[Ill.u.s.tration: FIG. 159.--Plate with star for testing effect of light.]

2. Pour twenty plate cultivations, ten of nutrient gelatine and ten of nutrient agar, each containing 0.1 c.c. of the bouillon culture.

3. Place one agar plate and one gelatine plate into the hot and cold incubators, respectively, as _controls_.

4. Fasten a piece of black paper, cut the shape of a cross or star, on the centre of the cover of each of the remaining plates (Fig. 159).

5. Expose these plates to the action of diffuse daylight (not direct sunlight) in the laboratory for one, two, three, four, five, six, eight, ten, twelve hours.

6. After exposure to light, incubate under optimum conditions.

7. Examine the plate cultivations after twenty-four and forty-eight hours' incubation, and compare with the two controls. Record results. If growth is absent from that portion of the plate unprotected by the black paper, continue the incubation and daily observation until the end of seven days.

8. Control the results.

(b) Direct Sunlight:

1. Prepare plate cultivations precisely as in the former experiments and place the two controls in the incubators.

2. Arrange the remaining plates upon a platform in the direct rays of the sun.

3. On the top of each plate stand a small gla.s.s dish 14 cm. in diameter and 5 cm. deep.

4. Fill a solution of potash alum (2 per cent. in distilled water) into each dish to the depth of 2 cm. to absorb the heat of the sun's rays and so eliminate possible effects of temperature on the cultivations.

5. After exposures for periods similar to those employed in the preceding experiment, incubate and complete the observation as above.

(c) Primary Colours: Each colour--violet, blue, green and red--must be tested separately.

1. Prepare plate cultivations, as in the previous "light" experiments, and incubate controls.

2. Fasten a strip of black paper, 3 cm. wide, across one diameter of the cover of each plate.

3. Coat the remainder of the surface of the cover with a film of pure photographic collodion which contains 2 per cent. of either of the following aniline dyes, as may be necessary:

Chrysoidin (for red).

Malachite green (for green).

Eosin, bluish (for blue).

Methyl violet (for violet).

4. Expose the plates, thus prepared, to bright daylight (but not direct sunlight) for varying periods, and complete the observations as in the preceding experiments. The bactericidal action of light appears to depend upon the more refrangible rays of the violet end of the spectrum and is noted whether the red yellow rays are transmitted or not.

5. Control the results.

NOTE.--The ultra-violet rays obtained from a quartz mercury vapour lamp destroy bacterial life with great rapidity under laboratory conditions.

(C) _Heat._--(_Vide_ Thermal Death-point, page 298.)

(D) _Antiseptics and Disinfectants._--The resistance exhibited by any given bacterium toward any specified disinfectant or germicide should be investigated with reference to the following points:

(A) ~Inhibition coefficient~--i. e., that _percentage of the disinfectant_ present in the nutrient medium which is sufficient to prevent the growth and multiplication of the bacterium.

(B) ~Inferior lethal coefficient~--i. e., the _time exposure_ necessary to kill _vegetative forms_ of the bacterium suspended in water at 20 to 25 C, in which the disinfectant is present in _medium_ concentration (concentration insufficient to cause plasmolysis). And if the bacterium is one which forms spores,

(C) ~Superior lethal coefficient~--i. e., the _time exposure_ necessary to kill the _spores_ of the bacterium under conditions similar to those obtaining in B.

The example here detailed only specifically refers to certain of the disinfectants:

viz:--Bichloride of mercury; Formaldehyde; Carbolic acid;

investigated with regard to B. anthracis, but the technique is practically similar for all other chemical disinfectants.

~Inhibition Coefficient.~--

_Apparatus Required:_

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

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

Sterile tubes or capsules for dilutions.

Tubes of nutrient bouillon each containing a measured 10 c.c. of medium.

Twenty-four-hour-old agar culture of a recently isolated B.

Anthracis.

_Germicides:_

1. Five per cent. aqueous solution of carbolic acid.

2. One per cent. aqueous solution of perchloride of mercury.

3. One-tenth per cent. aqueous solution of formaldehyde.

METHOD.--

1. Number six bouillon tubes consecutively 1 to 6. Inoculate each from the stock cultivation of B. anthracis and at once add varying quant.i.ties[10] of the carbolic acid solution, viz.:

To tube 1 add 2.0 c.c. (= 1:100) To tube 2 add 1.0 c.c. (= 1:200) To tube 3 add 0.6 c.c. (= 1:300) To tube 4 add 0.5 c.c. (= 1:400) To tube 5 add 0.4 c.c. (= 1:500) To tube 6 add 0.2 c.c. (= 1:1,000)

2. Prepare a similar series of tube cultivations numbered consecutively 7 to 12 and add varying quant.i.ties of the mercuric perchloride solution, viz.:

To tube 7 add 0.1 (= 1:1,000) To tube 8 add 0.05 (= 1:2,000) To tube 9 add 0.03 (= 1:3,000) To tube 10 add 0.025 (= 1:4,000) To tube 11 add 0.02 (= 1:5,000) To tube 12 add 0.01 (= 1:10,000)

3. Prepare a similar series of tube cultivations numbered consecutively 13 to 18 and add varying quant.i.ties of the formaldehyde solution, viz.: