1. Slant.

2. Stab.

3. Petri dish.

4. Esmarch tube.

5. Hanging drop.

6. Anaerobic.

1. Slant Cultures

A platinum wire is taken and heated in the flame. When cool it is inserted into the material to be examined. Then, without touching anything, not even the sides of the tube, the point of the wire is carefully drawn over the surface of the medium and the wire again sterilized. When the cotton plug is- removed, the end of the tube should be passed through the flame. Care should be taken at all times that the platinum wire is carefully sterilized before being laid anywhere.

2. Stab Cultures

Stab Cultures are made by carefully inserting the platinum wire, which should be straight, into the center of the culture-media. The same precautions as mentioned above should be observed.

3. The Petri Dish

The Petri Dish consists of a shallow glass dish with a cover. It is used to a large extent for the purpose of isolating colonies and obtaining pure growths. The tubes inoculated directly from the material examined usually contain several varieties of organisms. The method of isolating is as follows: Three tubes of agar-agar or gelatin are melted and then placed in a water-bath at a temperature between 400 and 42 ° C. A platinum wire with a small loop at the end is inserted into the infected substance and then a tube is inoculated. From this tube a loopful is carried over to tube No. 2, and a third tube is inoculated from the second, the platinum wire being sterilized each time. Three sterile Petri dishes are taken and a tube is inserted under the cover of one and its contents poured out. This is done with all three, care being taken to have the medium evenly distributed over the bottom of the dish. They are then incubated twenty-four hours at a temperature of 37° C.

The first tube will contain so many organisms that Petri dish No. 1 will be covered with colonies. The second tube, being diluted, will give fewer colonies on dish No. 2, while dish No. 3, obtained by pouring out tube No. 3, will have only a few scattered colonies. From this last dish the individual growths may be removed with a sterilized platinum needle and inoculated into a fresh tube, a pure culture thus being obtained.

4. The Esmarch Tube

The Esmarch Tube is made by taking an inoculated tube of melted agar or gelatin, laying it on a block of ice, and rotating till the medium is distributed in a thin coat on the inside. Care must be taken that the contents do not come in contact with the cotton plug. This method has been practically supplanted by the Petri dishes.

5. Hanging-Drop Cultures

Hanging-Drop Cultures are obtained by taking a slide in which there is a depression and a ring of vaselin is made around it. A sterilized cover-glass is taken, a drop of bouillon placed on it, and this is inoculated with the usual precautions. The cover-glass is inverted over the depression in the slide and pressed down upon the vaselin. This is put in the incubator for twelve to twenty-four hours and then examined.

6. Anaerobic Cultures

Anaerobic Cultures may be obtained in various ways, the essential point being the elimination of free oxygen. A test-tube half full of a medium that will become solid on cooling is boiled, rapidly cooled, and then inoculated by a deep stab. On top of this may be poured melted paraffin, oil, or vaselin to keep out the air. Buchner's method consists in the use of two tubes, a small one to contain the culture, and a larger one to contain a fluid that will absorb the atmospheric oxygen. The solution used consists of pyrogallic acid and sodium hydroxid, about 2 gm. of the former, 20 c.c.. of a 10 per cent, solution of the latter. This is poured into the larger tube, the smaller one placed within it, and it is then tightly corked. A simpler method is one in which a larger tube containing a liquid medium is used. Into this is placed a smaller tube with the closed end uppermost. During sterilization the air will be displaced, and when the liquid cools the smaller tube will be full of the medium. In this the anaerobic organisms will grow. This method is also of value in determining whether there is any gas formation, and may be used in place of Smith's fermentation tubes.

Fig. 126. - The "Hanging Drop" seen from Above and in Profile (McFarland).

The oxygen within the container may be replaced by an atmosphere of hydrogen, and the latter tightly sealed.