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 egg beaten into it, then gently heated again to boiling, and boiled about ten minutes, when it is to be filtered through a wet folded filter into a flask. Gelatin usually filters fairly rapidly, but time may be saved by using two filters at once. When filtered it is to be run into test-tubes and sterilized, as described on page 87. It is used both in the form of "slant" and "stab" cultures, as in the case of agar-agar (see page 76).

In the preparation of this medium it is important to subject it as little as possible to the boiling temperature, for prolonged exposure to this destroys its power of solidifying. Therefore in sterilizing, gelatin tubes should never be allowed to remain exposed to live steam longer than twenty minutes. It is also important to apply the heat slowly during the process of heating after the addition of the egg above mentioned, in order to avoid "burning."

This medium is essentially gelatin dissolved in glucose bouillon (see page 73), and is prepared in the same manner as the plain gelatin, except that glucose bouillon is used instead of plain bouillon.

Blood-serum (Löffler's Mixture).-Formula:

Glucose bouillon (see page 73),
Beef blood-serum,

I part;

3 parts.

This culture-medium consists of a mixture of the bloodserum of the bullock and glucose bouillon, which is run into test-tubes and coagulated by heat in such a way as to form a slanting surface for culture purposes-i. e. it is used in the form of "slants."

The blood-serum is collected at the slaughter-house in tall glass jars of the capacity of a gallon or more. These jars should be thoroughly clean, but sterilization is not necessary.

The blood which is obtained by the Jewish method of slaughter-viz. by severing the carotid artery—is the best for the purpose, because it clots more readily. As the blood runs from the vessels of the animal it is received in the glass jar, and immediately placed in a cool place for twenty-four to forty-eight hours to allow it to clot and the serum to separate. All unnecessary agitation of the fresh blood should be avoided, as this interferes with its proper clotting. It is well to inspect the blood after a few hours, and gently loosen with a clean glass rod any adhesions which the clot may have formed to the wall of the jar, thus allowing the clot to more readily contract and squeeze out the serum

from its meshes. After about twenty-four hours the serum is removed by the aid of a clean pipette and brought to the laboratory. If the clot is in good condition, more serum will appear after another twenty-four hours, and if necessary this also may be used.

The presence of red blood-corpuscles in the serum is of little importance. Three parts of the beef blood-serum thus obtained are to be thoroughly mixed with one part of glucose bouillon (vide supra), convenient quantities being 900 c.c. of blood-serum and 300 c.c. of glucose bouillon.

This mixture is then run into test-tubes as described on page 85. The quantity run into each test-tube should be sufficient to fill it to a depth of about 3-4 cm. The tubes containing the requisite amount of the mixture are next subjected to the action of heat while in a slanting position, so that the mixture in the tubes may become solid or coagulated, and so offer a smooth slanting surface for culture purposes extending from a point near the bottom of the tube to about halfway up the opposite side or higher.

The coagulation is effected either in the hot-air sterilizer by packing the tubes on their sides, the proper slant being secured by means of strips of cardboard placed between the layers of tubes, or better, in the blood-serum coagulator which may be obtained from dealers in bacteriological apparatus.

If the hot-air sterilizer is employed, the temperature should not exceed 90° C. nor fall below 85° C., and the door should be kept closed. It is optional whether the sterilizer be packed full of tubes or only a few layers of tubes be coagulated at a time, with careful watching to avoid overheating. In the former case two or three hours will be required to firmly coagulate the tubes in the middle layers, while the lower layers may be overheated. To avoid this overheating of the lower layers, a false bottom or one or two layers of empty tubes may be employed.

The blood-serum coagulator is much more convenient and gives much more satisfactory results. The temperature

of the interior should be kept at about 95° C. To save time in heating, the apparatus may be filled with hot water from the hot-water faucet.

Whichever apparatus is employed for coagulation, it is of the utmost importance that the coagulation of the mixture be a thorough one, and that the medium in the tubes becomes firm and solid, otherwise bubbles and cavities will form in it and destroy its smooth surface when it is subjected to the subsequent steam sterilization. When the tubes are firmly coagulated they are to be packed with the cotton stopper uppermost in a round wire basket and sterilized by steam three times, as indicated on page 87, after which they are ready for use.

This method of preparing blood-serum tubes is very different from the one usually described, a most tedious. and time-consuming procedure, requiring a high degree of technical skill, by which it is practically impossible to make use of blood-serum tubes for ordinary purposes.

With the method here detailed we think that the best culture-medium for the routine examination of pathological material is obtained. It is preferred by us for various reasons, chief of which are as follows:

First, the ease and facility with which it can be prepared, especially when a proper coagulating apparatus is available.

Secondly, the greater and more rapid growth of certain important pathogenic bacteria upon it than upon ordinary media.

In the method usually described the serum (which should be clear or free from blood-corpuscles) is obtained under all aseptic precautions, is carefully mixed with sterile glucose bouillon in the proportions given above, and the mixture then run into sterile test-tubes. During all the manipulations precautions are necessary to avoid contamination, the serum never being allowed to come in contact with any object which is not sterile, and exposure to the air during the processes of transference from one vessel to another avoided as much as possible.

The mixture now being in test-tubes, it is subjected for one hour on each of five successive days to a temperature of 68° to 70° C. in a chamber provided with a water-jacket. This tem

perature is sufficient to kill the vegetative forms of any bacteria which may be in it, but does not coagulate the medium. The intervals between the sterilizations are for the purpose of allowing any spores to develop into the vegetative form and thus become susceptible to the destructive action of heat.

After the fifth sterilization the medium is solidified in the tubes in the form of "slants" by slowly raising the temperature of the chamber to about 80° C., and keeping the tubes at this temperature for several hours. In solidifying the great object is to obtain a gelatin-like, fairly transparent medium and to prevent opacity. To attain this it is necessary to proceed very carefully with the heating and avoid overheating or too rapid heating, the tubes being inspected from time to time and removed from the chamber as soon as their contents have the proper consistency. When gelatinized the tubes are placed in the incubator for twenty-four hours to determine whether they are sterile, after which they are ready for use.

The blood-serum medium produced by this older method is especially suited for the cultivation of certain pathogenic bacteria -for instance, the bacillus tuberculosis and the bacillus diphtheriæ -but we do not think that its superiority in this respect over the more readily prepared, firmly coagulated form above described is · sufficiently marked to compensate for the great difficulties in its preparation.

Litmus-milk is a form of culture-medium used for determining certain of the physiological properties of bacteria. It consists of cow's milk which has been colored blue by litmus and containing a minimum amount of cream. A pint or so of strictly fresh milk is placed in a flask and steamed in the steam sterilizer for about half an hour. When it is removed it will be found that most of the cream has collected at the surface, and it is then easy to draw off the milk from the deeper layers with a pipette into a separate flask. To the milk from which most of the cream has been thus removed is added sufficient of an aqueous solution of litmus (freshly filtered) to give it a pale-blue color. The colored milk is then run into test-tubes (5 cm. deep in each tube) and sterilized, as indicated on page 87, after which it is ready for use. It is of great importance that the milk be fresh. If it is not, it may contain spore-bearing bacilli which it is practically impossible to kill by the steam sterilization.

Potato-cultures according to Bolton.-Potatoes-pref