der, and then with small scissors the hair is cut away over a space, approximately 1 cm. square, about the root of the tail. In the center of this a small opening is made through the skin 3 or 4 mm. long with small scissors, and through the opening the points of the scissors are passed anteriorly beneath the skin for a distance of about 1 cm., so as to make a "pocket" or cavity by separating the skin from the muscles. Into the cavity thus formed the material for inoculation is then to be introduced by means of the platinum wire. As a rule, white mice are to be preferred to the wild brown

variety, on account of the greater ease with which they may be handled.

Mice may also be inoculated in the peritoneal cavity by introducing a very few drops of a suspension or a bouillon culture of an organism with a hypodermic syringe.

The quantity of bacteria used for purposes of inoculation varies with the organism and with the end in view. In general, it may be said that in inoculating with the growth from a solid medium with the platinum wire one or two loopsful are used. If bouillon cultures are employed, the quantity. injected varies from c.c. to I c.c. in most cases.

In cases where a "suspension" of the growth on a solid medium is injected the same quantities are used as in the case of bouillon cultures, the density of the suspension depending upon the operator. A "suspension" may be con

veniently prepared by pouring 5 or 8 c.c. of sterile bouillon, sterilized water, or 0.6 per cent. sodium chlorid solution (sterilized) into the tube containing the growth upon solid medium, then breaking up the colonies of the growth with the platinum wire, and shaking the tube.

The Care of Animals.-Inoculated guinea-pigs should be kept in boxes or cages so arranged as to permit of cleaning and disinfection. Cages made of a combination of galvanized-iron wire netting and galvanized sheet iron are to be preferred. The bottom of the cage should contain sawdust, and the top may be made to open on hinges. Good dimensions for such cages are 16 inches long, 10 inches wide, and 10 inches high. They may be satisfactorily disinfected, in most instances at least, by washing with boiling

water.

Inoculated mice are well kept in large glass jars with perforated covers. A small amount of raw cotton should be provided for bedding.

The "stock" guinea-pigs and rabbits may be kept together in a pen which should have light and ventilation. Guineapigs breed readily and their young thrive, but this is not usually the case with rabbits. Mice may be kept for use in a woven-wire cage set in a sheet-iron pan, which will permit of the easy removal of excreta. Some raw cotton should be furnished for bedding. The young of white mice are difficult to raise to maturity.

Food.—Rabbits and guinea-pigs eat the same things. In summer-time, grass, green corn-husks, and green vegetables generally are good food for them. In winter, carrots and oats form a satisfactory diet. Fresh water should also be supplied.

Mice may be fed on stale bread soaked in water, oats, birdseed, and occasionally some cheese. Fresh water should be furnished, and, if possible, a little milk sometimes.

4. CULTIVATION WITHOUT OXYGEN (ANAEROBIC CULTURES).

Method of Liborius (Fig. 27).-This consists in cultivating the bacteria in the depths of solid media in test-tubes

filled to a considerable height, so that oxygen cannot penetrate to them through the thick layer of medium.

A test-tube is filled about three-quarters full of sterile glucose gelatin or glucose agar-agar, and its contents boiled for a few minutes to expel the excess of oxygen from the medium. The tube is then immersed in cold water to cool its contents rapidly, and then, before the medium becomes solid, the tube is placed in a water-bath at 38° to 40° C. for a few minutes. When the medium may be assumed to have

FIG. 27.-Liborius's method of
making anaerobic cultures.

FIG. 28.-Buchner's method of
making anaerobic cultures.

reached this temperature, it is inoculated with the material from which a growth is sought to be obtained, and then rapidly solidified in cold water. When solid the end of the tube is sealed up. The colonies of anaerobic bacteria develop only in the deeper layers of the culture-medium. In inoculating the tube care should be taken to secure a good distribution of the bacteria through the medium.

The glucose in the medium, being a reducing agent, favors the growth of the anaerobic bacteria. Formic acid may be used in place of the glucose for the same purpose, in the proportion of 0.3 to 0.5 per cent.

Method of Buchner (Fig. 28).—This method consists in cultivating the bacteria in ordinary “slant" cultures, which are kept in an atmosphere from which the oxygen has been absorbed by pyrogallic acid. The inoculated “slant” tube, provided with a loosely fitting cotton stopper, is placed in a larger tube or cylinder which is hermetically closed, and in the bottom of which is deposited a quantity of a mixture of pyrogallic acid and a solution of potassium hydroxid (6 grams to 1000 c.c.). In preparing this apparatus the pyrogallic acid (in powder) is placed in the bottom of the tube first with the culture-tube in position. Then the solution of potassium hydroxid is run in, the outer tube or cylinder quickly closed, and the stopper sealed with paraffin or some other material.

It is necessary to seal up the apparatus quickly in order to obtain the full benefit of the oxygen-absorbing power of the pyrogallic acid. The quantity of pyrogallic acid employed should be about 1 gram for each 100 c.c. of air-space to be exhausted of oxygen, and for every gram of pyrogallic acid 10 c.c. of the solution of potassium hydroxid should be used. The culture-tube is to be elevated above the surface of the reducing mixture by means of a bent wire. Culture-media containing glucose should be used on account of the reducing properties of the glucose.

The substitution of formic acid for glucose in the proportions of 0.3 to 0.5 per cent. is also recommended, for the same

reason.

Esmarch's Method. This consists in preparing an Esmarch roll-culture (see page 110), of the organism, and, while the layer of glucose gelatin is still cold from the ice, filling the tube with liquified gelatin and solidifying this rapidly in cold water. The end of the tube is then to be sealed airtight. The colonies develop in the layer of gelatin close to the side of tube, and thus can be readily studied with low magnifying power.

Bouillon Cultures under Hydrogen. These are prepared by displacing the air in an ordinary culture-tube or other vessel containing glucose bouillon by hydrogen, and her

metically sealing the tube. The bouillon-tube is closed by a rubber stopper through which two small glass tubes pass, one running to the bottom of the tube, the other extending just inside the rubber stopper.

The tubes outside the stopper are bent at right angles, and then, beyond the bends, are constricted by drawing out while hot, so as to be readily sealed with the Bunsen flame. In the outer ends of the tubes loose plugs of cotton are placed, and the apparatus thus prepared is sterilized by steam for half an hour. After sterilization and cooling the bouillon is infected with the organism, the stopper and its glass tubes being removed and replaced with care to avoid contamination. The stopper is then sealed in position with paraffin or sealing-wax, after which hydrogen from a hydrogen generator is passed through the longer tube leading into the bouillon, and thus displaces the air through the shorter

tube. After the hydrogen has flowed for five minutes the glass tubes are quickly sealed with the Bunsen flame at the constrictions, and the culture thus prepared placed in the incubator. In passing the hydrogen through the apparatus the cotton plugs must of course be kept in position.

The generation of hydrogen is best effected by means of the well-known apparatus of Kipp. It is important that no oxygen be present in the gas, or an explosion may occur during the sealing of the tubes in the Bunsen flame.

Pure granulated zinc and pure sulphuric. acid of 25 to 30 per cent. strength should be used. Before running the gas into the test-tube or other apparatus the reservoir of the generator should be allowed to fill with FIG. 29.-Fränkel's gas, and this then allowed to escape into the air by opening the outlet, care being taken that no flame be near by. This should be repeated, and then a sample of the hydrogen collected in an inverted test-tube by displacement of water in the well-known way. The same should then be ignited, and if no explosion occurs the hydrogen is fit to use.

method of making anaerobic cultures.