As has already been stated (p. 21), there are certain requirements known as Koch's laws, or postulates, that must be fulfilled in order that we can prove a certain organism to be the cause of a definite pathologic condition. These require, among other things, that the organism be grown outside of the body-on artificial culture-media; these to be composed of such substances in such proportion as will enable the bacteria to live and multiply. Inasmuch as the different bacteria vary greatly in their metabolic activities, it becomes necessary to have various kinds of media; there are, however, certain forms that are employed routinely. Such media should contain at least 80 per cent, of water, should be of neutral or feebly alkaline reaction, and of a composition which, for the pathogenic types at least, should closely approximate the juices of the animal body. Such nutritive materials may be either liquid or solid, and some of the most useful may be liquefied and solidified at will.


This medium is used by itself and also as the nutritive basis of certain solid media. It may be made up with lean beef or with 3 gm. of beef extract. If the former is used it must be freed from fat and gristle and finely minced; 500 gm. of it are mixed with 1000 c.c. of water and allowed to stand on ice for twelve hours. At the end of this time the liquor is poured off, that remaining in the meat squeezed through a cloth, and enough water added to bring the amount up to 1000 c.c. It is then filtered, and to the clear filtrate is added 10 gm. of Witte's peptone, 5 gm. of sodium chlorid, and enough water to bring the quantity up to 1000 c.c. This mixture is boiled until everything is dissolved, and it is then neutralized, as its reaction is very acid.

The neutralization should be very carefully carried out so that the final reaction is slightly alkaline. This is done by carefully adding a 10 per cent, solution of caustic soda and testing with litmus-paper. During this process the solution is kept boiling. After the materials are all dissolved and the solution titrated, it should be allowed to cool before filtering. If filtered while hot there will be a subsequent precipitation of meat-salts, which will cloud it.

Glucose bouillon is similar to the above, except that it contains 1 per cent, glucose in addition.


To 1000 c.c. of beef bouillon 15 gm. of agar-agar are added and boiled for one hour, constantly stirring. Water is added at various intervals to keep up the required volume. After the boiling is done the contents are allowed to cool to 6o° C, at which point an egg is beaten into the fluid, which is again boiled for about ten minutes. Then filter while hot through wet filter-paper. A jacketed filter kept warm by a gas flame facilitates the process. As the fluid cools while filtering, it has to be again heated until all passes through. The finished agar should be a colorless, nearly transparent, firm jelly.

The purpose of the agar-agar is to give a medium that will remain solid at a temperature equal to that of the body, which is the best for many bacteria, the agar itself not contributing any nourishment to the medium. The agar will melt at about 420 C, but will again solidify when cooled.


To 1000 c.c. of boiling beef bouillon add 100 gm. of golden seal French gelatin. When the gelatin is thoroughly dissolved boil for about five minutes and neutralize by the method described for bouillon. The mixture is cooled to 6o° C, an egg beaten in, boiled about ten minutes, and filtered through wet filter-paper. Sufficient water should be added to bring the quantity up to the original amount. It may have to be reheated a couple of times before filtration is complete. As the gelatin solution is strongly acid in reaction, it must be corrected carefully by titration. Care must be taken not to bring the mixture to the boiling temperature more frequently than is necessary, as the power of coagulation may be destroyed.

This medium melts at temperatures above 220 C.

Glucose gelatin is gelatin that has been dissolved in glucose bouillon.

Blood-Serum (Loffler's Mixture)

The blood-serum is obtained by collecting it at a slaughter-house. Jars holding about 1 gallon should be used. These should be clean and sterile. The collected blood is put aside in a cool place for twenty-four to forty-eight hours until the blood is completely clotted. If the clot adheres to the side of the jar, loosen it with a glass rod. The clear serum is removed with a pipet. This is then mixed with glucose bouillon.

Glucose bouillon (1 per cent.)................ 1 part.

Beef blood-serum............................ 3 parts.

The above is then run into test-tubes to a depth of about 4 cm. These are placed on an incline so that they will be on a slant when coagulated. In this position they are placed in a hot-air sterilizer and kept at a temperature between 850 and 900 C. for one hour. The thermostat should be carefully watched so as not to have the heat vary from the above figures. After the medium has become thoroughly coagulated the tubes are sterilized in steam for one-half hour on three successive days. When blood-serum tubes are not available a good substitute in the form of a hard-boiled egg may be used. Remove the egg from the water, and with a sterile instrument remove a part of the shell, leaving the coagulated albumen exposed. Inoculate this surface with the suspected material, cover with a glass, and then place near a stove or in a "Thermos" bottle, and allow to incubate for eighteen to twenty-four hours. The blood-serum is particularly useful in the cultivation of the Bacillus diphtheriae, which grows upon it rapidly and with a characteristic appearance.

Litmus Milk

To milk that has been freed from cream enough of a freshly prepared aqueous solution of litmus is added to give it a blue color. This is run into test-tubes which are treated by intermittent steam sterilization. Fresh milk should be used and the process quickly carried out to prevent as much as possible the growth of bacteria. This medium is the best for determining the formation of acids or alkalis by bacteria.

Potato Cultures

The potatoes should be thoroughly scrubbed with brush and water. Solid cylinders of a size to fit the test-tubes are cut with a cork-borer. They are then split obliquely and the pieces placed in running water for some twelve hours. The oblique pieces are then placed in test-tubes with the larger end downward. A few drops of water should be added to prevent drying. The tubes are then put through the fractional steam sterilization.

Dunham's Peptone Solution

Peptone................................. 10 gm.

Sodium chlorid........................... 5 "

Distilled water........................... 1000 c.c.

The peptone and sodium chlorid are dissolved by boiling and the mixture filtered. Test-tubes are filled and sterilized. This solution is commonly employed for the detection of indol.

Filling Of Test-Tubes

New test-tubes are best cleaned by washing in a very weak solution of nitric acid, then rinsing in water, and allowing to become dry or nearly so. Old tubes that have contained cultures are boiled for nearly one hour in a 6 per cent, solution of common soda.

The cleaned tubes are plugged with raw cotton and placed in the hot-air sterilizer at 1500 C. until the cotton has turned brownish. This is to mold the stopper to the shape of the tube.

To fill the tubes it is best to take a large funnel and by means of a short piece of rubber connect it to a piece of glass tubing a couple of inches in length. The supply of the medium is controlled by a pinch-cock on the rubber. The glass tube is inserted into the test-tube, the required amount of medium run in, and the cotton plug put back. Care should be taken not to get any of the culture-medium on the neck of the tube, as the cotton would stick to it. If "slant" cultures are to be made, run in about 5 c.c. of fluid; if "stab" cultures, about 8 to 10 c.c. should be used. The filled tubes are then sterilized. After the final sterilization, if "slant" cultures are to be made, the test-tubes are so placed that the medium will come about half-way up the side of the tube.

Instead of test-tubes, flasks of varying sizes may be used to contain the medium.

Sterilization Of Culture Media

This may be done by the intermittent method. In this the media are exposed to steam on three successive days for a period of thirty to forty-five minutes. A single sterilization will kill all bacteria except those that are in the spore stage. These bodies will-however, develop within the twenty-four hours into the adult form, and are then killed by the subsequent sterilization.

Instead of the above the autoclave may be used. It is a metal chamber so arranged as to allow sterilization under pressure. A temperature of no° C. is obtained, and in it all bacteria and spores are destroyed in twenty to thirty minutes.

After the final sterilization if "slant" cultures are to be made the test-tubes are so placed that the medium will come about half-way up the side of the tube.

When the media have solidified the tubes can be kept a longer time if the cotton is trimmed off and rubber caps put on.

Sterilization Of Apparatus

Metal bodies that will not be injured, platinum wires, forceps, etc., may be placed directly in the flame of a Bunsen burner. Glassware is sterilized by hot air, by steam, or by boiling. Chemical sterilization is not often employed.