d, 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 deg. to 20 deg. 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 deg. to 37 deg. 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
plati