Each bacteriological or bacterioscopical analysis of air, earth, sewage, various food-stuffs, etc., includes, as a general rule, two distinct investigations yielding results of very unequal value:

The first is purely quantitative and as such is of minor importance as it 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 and more important is both qualitative and quantitative in character since it seeks to accurately identify such pathogenic bacteria as may be present while, incidentally, the methods advocated are calculated to indicate, with a fair degree of accuracy, the numerical frequency of such bacteria, 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; and the technique there set out will be found to be capable of expansion and adaptation to any circumstance or set of circumstances which may confront the student.

Controls.—The necessity for the existence of adequate controls in all experimental work cannot be too urgently insisted upon. Every batch of plates that is poured should include at least one of the presumably "sterile" medium; plate or tube cultures should be made from the various diluting fluids; every tube of carbohydrate medium that is inoculated should go into the incubator in company with a similar but uninoculated tube, and so on.

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:

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):

(Later stage):

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.

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.

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

I. The Escherich Group.

II. The GÆrtner Group.

III. The Eberth Group.

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.

ANALYTICAL SCHEME FOR ISOLATION OF MEMBERS OF THE COLI AND TYPHOID GROUPS.

Method.—

1. Arrange four sterile capsules numbered I, II, III, and IV; pipette 9 c.c. sterile bouillon into the first capsule, and 9.9 c.c. into each of the remaining three.

2. Pipette 100 c.c. sterile bouillon into the Erlenmeyer flask.

3. Remove the cotton-wool plug from the flask and replace it by the sterile funnel.

4. Place flask and funnel on one pan of the scales, and counterpoise accurately.

5. Empty the sample of soil into the mortar and triturate thoroughly.

6. By means of the sterile spatula add 10 grammes of the earth sample to the bouillon in the flask.

The final results will be more reliable if steps 2, 3, 4, and 5 are performed under a hood—to protect from falling dust, etc.

7. Remove the funnel from the mouth of the flask; replace it by the rubber stopper and shake vigourously; then allow the solid particles to settle for about thirty minutes. One cubic centimetre of the turbid broth contains the washings from 0.1 gramme of soil.

8. Pipette off 1 c.c. of the supernatant bouillon, termed the "soil water," and add it to the contents of capsule I; mix thoroughly.

9. Remove 0.1 c.c. of the infected bouillon from capsule I and add it to capsule II, and mix.

10. 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. fluid from capsule I contains soil water from .01 gm. earth.
Then 1 c.c. fluid from capsule II contains soil water from .0001 gm. earth.
Then 1 c.c. fluid from capsule III contains soil water from .000001 gm. earth.
Then 1 c.c. fluid from capsule IV contains soil water from .00000001 gm. earth.

(A) Aerobes (Vegetative Forms and Spores).—

11. Pour a set of gelatine plates from the contents of each capsule—two plates in a set, and containing respectively 0.1 c.c. and 0.4 c.c. of the diluted soil water. Label and incubate.

12. Pour similar sets of wort gelatine plates from the contents of capsules II and III, label, and incubate at 20° C.

13. Pour similar sets of agar plates from the contents of capsules II and III; label and incubate at 37° C.

14. Weigh out a second sample of soil—10 grammes—dry over a water-bath until of constant weight and calculate the ratio

15. "Count" the plates after incubation for three, four, or five days, and correcting the figures thus obtained by means of the "wet" to "dry" soil ratio estimate—

(a) The number of aerobic micro-organisms present per gramme of the soil.

(b) The number of yeasts and moulds present per gramme of the soil.

(c) The number of aerobic organisms "growing at 37° C." present per gramme of the soil.

(B) Anaerobes (Vegetative Forms and Spores).—

16. Pour similar sets of plates in glucose formate gelatine and agar and incubate in Bulloch's anaerobic apparatus.

(C) Aerobes and Anaerobes (Spores Only).—

17. Pipette 5 c.c. soil water into a sterile tube.

18. Place in the differential steriliser at 80° C. for ten minutes.

19. Pour two sets of four gelatine plates containing 0.1, 0.2, 0.5, and 1 c.c. respectively of the soil water; label and incubate at 20° C., one set aerobically, the other anaerobically in Bulloch's apparatus.

20. "Count" the plates (delay the enumeration as long as possible) and estimate the number of spores of aerobes and anaerobes respectively present per gramme of the soil.

21. Calculate the ratio existing between spores and spores + vegetative forms under each of the two groups, aerobic and anaerobic micro-organisms.

Qualitative Examination.—The qualitative examination of soil is usually directed to the detection of one or more of the following:

Members of the Coli-typhoid group.

Streptococci.

Bacillus anthracis.

Bacillus tetani.

Bacillus oedematis maligni.

The nitrous organisms.

The nitric organisms.

1. Transfer the remainder of the soil water (88 c.c.) to a sterile Erlenmeyer flask by means of a sterile syphon.

2. Fix up the filtering apparatus as for the qualitative examination of water, and filter the soil water.

3. Suspend the bacterial residue in 5 c.c. sterile bouillon (technique similar to that described for the water sample, vide pages 434-436).

Every cubic centimetre of suspension now contains the soil water from nearly 1 gramme of earth.

The methods up to this point are identical no matter which organism or group of organisms it is desired to isolate; but from this stage onward the process is varied slightly for each particular bacterium.

I. The Coli-typhoid Group.—

II. Streptococci.—

III. Bacillus Anthracis.—

IV. Bacillus Tetani.—

The methods adopted for the isolation of these organisms are identical with those already described under water (page 437 et seq.).

V. Bacillus Œdematis Maligni.—Method precisely similar to that employed for the B. tetani.

VI. The Nitrous Organisms.—

1. Take ten tubes of Winogradsky's solution No I (vide page 198) and number them consecutively from 1 to 10.

2. Inoculate each tube with varying quantities of the material as follows:

Label and incubate at 30° C.

VII. The Nitric Organisms.—

3. Take ten tubes of Winogradsky's solution No II, number them consecutively from 1 to 10 and inoculate with quantities of soil water similar to those enumerated in section VI step 2. Label and incubate at 30° C.

4. Examine after twenty-four and forty-eight hours' incubation. From those tubes that show signs of growth make subcultivations in fresh tubes of the same medium and incubate at 30° C.

5. Make further subcultivations from such of those tubes as show growth, and again incubate.

6. If growth occurs in these subcultures, make surface smears on plates of Winogradsky's silicate jelly (vide page 198).

7. Pick off such colonies as make their appearance and subcultivate in each of these two media.

TESTING FILTERS.

Porcelain filter candles are examined with reference to their power of holding back all the micro-organisms suspended in the fluids which are filtered through them, and permitting only the passage of germ-free filtrates. In order to determine the freedom of the filter from flaws and cracks which would permit the passage of bacteria no matter how perfect the general structure of the candle might be, the candle must first be attached by means of a long piece of pressure tubing, to a powerful pump, such as a foot bicycle pump, fitted with a manometer. The candle is then immersed in a jar of water and held completely submerged whilst the internal pressure is gradually raised to two atmospheres by the action of the pump. Any crack or flaw will at once become obvious by reason of the stream of air bubbles issuing from it.

The examination for permeability is conducted as follows:

Apparatus Required:

Method.—

1. Prepare surface cultivations, on nutrient agar in a culture bottle, of the Bacillus mycoides, and incubate at 20° C., for forty-eight hours.

2. Pipette 5 c.c. sterile normal saline into the culture bottle and emulsify the entire surface growth in it.

3. Pipette the emulsion into the sterile measuring flask and dilute up to 1000 c.c. by the addition of sterile water.

4. Pour the emulsion into the filter reservoir and start the filtration.

5. When the filtration is completed, pour six agar plates each containing 1 c.c. of the filtrate.

6. Incubate at 37° C. until, if necessary, the completion of seven days.

7. If the filtrate is not sterile, subcultivate the organism passed and determine its identity with the test bacterium before rejecting the filter—since the filtrate may have been accidentally contaminated.

8. If the filtrate is sterile, resterilise the candle and repeat the test now substituting a cultivation of B. prodigiosus—a bacillus of smaller size.

9. If the second test is satisfactory, test the candle against a cultivation of a very small coccus, e. g., Micrococcus melitensis, in a similar manner; in this instance continuing the incubation of cultivations from the filtrate for fourteen days.

TESTING OF DISINFECTANTS.

Methods have already been detailed (page 310) for the purpose of studying the vital resistance offered by micro-organisms to the lethal effect of germicides. But it frequently happens that the bacteriologist has to determine the relative efficiency of "disinfectants" from the standpoints of the sanitarian and commercial man rather than from the research worker's point of view. In pursuing this line of investigation, it is convenient to compare the efficiency, under laboratory conditions, of the proposed disinfectant with that of some standard germicide, such as pure phenol. In so doing, and in order that the work of different observers may be compared, conditions as nearly uniform as possible should be aimed at. The method described is one that has been in use by the writer for many years past, modified recently by the adoption of some of the recommendations of the Lancet Commission on the Standardisation of Disinfectants—particularly of the calculation for determining the phenol coefficient.

This method has many points in common with that modification of the "drop" method known as the Rideal-Walker test.

General Considerations.—

These may be grouped under three headings: Test Germ, Germicide, and Environment.

1. Test Germ.—B. coli.

As disinfectants are tested for sanitary purposes, it is obvious that a member of the coli-typhoid group should be selected as the test germ. B. coli is selected on account of its relative nonpathogenicity, the ease with which it can be isolated and identified by different observers in various parts of the world, the stability of its fundamental characters, and evenness of its resistance when utilised for these tests; finally since the colon bacillus is an organism which is slightly more resistant to the lethal action of germicides than the more pathogenic members of this group, a margin of safety is introduced into the test which certainly enhances its value.

B. coli should be recently isolated from a normal stool, and plated at least twice to ensure the purity of the strain; and a stock agar culture prepared which should be used throughout any particular test. For any particular experiment prepare a smear culture on agar and incubate at 37° C. for 24 hours anaerobically. Then emulsify the whole of the surface growth in 10 c.c. of sterile water. Transfer the emulsion to a sterile test-tube with some sterile glass beads and shake thoroughly to ensure homogenous emulsion. Transfer to a centrifuge tube and centrifugalise the emulsion to throw down any masses of bacteria which may have escaped the disintegrating action of the beads. Pipette off the supernatant emulsion for use in the test.

2. Germicide.—

a. Disinfectant to be tested.—

The first essential point is to test the unknown disinfectant, which may be referred to as germicide-x, on the lines set out on page 311 to determine its inhibition coefficient.

This constant having been fixed, prepare various solutions of germicide-x with sterilised distilled water by accurate volumetric methods, commencing with a solution somewhat stronger than that representing the inhibition coefficient. The solutions must be prepared in fairly large bulk, not less than 5 c.c. of the disinfectant being utilised for the preparation of any given percentage solution.

b. Standard Control.—Phenol.

The standard germicide used for comparison should be one which is not subject to variation in its chemical composition, and the one which has obtained almost universal use is Phenol.

The following table shows the effect of different percentages of carbolic acid upon B. coli for varying contact times, compiled from an experiment conducted under the standard conditions referred to under Environment. The results closely correspond to those recorded by the Lancet Commission on Disinfectants, 1909.

- = No growth, i. e., bacteria killed.
+ = Growth, i. e., bacteria still living.

From this it will be seen that the following percentage solutions will need to be prepared, namely: 1.1 per cent., 1.0 per cent., 0.9 per cent., 0.75 per cent., 0.7 per cent., as controls for each experiment.

Prepare solutions of varying percentages by weighing out the quantity of carbolic acid required for each and dissolving in 100 c.c. of pure distilled water in an accurately standardised measuring flask. The solutions must be prepared freshly as required each day.

Environment.—

a. General.—

Close the windows and doors of the laboratory in which the investigation is carried out, to avoid draughts. Flush over the work bench and adjacent floor with 1:1000 solution of corrosive sublimate. Caution the assistant, if one is employed, to avoid unnecessary movement or speech.

b. Contact Temperature, 15-18° C.—

This is the temperature at which contact between the germicide and the test germ takes place, and is of importance, since some germicides (e. g., Phenol) appear to be more powerful at high temperatures. 18° C.—practically the ordinary room temperature—is a temperature at which the multiplication of B. coli is a comparatively slow process, but variation of a degree above this temperature or of two or three degrees below is of no moment. If the room temperature is below 15° C. when the experiments are in progress, arrange a water-bath regulated at 18° C. for the reception of the tubes containing the mixture of germ and germicide; if above 19° C. immerse the tubes in cold water, to which small pieces of ice are added from time to time to prevent the temperature rising above 18° C.

c. Relative Proportional Bulk of Test Germ and Germicide, 50:1.—

Five cubic centimetres is a convenient amount of germicidal solution to employ, and to this 0.1 c.c. of the emulsion of test germ should be added.

d. Bulk of Sample Removed from Germ + Germicide Mixture at Each of the Time Periods, 0.1 c.c.—

This is sufficient to afford a fair sample of the germ content of the mixture, and at the same time is insufficient to exert any inhibitory action when transferred to the subculture medium.

e. Subculture Medium. Bile Salt Broth.—

A fluid medium is essential in order to obtain immediate dilution of the germicide carried over; at the same time it is advantageous to employ a selective medium which favours the growth of the test germ to the exclusion of organisms likely to contaminate the preparation, and if possible one which affords characteristic cultural appearances.

Bile Salt Broth (page 180) combines these desiderata; it permits only the growth of intestinal bacteria, whilst the formation of an acid reaction and the production of gas in subcultures prepared from the germ-germicide mixture is fairly complete evidence of the presence of living B. coli.

The amount of medium present in each test-tube is a matter of importance, since the medium not only provides pabulum for the test germ, but also acts as a diluent to the germicide, to reduce its strength below its inhibition coefficient. For routine work each subculture tube contains 10 c.c. of medium, but it is obvious that if germicide-x possesses an inhibition coefficient of 0.1 per cent. the addition of 0.1 c.c. of a 10 per cent. solution to 10 c.c. of medium would effectually prevent the subsequent growth of the test germ after a contact period insufficient to destroy its vitality. Hence the preliminary tests may in some instances indicate the necessity for the presence of 12 c.c., 15 c.c. or more of the fluid medium in the culture tubes.

f. Incubation Temperature, 37° C.—

g. Observation Period of the Subcultivations, Seven Days.—

In order to determine whether or no the test germs have been destroyed, observations must always be continued—when growth appears to be absent—up to the end of seven days before recording "no growth."

h. Identification of the Organisms Developing in the Subcultivations after Contact in the Germ + Germicide Solution.—

This is based on the naked eye characters of the growth in the bile salt broth, supplemented where necessary by plating methods, further subcultivations upon carbohydrate media and agglutination experiments. The sign (+) is used to indicate that growth of the test organism occurred in the subcultivations, and the sign (-) to indicate that the test germs have been destroyed and no subsequent growth has taken place.

Method.—

Preliminary Tests.—

a. Inhibition Coefficient.—

Determine the lowest percentage of germicide-x which inhibits growth of B. coli in the bile salt broth, and the highest percentage which fails to inhibit (page 311). On the result of this experiment determine the bulk of medium required in the subculture tubes and the percentage solutions to be employed in the trial trip. Assuming the inhibition coefficient to be 1:1000, it will be quite safe to employ the ordinary culture tubes containing 10 c.c. medium in the subsequent experiments.

b. Trial Trip.—

Determine the lethal effect of a series of five solutions of germicide-x (say 1:100, 1:250, 1:300, 1:500, 1:600) at contact times of 2-1/2, 5, 25 and 30 minutes in the following manner:

1. Arrange five test-tubes marked A to E in the lower tier of the test-tube rack.

2. Into tube A pipette 5 c.c. germicide-x 1:100 solution.

Into tube B pipette 5 c.c. germicide-x 1:200 solution.

Into tube C pipette 5 c.c. germicide-x 1:300 solution.

Into tube D pipette 5 c.c. germicide-x 1:500 solution.

Into tube E pipette 5 c.c. germicide-x 1:600 solution.

3. Arrange 20 tubes of bile salt broth in the upper tier of the test-tube rack in two rows, those in the front row numbered consecutively from left to right 1-10, those in the back row 11-20.

4. Place a square wire basket of about 50 tubes capacity close to the left of the test-tube rack, for the reception of the inoculated tubes.

5. Take a sterile 1 c.c. pipette from the case, pick up a sterile rubber washer with forceps and push the point of the pipette into the central hole.

6. Put down the forceps on the bench with the sterile points projecting over the edge. Without taking the tube from the rack remove the cotton-wool plug from tube A, and lower the pipette, with the rubber washer affixed, on to the open mouth of the tube; with the help of the forceps to steady the washer, push the pipette on through the hole until the point of the pipette has reached to within a few millimetres of the bottom of the tube (see fig. 219).

7. Adjust in the same way a pipette and a washer in the mouth of each of the other tubes, B, C, D and E.

8. Set the electric signal clock to ring for the commencement of the experiment and at subsequent intervals of 2-1/2, 5, 25 and 30 minutes.

9. Take up 0.5 c.c. of B. coli emulsion in sterile pipette graduated in tenths of a cubic centimetre and stand by.

10. As soon as the bell rings lift the pipette from tube A with the left hand and from the charged pipette held in the right hand deliver 0.1 c.c. of B. coli emulsion into the 1:100 solution. Then replace the pipette and washer.

11. Raise the tube with the left hand and shake it to mix germ and germicide, whilst returning the delivery pipette in the right hand.

12. Repeat the process with tubes B, C, D and E; then drop the infected delivery pipette in the lysol jar. The inoculation of the five tubes can be carried out very expeditiously, but a period of 10 seconds must be allowed for each tube.

13. When the bell rings at 2-1/2 minutes blow through the pipette in tube A (this agitates the germ + germicide mixture and ensures the collection of a fair sample); allow the mixture to enter the pipette, and as the column of fluid extends well above the terminal graduation, the right forefinger adjusted over the butt-end of the pipette before it is lifted will retain more than 0.1 c.c. of the mixture within the bore when the point of the pipette is clear of the fluid in the tube. Touch the point of the pipette on the inner wall of the tube, and allow any excess of fluid to escape, only retaining 0.1 c.c. in the pipette.

14. At the same time, with the left hand remove Bile Salt Tube No. 1 from the upper tier of the rack, take out the cotton-wool plug with the hand already holding the pipette (the relative positions of pipette, plug and culture tubes being practically the same as those of platinum loop, plug and culture tube shown in Fig. 68, page 74).

15. Insert the point of the pipette into the subculture tube, and blow out the mixture into the medium—replug the tube and drop it into the wire basket. Replace the washer-pipette in tube A.

As soon as the point of the pipette has entered the mouth of tube A it may be released, since it has already been so adjusted that it just clears the bottom of the test-tube, and the elastic washer will prevent any damage to the tube.

Steps 13, 14 and 15 occupy on an average 10 seconds.

16. Repeat steps 13, 14 and 15 with each of the other tubes B, C, D and E.

17. Repeat these various steps 13-16 when the bell rings at 5, 25 and 30 minutes.

18. Place all the inoculated tubes in the incubator at 37° C.

19. Examine the tubes at intervals of 24 hours, and record the results in tabular form as shown in Table page 491 (the figures in the squares indicate the number of hours at which the changes in the medium due to the growth of B. coli first appeared).

20. If a consideration of the tabulated results indicates strengths of Germicide-x lethal at 2-1/2 and 30 minutes the final test can be arranged, but if this result has not been attained, sufficient evidence will probably be available to enable a second trial test to be planned which will give the required information.

Final Test.—

c. Determination of Phenol Coefficient.—

X-Disinfectant.—This comprises two distinct tests, one of the Germicide-x, the other of the standard phenol.

1. Arrange five test-tubes clearly marked in the lower tier of the rack.

2. Pipette into each 5 c.c. respectively of the five percentage solutions of x-disinfectant which the trial run has already shown will include those affording lethal values at 2-1/2 and 30 minutes.

3. Arrange 20 tubes of bile salt broth in the upper tier of the test-tube rack in two rows, those in the front row numbered consecutively from left to right 1-10, those in the back row 11-20.

4. Arrange further 20 tubes of bile salt broth numbered 21-40 in two rows in a second smaller rack which can be stood on the upper tier of the rack as soon as the first 20 tubes have been inoculated.

5. Place a square wire basket of about 50 tube capacity close to the left of the test-tube rack, for the reception of the inoculated tubes.

6. Adjust a sterile 1 c.c. pipette in the mouth of each of the tubes, A, B, C, D and E, by means of a washer, as previously described.

7. Set the electric signal clock to ring for the commencement of the experiment and subsequently at 2-1/2, 5, 10, 15, 20, 25, 30 and 35 minutes.

8. Complete precisely as indicated in Trial Runs, steps 9-19.

Control Phenol.—

Immediately the subculture tube from the 30-minute contact period have been inoculated, carry out a precisely similar experiment, in which five percentage strengths of Phenol, (e. g., 1.1, 1.0, 0.9, 0.75, 0.7) are arranged in the lower tier of the test-tube rack in place of the five strengths of Germicide-x.

Calculate the phenol coefficient by the following method:

(a) Divide the figure representing the percentage strength of the weakest lethal dilution of the carbolic acid control at the 2-1/2-minute contact period by the figure representing the percentage strength of the weakest lethal dilution of the x-disinfectant at the same period. The quotient = phenol coefficient at 2-1/2 minutes.

(b) Similarly obtain the phenol coefficient at 30 minutes contact period.

(c) Record the mean of the two coefficients obtained in (a) and (b) as the mean phenol coefficient, or simply as the Phenol Coefficient.

The details of the Final Test of an actual determination are set out in the accompanying table.

TABLE 27

Organism employed, B. Coli Communis.
Culture Medium, Nutrient Agar (+10). Age, 24 hrs. Temp. of Incubation, 37°C.