., but also if the water has been contaminated with excremental matter, 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 incubate the various cultivations at each of these temperatures.

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.

1. Quantitative.—

Collection of the Sample.—The most suitable vessels for the reception of the water sample are small glass bottles, 60 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 31).

(a) 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.

(b) 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 downward, 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.

(c) If the sample is obtained from a lake, river or the sea; or when it is desired to compare samples taken at varying depths, the apparatus designed by v. Esmarch (Fig. 203) is employed. In this the sterilised bottle is enclosed in a weighted metal cage which can be lowered, by means of a graduated line, until the required depth is reached. At this point the bottle is opened by a thin wire cord attached to the stopper; when the bottle is full (as judged by the air bubbles ceasing to rise) the pull on the cord is released and the tension of the spiral spring above the stopper again forces it into the neck of the bottle. When the apparatus is taken out of the water, the small bottles are filled from it, and packed in the ice-box mentioned below.

An inexpensive substitute for Esmarch's bottle can be made in the laboratory thus:

Select a wide-mouthed glass stoppered bottle of about 500 c.c. capacity (about 20 cm. high and 8 cm. in diameter).

Remove the glass stopper and insert a rubber cork with two perforations in its place.

Through one perforation pass a piece of glass tubing about 5 cm. long and through the other a piece 22 cm. long, reaching to near the bottom of the bottle, each tube projecting about 2.5 cm. above the rubber stopper. Plug the open ends of the tubes with cotton wool. Secure the stopper in place with thin copper wire.

Sterilise the fitted bottle in the autoclave. Remove the cotton wool plugs and connect the projecting tubes by a piece of loosely fitting stout rubber pressure tubing about 5 cm. long, previously sterilised by boiling.

Take a piece of stout rubber cord about 33 cm. long, and of 10 mm. diameter (such as is used for door springs) thread a steel split ring upon it and secure the free ends tightly to the neck of the bottle by cord or catgut.

Attach the cord used for lowering the bottle into the water to the split ring on the rubber suspender. The best material for this purpose is cotton insulated electric wire knotted at every metre.

Connect the split ring also with the short piece of rubber tubing uniting the two glass tubes by a piece of catgut (or thin copper wire) of such length that when the bottle is suspended there is no pull upon the rubber tube, but which, however, will be easily jerked off when a sharp pull is given to the suspending cord.

Now wind heavy lead tubing about 1 cm. diameter around the upper part of the bottle, starting at the neck just above the shoulder. This ensures the sinking of the bottle in the vertical position (Fig. 204).

The apparatus being arranged is lowered to the required depth, a sharp jerk is then given to the suspending cord, which detaches the rubber tube and so opens the two glass tubes. Water enters through the longer tube and the air is expelled through the shorter tube. The bubbles of air can be seen or heard rising through the water, until the bottle is nearly full, a small volume of compressed air remaining in the neck of the bottle.

As the apparatus is raised, the air thus imprisoned expands, and prevents the entry of more water from nearer the surface.

Transport of Sample.—If the examination of the sample cannot be commenced immediately, steps must be taken to prevent the multiplication of the bacteria contained in the water during the interval occupied in transit from the place of collection to the laboratory. To this end an ice-box such as that shown (in Fig. 205) is essential. It consists of a double-walled metal cylinder into which slides a cylindrical chamber of sufficient capacity to accommodate four of the 60 c.c. bottles; this in turn is covered by a metal disc—the three portions being bolted together by thumb screws through the overhanging flanges. When in use, place the bottles, rolled in cotton-wool, in the central chamber, pack the space between the walls with pounded ice, securely close the metal box by screwing down the fly nuts, and place it in a felt-lined wooden case. (It has been shown that whilst bacteria will survive exposure to the temperature of melting ice, 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.

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.
Bunsen burner.
Grease pencil.
Water-bath regulated at 42° C.

Method.—

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

2. Number the agar tubes, consecutively, 1 to 6; 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 water-bath 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. Tilt the bottle containing the water sample on the bench holding the neck between the middle and ring fingers of the left hand; grasp the head of the stopper between the forefinger and thumb, and remove it from the bottle.

6. Pass the pipette into the mouth of the bottle, holding its point well below the surface of the water (Fig. 206). Suck up rather more than 1 c.c. into the pipette and allow the pipette to empty; this moistens the interior of the pipette and renders accurate measurement possible. Now draw up exactly 1 c.c. into the pipette. Withdraw the pipette from the bottle, replace the stopper, and stand the bottle upright.

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 replace it in the water-bath. 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. In a similar manner add 1 c.c. of the sample to the contents of the fourth tube.

9. Similarly, add 0.5 c.c. and 0.1 c.c. respectively to the contents of the fifth and sixth tubes.

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

11. 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 number of the sample, (b) the quantity of water sample it contains, and (c) the date.

12. Pour the contents of a tube of liquefied agar—not inoculated—into a Petri dish to act as a control to demonstrate the sterility of the batch of agar employed.

13. Allow the plates to set, and incubate at 37° C.

14. Empty the water chamber of the levelling apparatus and refill it with ice-water.

15. By means of the sterile 10 c.c. pipette deliver 9.9 c.c. sterile distilled water into a sterile glass capsule.

16. Add 0.1 c.c. of the water sample to the 9.9 c.c. sterile water in the capsule. This will give a dilution of 1 in 100.

17. Plant the six tubes of nutrient gelatine in the following manner: To the first tube add 0.5 c.c. of the water sample direct from the bottle; to the second, 0.3 c.c.; and to the third, 0.2 c.c.; and pour a plate of each tube. To the fourth tube add 0.5 c.c. of the diluted water sample from the capsule; to the fifth, 0.3 c.c.; and to the sixth, 0.2 c.c.; and pour a plate from each.

18. Label each plate with the quantity of the water sample it contains—that is, 0.5 c.c., 0.3 c.c., 0.2 c.c., 0.005 c.c., 0.003 c.c., and 0.002 c.c.

19. Pour a control (uninoculated) gelatine plate.

20. Allow the plates to set, and incubate at 20°C.

21. To the first tube of liquefied wort gelatine add 0.5 c.c. water sample; to the second, 0.3 c.c.; and to the third, 0.2 c.c.

22. Label the plates, allow them to set, and incubate at 20° C.

23. Count and record the number of colonies that have developed upon the agar at 37° C. after forty-eight hours' incubation.

24. Note the number of colonies present on each of the gelatine and wort gelatine plates after forty-eight hours' incubation.

25. Replace the gelatine and wort plates in the incubator; observe again at three days, four days, and five days.

26. Calculate and record the number of organisms present per cubic centimetre of the original water from the average of the six gelatine plates at the latest date possible up to seven days—the presence of liquefying bacteria may render the calculation necessary at an earlier date, hence the importance of daily observations.

Method of Counting.—The most accurate method of counting the colonies on each of the plates is by means of either Jeffery's or Pakes' counting disc. Each of these discs consists of a piece of paper, upon which is printed a dead black disc, subdivided by concentric circles and radii, printed in white. In Jeffery's counter (Fig. 207), each subdivision has an area of 1 square centimetre; in Pakes' counter (Fig. 208), radii divide the circle into sixteen equal sectors, and counting is facilitated by concentric circles equidistant from the centre.

(a) In the final counting of each plate, place the plate over the counting disc, and centre it, if possible, making its periphery coincide with one or other of the concentric circles.

(b) Remove the cover of the plate, and by means of a hand lens count the colonies appearing in each of the sectors in turn. Make a note of the number present in each.

(c) If the colonies present are fewer than 500, the entire plate should be counted. If, however, they exceed this number, enumerate one-half, or one-quarter of the plate, or count a sector here and there, and from these figures estimate the number of colonies present on the entire plate. In practice it will be found that Pakes' disc is more suitable for the former class of plate; Jeffery's disc for the latter. It should be recollected however that unless the plates have been carefully leveled and the medium is of equal thickness all over it is useless to try and average from small areas—since where the medium is thick all the bacteria will develop, where the layer is a thin one, only a few bacteria will find sufficient pabulum for the production of visible colonies.

It will be noted that the quantities of water selected for addition to each set of tubes of nutrient media have been carefully chosen in order to yield workable results even when dealing with widely differing samples. Plates prepared in agar with 0.1 c.c. and in gelatin with 0.02 c.c. can be counted even when large numbers of bacteria are present in the sample; whereas if micro-organisms are relatively few, agar plate 4 and gelatine plate 1 will give the most reliable counts. Again the counts of the plates in a measure control each other; for example, the second and third plates of each gelatine series should together contain as many colonies as the first, and the second should contain about half as many more than the third and so on.

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 quantitative 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 canal, and so to constitute 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 quantity 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 choleræ 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 quantity 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 42°C.

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 42°C.

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.

12. Prepare surface plate cultivations upon nutrose agar from each tube that shows growth either macroscopically or microscopically, and incubate for twenty-four hours aerobically at 37° C.

13. Examine the growth on the plates either with the naked eye or with the help of a small hand lens. Practice will facilitate the recognition of colonies of the coli group, the typhoid group and the paratyphoid group; also those due to the growth of streptococci. The investigation from this stage proceeds along two divergent lines of enquiry—the first being concerned with the identity of the bacilli—typhoid bacilli, the second with that of the cocci.

A. B. Coli and its allies.

14. Pick off coliform or typhiform colonies; make streak or smear subcultivations upon nutrient agar; incubate aerobically for twenty-four hours at 37° C.

15. Examine the growth in each tube carefully both macroscopically and microscopically. If the growth is impure, replate on nutrose agar, pick off colonies and subcultivate again. When the growth in a tube is pure, add 5 c.c. sterile normal saline solution or sterile broth, and emulsify the entire surface growth with it.

16. Utilise the emulsion for the preparation of a series of subcultivations upon the media enumerated below, using the ordinary loop to make the subcultures upon solid media, but adding one-tenth of a cubic centimetre of the emulsion to each of the fluid media by means of a sterile pipette.

Gelatine streak.
Agar streak.
Potato.
Nutrient broth.
Litmus milk.
Dextrose peptone solution.
Lævulose peptone solution.
Galactose peptone solution.
Maltose peptone solution.
Lactose peptone solution.
Saccharose peptone solution.
Raffinose peptone solution.
Dulcite peptone solution.
Mannite peptone solution.
Glycerin peptone solution.
Inulin peptone solution.
Dextrin peptone solution.

17. Differentiate the bacilli after isolation by means of their cultural reactions and biological characters into members of:

I. The Escherich Group.

B. coli communis.
B. coli communior.
B. lactis aerogenes.
B. cloacæ.

II. The Gærtner Group.

Bacillus enteritidis (of Gærtner).
B. paratyphosus A.
B. paratyphosus B.
Bacillus choleræ suum.

III. The Eberth Group.

B. typhosus.
B. dysenteriæ (Shiga).
B. dysenteriæ (Flexner).
B. fæcalis alcaligines.

18. Confirm these results by testing the organisms isolated against specific agglutinating sera obtained from experimentally inoculated animals.

If a positive result is obtained when using this method, it only needs a simple calculation to determine the smallest quantity (down to 0.1 c.c.) of the sample that contains at least one of the microbes of indication. For instance, if growth occurs in all the tubes from 4 to 10, and that growth is subsequently proved to be due to the multiplication of B. coli, then it follows that at least one colon bacillus is present in every 10 c.c. of the water sample, but not in every 5 c.c. If, on the other hand, the presence of the B. coli can only be proved in flask No. 7, then the average number of colon bacilli present in the sample is at least one in every 50 c.c. (i. e., twenty per litre), but not one in every 25 c.c. and so on.

The general outline of the method of identifying the members of the coli-typhoid group is given in the form of an analytical schema—whilst the full differential details are set out in tabular form.