the steam sterilizer for thirty minutes on three successive days.

VI. Dextrose, Lactose, and Saccharose Bouillon.

(a) After filtration of the meat-infusion, prepared as above described, place in an Erlenmeyer flask, and inoculate with a fluid culture of the Bacillus coli or an allied gas-producer.

(b) Place in the incubator at 37.5° for twentyfour hours.

mentation-tube.

This removes the meat-sugar.

mentation-tube.

(c) From this infusion prepare bouillon in the ordinary way.

(d) To the completed broth add 1 per cent. of the required sugar.

(e) Distribute in test-tubes or in fermentationtubes,' and sterilize in the steam sterilizer on three successive days.

A small test-tube inverted inside a large one will answer for a fermentation-tube.

VII. Milk and Litmus Milk.

(a) Heat fresh milk for fifteen minutes in the steam sterilizer.

(b) Place in the ice-box over night.

(c) Siphon off the milk, without cream or sedi

(e) If less than 2 per cent. acid to phenolphthalein, place in tubes and sterilize for twenty minutes on four successive days in the steam sterilizer. If more than 2 per cent. acid, adjust to + 1.5 per cent.

by the addition of

n

sodium hydroxid.

(A) A solution of litmus may be added just

rubber

glass

40 ст.

FIG. 34.-Siphon, with one-way valve for starting the flow of serum. The end of the glass tube is turned over to prevent the clot from entering.

Glass and
Asbestos cover

Thermometer.

I

Adjustable legs for
regulating slant.

Water gauge

FIG. 35.-Coagulator for blood-serum tubes. Providence Health Department pattern, with wooden rack for holding tubes away from sides. When in use, the four sides and the space below are covered with asbestos boards.

previous to its distribution in tubes, sufficient to give the milk a pale-blue color.

VIII. Blood-serum (Löffler's).

(a) Receive freshly drawn beef blood in sterile jars.

(6) Allow twenty minutes for coagulation to begin, then with a sterile glass-rod break up any adhesions between the coagulum and the jar.

(c) Allow the jars to stand twenty-four hours in the ice-box.

(d) Siphon off the clear serum.1

(e) To 3 parts of serum add 1 part of 1 per cent. dextrose bouillon.2

(f) Adjust to + 0.8 per cent.

(g) Distribute in tubes and solidify with a slanting surface by heating for three hours in a bloodserum coagulator.

(h) Sterilize in the steam sterilizer for twenty minutes on three successive days.

1 If the serum is not clear, filter through the coagulum left after the filtration of bouillon, as suggested by Hill.

2 If 1.25 per cent. glycerin is added also, it seems to prevent the tubes from drying.

CHAPTER VII.

CULTURES OF BACTERIA.

OWING to their small size and to their similarity of form, the different species of bacteria cannot be recognized by microscopic examination alone. Recourse must be had to a study of their biologic peculiarities. For this purpose it is necessary to grow them on artificial culture-media. If a single germ is planted on a suitable nutrient medium, and is protected from contamination, it multiplies rapidly and forms a colony. Such a colony is composed of but one species, since all its members are the descendants of a single germ. Cultures made from such a colony are known as "pure cultures," since they contain but one species. From a study of the behavior of these pure cultures, under different conditions, the diagnostic characters of the species are determined.

I. BOUILLON CULTURES.

I. Inoculate a tube of bouillon by touching the culture to be studied with a sterile platinum needle, and then dipping the needle in the bouillon.

2. Place at the room-temperature or in the incubator, and examine from day to day, and note any changes as follows: