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. 2. Select two white mice, as nearly as possible of the same age, size, and weight. 3. Inoculate the first mouse, subcutaneously at the root of the tail, with an amount of cultivation equivalent to 1 per cent. of its body-weight. 4. Inoculate the second mouse intraperitoneally with a similar dose. 5. Observe carefully until death occurs, or until the lapse of twenty-eight days. 6. If the inoculated animals succumb, make complete post-mortem examination. If death follows shortly after the injection of cultivations of bacteria, the inoculation experiments should be repeated two or three times. Then, if the organism under observation invariably exhibits pathogenic effects, steps should be taken to ascertain, if possible, the minimal lethal dose (_vide infra_) of the growth upon solid media for the frog or white mouse respectively. Other experimental animals--_e. g._, the white rat, guinea-pig, and rabbit--should next be tested in a similar manner. 7. If the inoculated mice are unaffected, test the action of the organism in question upon white rats, guinea-pigs, rabbits, etc. _Minimal Lethal Dose_ (_m. l. d._); If the purpose of the inoculation is to determine the minimal lethal dose, a slightly different procedure must be followed. For this and other exact experiments a special platinum loop is manufactured, some 2.5 mm. by 0.75 mm., with parallel sides, and calibrated by careful weighing, to determine approximately the amount of moist bacterial growth, the loop will hold when filled. 1. The cultivation must be prepared on a solid medium of the optimum reaction, incubated at the optimum temperature, and injected at the period of greatest activity and vigour, of the particular organism it is desired to test. 2. Arrange four sterile capsules in a row and label them I, II, III, and IV. Into the first deliver 10 c.c. sterile bouillon by means of a sterile graduated pipette; and into each of the remaining three, 9.9 c.c. 3. Remove one loopful of the bacterial growth from the surface of the medium in the culture tube, observing the usual precautions against contamination, and emulsify it evenly with the bouillon in the first capsule. Each cubic centimetre of the emulsion will now contain one-tenth of the organisms contained in the original loopful (written shortly 0.1 loop).