Differential Table of Coli-Typhoid Group

Motility Dextrose Lævulose Galactose Maltose Lactose Sacchrarose Raffinose Dextrin

A = acid reaction
G = gas formation A G A G A G A G A G A G A G A G

The Escherich Group.

B. coli communis + + + + + + + + + + + O + + + +

B. coli communior + + + + + + + + + + + + + + + + +

B. lactis aerogenes - + + + + + + + + + + O O + +

B. acidi lactici - + + + + + + + + + + O O O

B. pneumoniæ - + + + + + + + + + + + + + + + +

B cloaceæ(A) + + + + + + + + + + + + + + + + +

The Gærtner Group.

B. enteritidis + + + + + + + + + O O O O

B. paratyphosus A + + + + + + + + + O O O O

B. paratyphosus B + + + + + + + + + O O O O

B. choleræ suum + + + + + + + + + O O O

B. suipestifer + + + + + + + + + O O O

The Eberth Group.

B. typhosus + + + + + O O O +

B. dysenteriæ (Shiga) - + + + O O O O O

B. dysenteriæ (Flexner) - + + + + O O ± O

B. fæcalis alkaligines + O O O O O O O O

Table Notes: (B) (C)

Inulin Salicin Glycerine Dulcite Mannite Sorbite Indol Litmus Milk

A=acid reaction G=gas formation A G A G A G A G A G A G Early Late

The Escherich Group

B. coli communis O O + + + + + + + + + + + C

B. coli communior O O + + + + + + + + + + + C

B. lactis aerogenes O O O O + + + + - + + C

B. acidi lactici O O O + + + + + + + + + C

B. pneumoniæ O O + + + + + + + + - + + C

B cloaceæ[A] O O + + O + + - + + + + C

The Gærtner Group.

B. enteritidis O O O + + + + + + - ± -

B. paratyphosus A O ± O + + + + + + - + O

B. paratyphosus B O O O + + + + + + - + -

B. choleræ suum O O O O O + + ± + -

B. suipestifer O O O + + + + + + - + -

The Eberth Group.

B. typhosus O O O O + + - + +

B. dysenteriæ (Shiga) O O O O O O - + -

B. dysenteriæ (Flexner) O O O O + O ± + -

B. fæcalis alkaligines O O O O O O - - -

Table Notes: (D) (E)

Table Notes:

(A) * Liquefies gelatine.

(B) + = motile. - = non-motile.

(C) + = acid or gas production. ± = slight acid production. O = no change.

(D) + = indol production. ± = slight indol production. - = no indol formed.

(E) + = acid production. - = alkali production. O = no change in reaction. C = clot.

21. Determine the pathogenicity for mice (subcutaneous inoculation) and rabbits (intravenous inoculation) of the streptococci isolated.

On the facing insert page is reproduced a blank from the author's Laboratory Water Analysis Book, by means of which an exact record can be kept, with a minimum of labour, of every sample examined.

B. Concentration Method.

The remaining organisms referred to on page 426 are more conveniently sought for by the concentration method.

Collection of the Sample.—The quantity of water required for this method of examination is about 2000 c.c., and the vessel usually chosen for its reception is an ordinary blue glass Winchester quart bottle, sterilised in the hot-air oven, and over this a paper or parchment cap fastened with string. The bottle may be packed in a wooden box or in an ordinary wicker case. The method of collecting the sample is identical with that described under the heading of Quantitative Examination; there is, however, not the same imperative necessity to pack the sample in ice for transmission to the laboratory.

Apparatus required:

Sterile Chamberland or Doulton "white" porcelain open mouth filter candle, fitted with rubber washer.

Rubber cork to fit mouth of the filter candle, perforated with one hole.

Kitasato serum flask, 2500 c.c. capacity.

Geryk air pump or water force pump.

Wulff's bottle, fitted as wash-bottle, and containing sulphuric acid (to act as a safety valve between filter and pump).

Pressure tubing, clamps, pinch-cock.

Retort stand, with ring and clamp.

Rubber cork for the neck of Winchester quart, perforated with two holes and fitted with one 6 cm. length of straight glass tubing, and one V-shaped piece of glass tubing, one arm 32 cm. in length, the other 52 cm., the shorter arm being plugged with cotton-wool. The rubber stopper must be sterilised by boiling and the glass tubing by hot air, before use.

Flask containing 250 c.c. sterile broth.

Test-tube brush to fit the lumen of the candle, enclosed in a sterile test-tube (and previously sterilised by dry heat or by boiling).

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

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

Case of sterile pipettes, 1 c.c. in hundredths.

Tubes of various nutrient media (according to requirements).

Twelve Buchner's tubes with rubber stoppers.

Pyrogallic acid tablets.

Caustic soda tablets.

Method.—

1. Fit up the filtering apparatus as in the accompanying diagram (Fig. 209), interposing the wash-bottle with sulphuric acid between the filter flask and the force-pump (in the position occupied in the diagram by the central vertical line), and placing another screw clamp on the rubber tubing connecting the lateral arm of the filter flask with the wash-bottle.

2. Filter the entire 2000 c.c. of water through the filter candle.

3. When the nitration is completed, screw up the clamps and so occlude the two pieces of pressure tubing.

4. Reverse the position of the glass tubes in the Wulff's bottle so that the one nearest the air pump now dips into the sulphuric acid.

5. Slowly open the metal clamps and allow air to gradually pass through the acid, and enter filter flask, and so restore the pressure.

6. Unship the apparatus, remove the cork from the mouth of the candle.

7. Pipette 10 c.c. of sterile broth into the interior of the candle, and by means of the sterile test-tube brush (Fig. 210) emulsify the slimy residue which lines the candle, with the broth.

Practically all the bacteria contained in the original 2000 c.c. of water are now suspended in 10 c.c. of broth, so that 1 c.c. of the suspension is equivalent, so far as the contained organisms are concerned, to 200 c.c. of the original water. (Some bacteria will of course be left behind on the walls of the filter and in its pores.)

Up to this point the method is identical, irrespective of the particular organism whose presence it is desired to demonstrate; but from this point onward the methods must be specially adapted to the isolation of definite groups of organisms or of individual bacteria.

The Coli-Typhoid Group.—

1. Number nine tubes of bile salt broth (vide page 180), consecutively from 1 to 9.

2. To No 1 add 1 c.c. } of the original water sample
2 add 2 c.c. } before the nitration is commenced.
3 add 5 c.c. }

3. To the remaining tubes of bile salt broth add varying quantities of the suspension by means of suitably graduated sterile pipettes, as follows:

No. 4 0.05 c.c. (equivalent to 10 c.c. of the original water sample).
No. 5 0.125 c.c. (equivalent to 25 c.c. of the original water sample).
No. 6 0.25 c.c. (equivalent to 50 c.c. of the original water sample).
No. 7 0.5 c.c. (equivalent to 100 c.c. of the original water sample).
No. 8 1.0 c.c. (equivalent to 200 c.c. of the original water sample).
No. 9 2.5 c.c. (equivalent to 500 c.c. of the original water sample).

4. Put up each tube anaerobically in a Buchner's tube and incubate at 42° C.

5. The subsequent steps are identical with those described under the Enrichment method (see page 428 to 431; Steps 8 to 18).

Alternative Methods.—

A few of the older methods for the isolation of the members of the coli-typhoid groups are referred to but they are distinctly inferior to those already described.

(A) The Carbolic Method:

1. Take ten tubes of carbolised bouillon (vide page 202) and number them consecutively from 1 to 10.

2. Inoculate each tube with a different amount of the water sample or suspension, as in the previous method.

3. Incubate aerobically at 37° C.

4. Examine the culture tubes after twenty-four hours' incubation.

5. From those tubes which shows signs of growth, pour plates in the usual manner, using carbolised gelatine (vide page 202) in place of the ordinary gelatine, and incubate at 20° C. for three, four, or five days as may be necessary.

6. Subcultivate from any colonies that make their appearance, and determine their identity on the lines laid down in the previous method.

(B) Parietti's Method:

1. Take nine tubes of Parietti's bouillon (vide page 202)—i. e., three each of those containing 0.1 c.c., 0.2 c.c., and 0.5 c.c. of Parietti's solution respectively. Mark plainly on the outside of each tube the quantity of Parietti's solution it contains.

2. To each tube add a different amount of the original water, or of the suspension, and incubate at 37° C.

3. Examine the culture tubes after twenty-four and forty-eight hours' incubation, and plate in nutrient carbolised or potato gelatine from such as have grown.

4. Pick off suspicious colonies, if any such appear on the plates, subcultivate them upon the various media, and identify them.

(C) Elsner's Method: This method simply consists in substituting Elsner's potato gelatine (vide page 204) for ordinary nutrient gelatine in any of the previously mentioned methods.

(D) Cambier's Candle Method:

Treat a large volume of the water sample by the concentration method (vide page 434).

1. Remove the rubber stopper from the mouth of the filter candle, introduce 10 c.c. sterile bouillon into its interior, and emulsify the bacterial sediment; replug the mouth of the candle with a wad of sterile cotton-wool.

2. Remove the filter candle from the filter flask and insert it into the mouth of a flask or a glass cylinder containing sterile bouillon sufficient to reach nearly up to the rubber washer on the candle.

3. Incubate for twenty-four to thirty-six hours at 37° C.

4. From the now turbid bouillon in the glass cylinder pour gelatine plates and incubate at 20° C.

5. Subcultivate and identify any suspicious colonies that appear.

(The method depends upon the assumption that members of the typhoid and coli groups find their way through the porcelain filter from the interior to the surrounding bouillon at a quicker rate than the associated bacteria.)

B. Enteritidis Sporogenes.—

1. Transfer 5 c.c. of the emulsion from the filter candle to a sterile test-tube and plug carefully.

2. Place the test-tube in the interior of the benzole bath employed in separating out spore-bearing organisms (vide page 257), and expose to a temperature of 80° C. for twenty minutes.

3. Number ten tubes of litmus milk consecutively from 1 to 10.

4. Remove the test-tube from the benzole bath and shake well to distribute the spores evenly through the fluid.

5. To each tube of litmus milk add a measured quantity of the suspension corresponding to the amounts employed in isolating the coli group (vide page 437).

6. Incubate each tube anaerobically at 37° C. Anaerobic conditions can be obtained by putting the cultures up in Buchner's tubes or in Bulloch's apparatus. If, however, whole milk has been used in making the litmus milk the layer of cream that rises to the surface will be sufficient to ensure anaerobiosis; whilst if separated milk has been employed it will be sufficient to pour a layer of sterile vaseline or liquid paraffin on the surface of the fluid.

7. Examine after twenty-four hours' incubation. Note (if B. enteritidis sporogenes is present)—

(a) Acid reaction of the medium as indicated by the colour of the litmus or its complete decolourisation.

(b) Presence of clotting, and the separation of clear whey.

(c) Presence of gas, as indicated by fissures and bubbles in the coagulum, and possibly masses of coagulum driven up the tube almost to the plug.

8. Replace the tubes which show no signs of growth in the incubator for a further period of twenty-four hours and again examine with reference to the same points.

9. Remove those tubes which give evidence of growth from the Buchner's tubes and carefully pipette off the whey; examine the whey microscopically.

10. Inoculate two guinea-pigs each subcutaneously with 0.5 c.c. of the whey and observe the result.

Vibrio Choleræ.—

1. Number ten tubes of peptone water consecutively from 1 to 10.

2. To each of the tubes of peptone water add a measured quantity of the suspension, corresponding to those amounts employed in isolating the members of the coli group (vide page 437).

3. Incubate aerobically at 37° C. for twenty-four hours. Examine the tubes carefully for visible growth, especially delicate pellicle formation, which if present should be examined microscopically for vibrios, both by stained preparations or by fresh specimens with dark ground illumination.

4. Inoculate fresh tubes of peptone water from such of the tubes as exhibit pellicle formation—from the pellicle itself—and incubate at 37° C. for twenty-four hours.

5. Test the peptone water itself for the presence of indol and nitrite by the addition of pure concentrated H2SO4.

5. Prepare gelatine and agar plates in the usual way from such of these tubes as show pellicle formation.

6. Pick off from the plates any colonies resembling those of the Vibrio choleræ and subcultivate upon all the ordinary laboratory media.

7. Test the vibrio isolated against the serum of an animal immunised to the Vibrio choleræ for agglutination.

B. Anthracis.—

1. Transfer 5 c.c. of the emulsion from the filter candle to a sterile test-tube and plug carefully.

2. Place the test-tube in the interior of the benzole bath employed in separating out spore-bearing organisms (vide page 257), and expose to a temperature of 80° C. for twenty minutes.

3. Inoculate a young white rat subcutaneously (on the inner aspect of one of the hind legs) with 1 c.c. of the emulsion. Observe during life, and, if the animal succumbs, make a complete post-mortem examination.

4. Melt three tubes of nutrient agar in boiling water and cool to 42° C.

5. Number the tubes 1, 2, and 3. To No. 1 add 0.2 c.c., to No. 2 add 0.3 c.c., and to No. 3 add 0.5 c.c. of the suspension, and pour plates therefrom.

6. Incubate at 37° C. for twenty-four or forty-eight hours.

7. Pick off any colonies resembling those of anthrax and subcultivate on all the ordinary laboratory media.

8. Inoculate another young white rat as in 3, using two loopfuls of the agar subcultivation emulsified with 1 c.c. sterile bouillon. Observe during life, and if the animal succumbs, make a complete post-mortem examination.

B. Tetani.—

1. Proceed as detailed above in steps 1 and 2 for the isolation of the B. anthracis.

2. Add 1 c.c. of the suspension to each of three tubes of glucose formate broth, and incubate anaerobically in Buchner's tubes at 37° C.

3. From such of the tubes as show visible growth (with or without the production of gas) after twenty-four hours' incubation inoculate guinea-pigs, subcutaneously (under the skin of the abdomen), using 0.1 c.c. of the bouillon cultivation as a dose. Observe carefully during life, and, if death occurs, make a complete post-mortem examination.

4. From the same tubes pour agar plates and incubate anaerobically in Bulloch's apparatus, at 37° C.

5. Subcultivate suspicious colonies on the various media, incubate anaerobically, making control cultivations on glucose formate agar, stab and streak, to incubate aerobically and carry out further inoculation experiments with the resulting growths.