longer; the egg will produce a clearer solution and save much trouble. A small portion, while hot, is now filtered into a test-tube and tested for alkalinity, and then re-heated several times, watching if a cloudy ppt. forms.

If the fluid remains clear upon cooling, the remainder of the material can be filtered. It must be accomplished while hot, else the gelatine will coagulate and prevent further filtration.

This can be carried on either by keeping hot the solution continually in waterbath, and only filtering a small quantity at a time through the filter, or keeping the filter itself hot, either with a hot water filter or placing the filter in steam chest. (Fig. 24.)

Clouding of Gelatine. If the gelatine does not come out clear, or becomes turbid on cooling, it may be due to several things1. The filter-paper too thin or impure.

2. Too strongly alkaline.

3. Cooked too long or not long enough.

The addition of the white of an egg, as before mentioned, will often clear it up; if this avails not, re-filtering several times, and attention to the few points mentioned.

Sterilizing the Gelatine. The gelatine is kept in little flasks or poured at once into sterile test-tubes, careful not to wet the neck where the cotton enters, lest when cool the cotton plug stick to the tube.

The tubes are then placed in steam-chest for three successive

days, 15 minutes each day (or in water-bath 1 hour a day for three days). Then set aside in a temperature of 15° to 20° C., and if no germs develop and the gelatine remains clear, it can be used for cultivation purposes.

Modifications. The amount of gelatine added to the meatwater can be variously altered, and instead of making gelatine bouillon milk, blood, serum, urine, and agar can be added. Glycerine (4 to 6%) is a common addition, and sometimes reducing agents to absorb the oxygen are mixed with it.

Agar-Agar. This agent, which is of vegetable origin, derived from sea-plants gathered on the coasts of India and Japan, has many of the properties of gelatine, retaining its solidity at a much higher temperature; it becomes liquid at 90° C. and congeals again at 45° C. Gelatine will liquefy at 35° C.

It is not affected very much by the peptonizing action of the bacteria.

Preparation of Agar-Agar Bouillon or Nutrient Agar. The ordinary bouillon is first made, and then the agar cut in small pieces, added to the bouillon (15 grammes of agar to 1000 grammes bouillon. It is allowed to stand several minutes until the agar swells, and then placed in water-bath or steam-chest for six hours or more. It is then neutralized, very little of the alkali being sufficient.

A white of an egg added, and boiled for several hours longer, when, even if not perfectly clear, it is filtered.

The filtering process, very difficult because of the readiness with which the agar solidifies, must be done in steam-chest or with hot-water filter, and very small quantities passed through at a time, changing the filter-paper often.

Cotton can be used instead of filter-paper, or filtering entirely dispensed with, simply decanting.

As agar is seldom clear, a little more or less opaqueness will not harm. The test-tubes are filled as with the gelatine, and sterilized in the same manner. While cooling, some of the tubes can be placed in a slanting position, so as to obtain a larger surface to work upon.

Water of condensation will usually separate and settle at the

bottom, or a little white sediment remain encysted in the centre; this cannot easily be avoided, nor does it form any serious obstacle.

The crude agar should first be rinsed in water, and then in 5% acetic acid and clear water again, to rid it of impurities.. If agar is boiled thoroughly over a hot flame or in an autoclave, it can be filtered much more readily. The main point is to see that all the agar is dissolved.

It has been suggested to pour the hot agar into high cylindrical glass vessels and allow it to cool slowly in the steam oven, the flame having been gradually lowered and then turned out. After a time the cloudy portion will form a sediment at the bottom; the agar can then be shaken out as a long cylinder and the cloudy portion cut off.

The Japanese Method.-Yokote prepares agar as follows: the meat is cooked in water over a sand bath 14 hours. Filtered, chopped agar is then added and the mixture cooked 1 hour longer; peptone and salt added next. Neutralization. After the mixture has cooled to about 50° C. whites of 2 eggs are added and the mixture shaken thoroughly.

Again the mixture is placed on the sand-bath and heated to 110° C. and over for 1 to 2 hours, and then filtered through ordinary filter-paper. Yokote claims that by this procedure the agar can be filtered as easily as bouillon and without any loss. (The water evaporated in boiling must be added before filtering.)

Glycerine Agar. The addition of 4 to 6% of glycerine to nutrient agar greatly enhances its value as a culture medium. Gelatine-Agar. A mixture of 5 per cent. gelatine and 0.75 per cent. agar combines in it some of the virtues of both agents.

Blood Serum. Blood serum being rich in albumen coagulates very easily at 70° C., and if this temperature is not exceeded, a transparent, solid substance is obtained upon which the majority of bacteria develop, and some with preference.

Preparation of Nutrient Blood Serum. If the slaughter of the animal can be supervised, it were best to have the site of the wound and the knife sterilized, and sterile flasks to receive the blood directly as it flows.

It is placed on ice forty-eight hours, and the serum is drawn

out with sterile pipettes into test-tubes; these are placed obliquely in an oven where [the temperature can be controlled and maintained at a certain degree. See Fig. 26.

FIG. 25.

1

Flask to receive blood serum.

Incubators or Brood-ovens. Incubators or brood-oyens consist essentially of a doublewalled zinc or copper chest, the space between the walls being filled with water.

The oven is covered with some impermeable material to prevent the action of the surrounding atmosphere. (Fig. 27.) It is supplied with a thermometer and a regulator. The regulator is connected with a Bunsen burner, and keeps the temperature at a certain height.

There are several forms of regulators in use, and new ones are invented continually. The size of the flame in some is regulated by the expansion of mercury, which, as it rises, lessens the opening of the gas supply. The mercury contracting on cooling allows more gas to enter again. (Fig. 28.)

Koch has invented a safety burner, by which the gas supply is shut off should the flame accidentally have gone out.

Coagulation of Blood Serum. The tubes of blood serum having been placed in the oven, are kept at a temperature of 65° to 68° C., until coagulation occurs; then removed and sterilized.

Sterilization of Blood Serum. The tubes are placed 3 to 4 days in incubation at 58° C., and those tubes which show any evidences of organic growth are discarded.

If now, at the end of a week, the serum remains sterile at the ordinary temperature of the room, it can be used for experimental purposes.

Perfectly prepared blood serum is transparent, of a gelatinelike consistence, and straw-color. It will not liquefy by heat, though bacteria can digest it. Water of condensation always forms, which prevents the drying of the serum. Blood serum

may be prepared in a shorter way by coagulating the serum at a temperature short of boiling-point. Sterilization is completed in three days by exposing the tubes to a temperature of about 90° C. cach day for five minutes. Tubes so prepared are opaque and white.

Preservation of Blood Serum in Liquid State. Kirchner advises the use of chloroform. To a quantity of serum in a well-stoppered flask a small amount of chloroform is added-enough to form about a 2 mm. layer on the bottom. If the chloroform is not allowed to evaporate, the serum remains sterile for a long time. When needed for use test-tubes are filled and placed in a water-bath at 50° C. until all chloroform has been driven off (determined by absence of characteristic odor); the serum is then solidified and sterilized as in the ordinary way.