To tube No. 13 add 1.0 c.c. (= 1:1,000) To tube No. 14 add 0.4 c.c. (= 1:2,500) To tube No. 15 add 0.2 c.c. (= 1:5,000) To tube No. 16 add 0.1 c.c. (= 1:10,000) To tube No. 17 add 0.075 c.c. (= 1:15,000) To tube No. 18 add 0.05 c.c. (= 1:20,000)

4. Incubate all three sets of cultivations under optimum conditions as to temperature and atmosphere.

5. Examine each of the culture tubes from day to day, until the completion of seven days, and note those tubes, if any, in which growth takes place.

6. From such tubes as show growth prepare subcultivations upon suitable media, and ascertain that the organism causing the growth is the one originally employed in the test and not an accidental contamination.

~Inferior Lethal Coefficient.~--

_Apparatus Required:_

Highly concentrated solutions of the disinfectants.

Sterile test-tubes in which to make dilutions from the concentrated solutions of the disinfectants.

Hanging-drop slides.

Cover-slips.

Erlenmeyer flask containing 100 c.c. sterile distilled water.

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

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

METHOD.--

1. Prepare a surface cultivation of the "test" organism B. anthracis upon nutrient agar in a culture bottle and incubate under optimum conditions for twenty-four hours; then examine the cultivation microscopically to determine the absence of spores.

2. Prepare solutions of different percentages of each disinfectant.

3. Make a series of hanging-drop preparations from the agar culture, using a loopful of disinfectant solution of the different percentages to prepare the emulsion on each cover-slip.

4. Examine microscopically and note the strongest solution which does not cause plasmolysis and the weakest solution which does plasmolyse the organism.

5. Make control preparations of these two solutions and determine the percentage to be tested.

6. Pipette 10 c.c. sterile water into the culture bottle and suspend the entire surface growth in it.

7. Transfer the suspension to the Erlenmeyer flask and mix it with the 90 c.c. of sterile water remaining in the flask.

8. Pipette 10 c.c. of the diluted suspension into each of ten sterile test-tubes.

9. Label one of the tubes "Control" and place it in the incubator at 18 C.

10. Add to each of the remaining tubes a sufficient quant.i.ty[11] of a concentrated solution of the disinfectant to produce the percentage previously determined upon (_vide_ step 5).

11. Incubate the tubes at 18 C. to 20 C.

12. At hourly intervals remove the control tube and one of the tubes with added disinfectant from the incubator.

13. Make a subcultivation from both the control and the test suspension, upon the surface of nutrient agar; incubate under optimum conditions.

14. Observe these culture tubes from day to day until the completion of seven days, and determine the shortest exposure necessary to cause the death of vegetative forms.

~Superior Lethal Coefficient.~--

1. Prepare surface cultivations of the "test" organisms upon nutrient agar in a culture bottle, and incubate under optimum conditions, for three days, for the formation of their spores.

2. Transfer the emulsion to a sterile test-tube and heat in the differential steriliser for ten minutes at 80 C. to destroy all vegetative forms.

3. Employing that percentage solution of the disinfectant determined in the previous experiment, and complete the investigations as detailed therein, steps 7 to 14, increasing the interval between planting the subcultivations to two, three, or five hours if considered advisable.

NOTE.--Where it is necessary to leave the organisms in contact with a strong solution of the disinfectant for lengthy periods, some means must be adopted to remove every trace of the disinfectant from the bacteria before transferring them to fresh culture media; otherwise, although not actually killed, the presence of the disinfectant may prevent their development, and so give rise to an erroneous conclusion. Consequently it is essential in all germicidal experiments to determine first of all the inhibition coefficient of the germicide employed. Under the circ.u.mstances referred to above it is usually sufficient to prepare the subcultures in such a volume of fluid nutrient medium as would suffice to reduce the concentration of the germicide to about one hundredth of the inhibition percentage, a.s.suming that the entire bulk of inoculum was made up of that strength of germicide employed in the test.

In some cases it is a simple matter to neutralise the germicide and render it inert by washing the organisms in some non-germicidal solution (such for example as ammonium sulphide when using mercurial salts as the germicide). When, however, it is desired to remove the last traces of germicide proceed as follows:

1. Transfer the suspension of bacteria to sterile centrifugal tubes; add the required amount of disinfectant, and allow it to remain in contact with the bacteria for the necessary period.

2. Centrifugalise thoroughly, pipette off the supernatant fluid; fill the tube with sterile water and distribute the deposit evenly throughout the fluid.

3. Centrifugalise again, pipette off the supernatant fluid; fill the tube with sterile water; distribute the deposit evenly throughout the fluid, and transfer the suspension to a litre flask.

4. Make up to a litre by the addition of sterile water; filter the suspension through a sterile porcelain candle.

5. Emulsify the bacterial residue with 5 c.c. sterile bouillon.

6. Prepare the necessary subcultivations from this emulsion.

PATHOGENESIS.

_Living Bacteria._--

(a) Psychrophilic Bacteria: When the organism will only grow at or below 18 to 20 C.,

1. Prepare cultivations in nutrient broth and incubate under optimum conditions.

2. After seven days' incubation inject that amount of the culture corresponding to 1 per cent. of the body-weight of a healthy frog, into the reptile's dorsal lymph sac.

3. Observe until death takes place, or, in the event of a negative result, until the completion of twenty-eight days (_vide_ Chapter XVIII).

4. If, and when, death occurs, make a careful post-mortem examination (_vide_ Chapter XIX).

(b) Mesophilic Bacteria: When the organism grows at 35 to 37 C.,

1. Prepare cultivations in nutrient broth and incubate under optimum conditions for forty-eight hours.