Gelatin medium may be prepared by adding the proper amount of gelatin (10 to 15 per cent. by weight) broken into small pieces (powdered gelatin in the same proportion may be used) to broth, gently warming until the gelatin is dissolved, standardizing as for broth, filtering and sterilizing. It is usually cleared before filtering by stirring into the gelatin solution, cooled to below 60°, the white of an egg for each 1000 cc., and then thoroughly boiling before filtering. The coagulation of the egg albumen entangles the suspended matter so that the gelatin filters perfectly clear, though with a slight yellowish color. The filtering may be done through filter paper if the gelatin is well boiled and filtered boiling hot, but is more conveniently done through absorbent cotton, wet with boiling water.
Or, the gelatin may be added to meat juice before it is boiled, then this is heated to about body temperature (not too hot, or the proteins will be coagulated too soon) until the gelatin is dissolved. Then the material is standardized and thoroughly boiled and filtered. The proteins of the meat juice coagulate and thus clear the medium without the addition of egg white. Commercial gelatin is markedly acid from the method of manufacture, hence the medium requires careful titration, even when made from a standardized broth.
Gelatin should be sterilized by discontinuous heating at 100° on three successive days, because long boiling or heating above 100° tends to hydrolyze the gelatin into gelatin proteose and peptone and it will not gelatinize on cooling. It may be heated in the autoclave for ten to fifteen minutes at 10 pounds’ pressure and sometimes not be hydrolyzed, but the procedure is uncertain and very resistant spores may not be killed. The medium should be put into the culture tubes in which it is to be used as soon as filtered, and sterilized in these, since, if put into flasks these must be sterilized
, and then when transferred to tubes for use, it must be again sterilized unless great care is taken to have the tubes plugged and sterilized first, and in transferring aseptically to these tubes. These repeated heatings are very apt to decompose the gelatin, so it will not “set” on cooling. The prepared and sterilized tubes of gelatin should be kept in an ice-box or cool room, as they will melt in overheated laboratories in summer or winter.
Agar Medium.—Agar agar, usually called agar, is a complex carbohydrate substance of unknown composition obtained from certain seaweeds along the coast of Japan and Southeastern Asia. It occurs in commerce as thin translucent strips or as a powder. It resembles gelatin only in the property its solutions have of gelatinizing when cooled. Gelatin is an albuminoid closely related to the proteins, agar a carbohydrate. Agar is much less soluble in water, 1 or 1.5 per cent. of agar giving a jelly as dense as 10 to 15 per cent. of gelatin. It dissolves only in water heated to near the boiling-point (98° to 99°) and only after much longer heating. This hot solution “jells,” “sets” or gelatinizes at about 38° and remains solid until again heated to near boiling. Hence bacteria may be grown on agar at the body temperature (37°) and above, and the agar will remain solid, while gelatin media are fluid above about 25°. No pathogenic bacteria and none of the saprophytes liable to be met with in the laboratory are able to “liquefy” agar.
An agar medium is conveniently prepared from broth by adding 1 or 1.5 per cent. of the finely divided agar to the broth and boiling until dissolved, standardizing, clearing, filtering, and sterilizing. The agar must be thoroughly boiled, usually for ten to fifteen minutes, and the water loss made up by the addition of distilled water before titration. Agar is practically neutral so that there is little difference between the titration of the dissolved agar and the original broth. The agar solution should be kept hot from the beginning to the end except the cooling down to below 60°, when the egg white for clearing is added. Though filtration through paper is possible as with gelatin, if the agar solution is thoroughly boiled and filtered boiling hot, it is more satisfactory for beginners to use absorbent cotton wet with boiling water and to pour the hot agar through the same filter if not clear the first time. The solidified agar medium is never perfectly clear, but always more or less opalescent. The agar medium may be sterilized in the autoclave for fifteen minutes at 15 pounds pressure as the high temperature does not injure the agar.
Potato Media.—Potatoes furnish a natural culture medium which is very useful for the study of many bacteria. The simplest, and for most purposes the best, way to use potatoes is in culture tubes as “potato tube cultures” (No. 8, [Fig. 119]). These are prepared as follows: Large tubes are used. Large healthy potatoes are selected. Each end of the potato is sliced off so as to have parallel surfaces. With a cork-borer of a size to fit the tubes used, cylinders about one and one-half inches long are made. Each cylinder is cut diagonally from base to base. This furnishes two pieces each with a circular base and an oval, sloping surface. The pieces are then washed clean and dropped for a minute into boiling water to destroy the oxidizing enzyme on the surface which would otherwise cause a darkening of the potato. (The darkening may also be prevented by keeping the freshly cut potatoes covered with clean water until ready to sterilize.) A bit of cotton one-fourth to one-half inch in depth is put into each of the test-tubes to retain moisture and a piece of potato dropped in, circular base down. The tubes are then plugged with cotton and sterilized in the autoclave at 15 pounds pressure for not less than twenty-five minutes, since potatoes usually harbor very resistant spores, and it is not unusual for a few tubes to spoil even after this thorough heating.
Potatoes are sometimes used in “potato plate cultures.” The term “plate culture” is a relic of the time when flat glass plates were used for this and other “plate cultures.” Now glass dishes of the general form shown in [Fig. 115], called “Petri dishes,” or plates are used for practically all plate culture work. For “potato plates” slices from potatoes are cut as large and as thick as the relative sizes of potato and dish permit ([Fig. 116]). The slices should be thin enough not to touch the lid and thick enough to be firm.
Fig. 115.—Petri dish with the lid partly raised. × ½