bacteria develop only in the deeper layers of the culturemedium. These colonies may be made accessible for subcultures either by breaking the tube or by removing the overlying portions of the culture-media by means of a stout platinum wire, previously sterilized in a flame. For taking out colonies for transplantation, a capillary glass tube, sterilized in a flame, may be found useful in place of the platinum wire. In inoculating the tube, care should be taken to secure a good distribution of the bacteria through the medium by manipulating the platinum wire.

FIG. 23-Liborius's method of
making anaerobic cultures.

FIG. 24.-Buchner's method of
making anaerobic cultures.

This method will be found very practical for obtaining pure cultures from mixed growths if dilutions (see page 111) be made. In making dilutions it is well to use a tube of bouillon or sterilized water for the first tube, thus economizing medium, for the first tube will usually have so many colonies that no colonies suitable for sub-cultures will be available.

The microscopical appearances of the colonies may be studied by placing thin slices of the medium, containing the colonies, on a slide. These slices may be easily ob

tained with the aid of a stout platinum wire with a flattened end, more or less bent.

Anaerobic bacteria grow readily in "deep stab" cultures. In these cultures the medium should fill the tube to almost half its height at least. After inoculation some melted medium may be poured in so as to fill the tube to an additional height of some centimeters, but this is not necessary.

Simple Anaërobic Plate-cultures.-These are prepared like the ordinary Petri plate-cultures (see page 111) except that the melted culture-medium is poured into the upturned larger dish, or cover, of the pair, while the smaller dish is then placed, bottom surface downward, in the melted culturemedium, and allowed to settle by its own weight into the fluid medium. The dishes are not disturbed until the medium has hardened. Sufficient medium should be used to fill the space between the sides of the dishes. This quantity will be about 10 c.c. By slightly inclining the smaller dish in placing it in the melted medium, air-spaces can be easily avoided.

By this method the colonies develop in a thin layer of culture-medium enclosed between glass surfaces. The method gives a good chance to study the microscopical characters of the colonies. Surface colonies are, of course, not obtained by this method. The colonies are easily made accessible for transplantation by separating the dishes from one another. The layer of culture-medium will adhere to one dish or the other.

In order to avoid contamination, the dishes should be arranged in the manner above described during their sterilization previous to using.

Buchner's Method. This method consists in cultivating bacteria in an atmosphere from which the oxygen has been absorbed by a mixture of alkali and pyrogallic acid. Tubecultures, or cultures in Petri dishes, may be used. They should be placed in some form of a glass chamber, which is closed air-tight, along with the necessary quantity of alkali and pyrogallic acid mixture. In preparing the apparatus, the pyrogallic acid (in powder) is placed first in the chamber along with the culture tubes or plates, then the necessary

quantity of a solution of potassium hydroxid (1 : 10) is run in, and the chamber quickly closed. For single tube-cultures a large test-tube provided with a tightly fitting rubber stopper, which is sealed in position with wax, may be used for the air-tight chamber (see Fig. 24). The culture-tube is to be elevated above the surface of the reducing mixture by means of a bent wire.

If a number of tube-cultures or Petri plate-cultures are desired, the glass chambers known as Novy's jars are very satisfactory to use. The joints of this apparatus should be well smeared with vaselin. To avoid breakage the testtube containing the inoculated culture-medium may be held in a beaker, with some cotton at the bottom, while in the apparatus. Petri plate-cultures may be placed one above another in the jar, the bottom plate being supported above the level of the reducing fluid by some sort of wire frame.

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.

Hans Zinsser's Method for Anaerobic Plate-cultures.The method is described by Dr. Zinsser as follows:

"The apparatus used consists of two circular glass dishes, fitting one into the other, as do the halves of a Petri dish, and similar to these in every respect except that they are higher, and that a slightly greater space is left between their sides. when they are placed together. The dishes should be about to 1 inch in height; they need be of no particular diameter, although those of about the same size as the usual Petri dishes are most convenient. The sole requirement necessary for successful plating is that the trough left between the two plates when put together shall not be too broad, a quarter of an inch being most favorable.

"Into the smaller of these plates the inoculated agar is poured, exactly as is done into a Petri dish in the ordinary

aerobic work.

Prolonged boiling of the agar before plating is not essential. When the agar-film has become sufficiently hard on the bottom of the smaller dish, the entire apparatus is inverted. The smaller dish is now lifted out of the larger, and placed, still inverted, over a moist surface-a towel or the wet surface of the table-to prevent contamination. Into the bottom of the larger dish, which now stands open, there is placed a quantity (1 to 2 drachms) of dry pyrogallic acid. Into this, over the pyrogallic acid, the smaller dish, still inverted, is then placed. A strong solution of sodium hydrate is poured into the space left between the sides of the two dishes, in quantity sufficient to fill the receiving one half full. While this is gradually dissolving the pyrogallic acid (and this is the only step which requires speed), albolene, or any other oil, is dropped from a pipette, previously filled and placed in readiness, into the same space, thus completely sealing the chamber formed by the two dishes.

"If these steps have been performed successfully, the pyrogallic solution will at this time appear of a light brown color, and the smaller plate, with its agar-film, will float unsteadily above the other. Very rapidly, as the pyrogallic acid absorbs the free oxygen in the chamber, this plate is drawn down close to the other, and the acid assumes a darker hue, which remains without further deepening even after three or four days' incubation."

Wright's Method. The method depends upon the absorption of oxygen by an alkaline solution of pyrogallic acid, as in the well-known method of Buchner. It is applicable to culture in test-tubes and in flasks. The details of the method are as follows:

After the culture-medium in the test-tube has been inoculated, the cotton stopper is thrust sufficiently far down into the test-tube so that the upper end of the cotton stopper lies about 15 mm. below the mouth of the test-tube. It is usually desirable to cut off a part of the protruding portion of the cotton before doing this. Now fill the space in the tube above the cotton stopper with dry pyrogallic acid. Next pour quickly onto this pyrogallic acid enough of a strong watery solution of sodium hydrate to dissolve it all;

avoid pouring on an excess; for a test-tube of an inch in diameter about 2 c.c. will be an ample quantity. Then, as quickly as possible, insert firmly a rubber stopper in the mouth of the tube so as to close it tightly. The culture is then ready to be set aside for development.

The cotton of the stopper should be of a kind that will readily absorb fluids.

The solution of sodium hydrate consists of one part of sodium hydrate in sticks and two parts of water.

It may be thought that there is danger of contaminating the culture-medium from the alkaline pyrogallic acid mixture running down the sides of the tube. This does not occur, because the mass of the cotton stopper is sufficiently large to absorb completely the quantity of fluid in it, with a good margin to spare.

This simple method has given satisfactory cultures of the tetanus bacillus obtained from cases of tetanus in the Massachusetts General Hospital and of other obligate anaërobic bacteria. It can be applied to all forms of test-tube cultures, both in solid and fluid media, including Esmarch roll-cultures. In applying the method to Esmarch roll-cultures the mixture of pyrogallic acid and alkali should be placed in the cotton, and the rubber stopper inserted before the tube is rolled on the ice. Glucose-agar readily lends itself to Esmarch rollcultures if the tubes are kept in a slanting position during growth.

The accompanying photograph shows the appearance of a bouillon tube prepared according to this method (Fig. 25).