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 ident.i.ty 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 quant.i.ty 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 subst.i.tuting 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 gla.s.s 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 gla.s.s cylinder pour gelatine plates and incubate at 20 C.

5. Subcultivate and identify any suspicious colonies that appear.

(The method depends upon the a.s.sumption 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 a.s.sociated 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 quant.i.ty 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 ma.s.ses 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 Cholerae.~--

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

2. To each of the tubes of peptone water add a measured quant.i.ty 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 H_{2}SO_{4}.

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 cholerae and subcultivate upon all the ordinary laboratory media.

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

~B. Anthracis.~--

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