time, while the mixture is kept boiling, the reaction being tested between each addition of alkali after thorough stirring with a glass rod.

The test of the reaction is best made by placing a drop of the mixture on a piece of litmus-paper by means of the glass rod and then moistening the paper at the water-faucet. In this way the best judgment can be formed of changes in the color of the paper. If the mixture becomes too alkaline, dilute hydrochloric acid is to be added to correct this.

When the proper reaction has been obtained the mixture is to be filtered through filter-paper into a flask, and sufficient water added to bring the volume of the filtrate up to 1000 c.c., thus replacing the loss by evaporation. The filtrate in the flask is now bouillon. If the bouillon be heated to the boiling-point, it will usually become more or less. clouded by a precipitate of phosphates. As a rule, subsequent heatings do not cause any further precipitations. Therefore it is advisable, if the bouillon is designed for use as bouillon cultures, to steam the flask containing the freshly prepared bouillon in the steam sterilizer for about half an hour, and then, if the bouillon be clouded, to again filter, so that the subsequent sterilizations in the test-tubes will not cause precipitates.

For bouillon cultures the bouillon is run into test-tubes, each tube being filled to a depth of about 4 cm., and sterilized immediately and on the two following days, according to the general directions given on page 87, after which it is ready for use.

Bouillon may also be made as above indicated by using three grams of Liebig's extract of beef to the liter, instead of the beef-infusion.

Glucose Bouillon.-Formula:

This medium is identical with the preceding, except that it contains 10 grams of glucose to the liter (I per cent.) in addition to the other ingredients. The preparation of glucose bouillon is the same as that of plain bouillon, the glucose being added with the pepton and sodium chlorid. Agar-agar (plain).—Formula for 1000 c.c.:

Agar-agar is essentially bouillon in which agar-agar has been dissolved so that a transparent jelly is formed. The function of the agar-agar is merely to give the medium the property of becoming liquid when heated and solid when cool; it is not nutritive. The nutritive substances are in the bouillon.

To make one liter, 15 grams of agar-agar are placed in a liter of bouillon and boiled for one hour in a saucepan. The agar-agar dissolves slowly, and continuous boiling is necessary to ensure its subsequent filtration.. Before boiling about. 200 c.c. of water should be added to compensate for evaporation, and later, as the level of the liquid falls, more water should be added from time to time. It is well to have some mark on the side of the saucepan which will indicate the level of a liter.

After the boiling is completed the saucepan is to be placed in cold water until the temperature of its contents falls to about 60° C., as shown by the thermometer, the cooling being facilitated by stirring with a glass rod. When this temperature is reached, an egg is beaten into the mass and the saucepan with its contents replaced on the stove, where it is slowly brought to boiling and boiled for about ten minutes. The object of the adding of the egg is to clarify the medium. It is then filtered, boiling hot, through wet folded

filter-paper into a flask. A funnel with corrugations on its sides is best to use. With this the folding of the filter-paper is not necessary.

In order to save time, it is best to use two filters and two flasks at once, for the filtration rapidly becomes slow as the mass cools, and several heatings of the residue on the filter

a

"

'slant" culture (b).

are necessary. As soon as the filtrate begins to appear slowly, drop by drop, the mass remaining on the filter should be turned back into the saucepan-which can best be done by making a hole in the bottom of the filter with the glass rod-and brought again to boiling. While boiling hot it is again poured on a fresh filter. This preparation of fresh filters and reheating may have to be repeated several times before all of the mixture is filtered. The filtration may also be carried on in the steam sterilizer to prevent the cooling of the medium.

When the amount of coagulated egg-albumin and medium remaining on the filter does not exceed a volume of 50-100 c.c., the filtration may be considered complete. To the filtrate, which is now agaragar, is next added sufficient water to make up the loss by evaporation, and the medium is then to be run

into test-tubes and sterilizd, as described on page 86.

Precipitates of phosphates in the medium frequently occur after the first sterilization, but if these be removed they do not usually appear again in subsequent heatings. Therefore, if it be desirable to obtain a very clear agar-agar, it is well to place the flask containing the freshly-prepared medium in the steam sterilizer for half an hour, and then filter again to

remove any precipitate which may have appeared. The subsequent sterilization in the test-tubes will then cause no precipitation.

In filling the test-tubes it is customary to fill some tubes to a depth of about 3 cm. and others to a depth of about 5 cm. After the complete sterilization of the medium in the tubes as described on page 87, the first-mentioned tubes are placed on their sides with their mouths slightly elevated while the medium is still fluid, so as to form, after solidification, a slanting surface extending from near the bottom of one side of the tube to about half the length of the tube on the opposite side. The solidification of the agar-agar takes place in a short time, and as soon as it occurs the tubes are ready for use, this form of culture being known as a "slant" tube or culture (Fig. 17, b). It is well, however, to allow the tubes to remain in their slanting position for a day or two to permit the medium to become more or less adherent to the walls of the tube, and thus avoid its tendency to slide downward when the tubes are placed in the upright position.

The tubes filled to a depth of 5 cm. are to be allowed to cool and solidfy while in an upright position, and the form of culture-tube thus obtained is called a "stab" culture (Fig. 17, a), because the medium in the tube is inoculated for culture purposes by inserting an infected platinum wire into its depths.

Glucose Agar-agar.-Formula for 1000 c.c.:

This medium differs from plain agar-agar only in the addition of 10 grams (1 per cent.) of glucose. The glucose should be obtained in the form of solid masses, not as a thick fluid, and it is to be added with the pepton and sodium

chlorid. In short, glucose agar-agar is made with glucose bouillon in identically the same manner that plain agar-agar is made with plain bouillon.

Lactose-litmus agar-agar consists of plain agar-agar to which has been added 2 or 3 per cent. of lactose and sufficient litmus tincture to give it a pale-blue color.

Glycerin Agar-agar.-Formula for 1000 c.c.:

This medium is prepared by adding to plain agar-agar after its final filtration, and before running it into the test-tubes, 60 c.c. (6 per cent.) of glycerin c. p., and mixing thoroughly. Gelatin (plain).-Formula for 1000 c.c.:

Gelatin is essentially bouillon in which gelatin has been dissolved, so that a transparent jelly is produced which is solid at ordinary temperatures and fluid when slightly warmed. To prepare one liter, 100 grams (10 per cent.) of golden seal French gelatin are dissolved in a liter of the hot bouillon which has been heated to boiling in a saucepan. When the gelatin is thoroughly dissolved the mixture is boiled for about five minutes, and the marked acidity of the gelatin then carefully neutralized by the addition of caustic soda, in 10 per cent. solution, to a very faint alkalinity, as has been described in the preparation of bouillon. As in the case of agar-agar, the mass is then cooled to 60° C., an