arate sterile capsules.

8. Inoculate a series of animals with these measured doses, filling the
syringe first from that capsule containing the smallest dose, then from
the capsule containing the next smallest, and so on. If care is taken,
it will not be found necessary to sterilise the syringe during the
series of inoculations.

9. Plant tubes of gelatine or agar, liquefied by heat, from each of the
higher dilutions, say from 0.0000001 loop to 0.01 loop; pour plates and
incubate. When growth is visible enumerate the number of organisms
present in each, average up and calculate the number of bacteria present
in one loopful of the inoculum.

10. The smallest dose which causes the infection and death of the
inoculated animal is noted as the minimal lethal dose.

_Toxins._--

Prepare flask cultivations of the organism under observation in glucose
formate broth, and incubate for fourteen days under optimum conditions.

(a) Intracellular or Insoluble Toxins:

1. Heat the fluid culture in a water-bath at 60 deg. C. for thirty
minutes. (The resulting sterile, turbid fluid is often spoken of as
"killed" culture,)

2. Inoculate a tube of sterile bouillon with a similar quantity, and
incubate under optimum conditions. This "control" then serves to
demonstrate the freedom of the toxin from living bacteria.

[Illustration: FIG. 160.--Apparatus arrange for toxin filtration.]

3. Inject intraveneously that amount of the cultivation corresponding to
1 per cent. of the body-weight of the selected animal, usually one of
the small rodents.

4. Observe during life or until the completion of twenty-eight days, and
in the event of death occurring during that period, make a complete
post-mortem examination.

5. Repeat the experiment at least once. In the event of a positive
result estimate the minimal lethal dose of "killed" culture for each of
the species of animals experimented upon.

(b) Extracellular or Soluble Toxins:

1. Filter the cultivation through a porcelain filter candle (Berkefeld)
into a sterile filter flask, arranging the apparatus as in the
accompanying figure (Fig. 160).

2. Inoculate mice, rats, guinea-pigs, and rabbits subcutaneously with
that quantity of toxin corresponding to 1 per cent. of the body-weight
of each respectively, and observe, if necessary, until the completion of
one month.

3. Inoculate a "control" tube of bouillon with a similar quantity and
incubate, to determine the freedom of the