gelatin. In this way a gelatin stab-culture (Fig. 26, a) is obtained.

Agar, and often also gelatin, is generally solidified so as to yield a slanting surface for inoculation, the point of the needle being passed from below upward upon the surface of the culture-medium (streak-culture). (Fig. 26, b.) Inoculations upon potatoes are made in exactly the same way.

In the preparation of a bouillonculture or a milk-culture the mass of bacteria is rubbed simply on the inner surface of the tube below the level of the bouillon or the milk, the loop used for the inoculation being then thoroughly shaken.

In order to inoculate one testtube from another, to continue the pure culture further, the two tubes are grasped between the fingers (Fig. 20) (the first tube between the thumb and the index-finger, and the second tube between the index-finger and the middle finger), their stoppers are removed (the one being held between the third and fourth, and the other between the fourth and fifth fingers), and from the first tube there is taken, with the platinum needle previously sterilized in the flame, or, in the case of fluid culturemedia, with the platinum loop, a small amount of the culture, which is then conveyed upon or into the new culture-medium. The reinoculation of pure cultures that it is desired to maintain must be repeated at intervals of four weeks.

The development of gelatin-cultures is permitted to take place at room-temperature, or in the thermostat at a temperature of 24° C. (75.2° F.) or 26° C. (78.8° F.). The remaining cultures are, however, usually kept in the thermostat at a temperature of 37° C. (98.6° F.). A thermo

stat or incubator consists of a double-walled copper or sheetiron chamber surrounded with felt or asbestos. The space between the two walls is filled with water, which is kept constantly at an equable temperature by means of a thermo-regulator. The layer of water transmits its heat to the interior of the thermostat, the actual culture-chamber, which constantly should have a temperature of 37° C.

(98.6° F.) for agar, or from 24° C. (75.2° F.) to 26° C. (78.8° F.) for gelatin. The culture-tubes, which are to be kept for a considerable length of time in the thermostat, must be provided with rubber caps sterilized with mercuric chlorid, in order to protect them from evaporation.

Fig. 27.-Method of inocusterile nutrient jelly.

The growth in a number of these cultures constitutes for many bacteria a definite characteristic, which is of the highest significance in their identification. Least typical, as a rule, is the growth upon agar. Attention must be directed to the thickness, the transparency, and the color of lating a test-tube containing the deposit, etc. In bouillon-cultures there may be distinguished bacteria that develop only upon the surface in the form of a thick or a thin membrane, others that render the bouillon more or less homogeneously turbid, and still others that grow only at the bottom as a crumbling or a viscid sediment. Milk is unaltered by many bacteria, while others cause coagulation through the formation of acid. The most important peculiarities are furnished by gelatin-cultures. In the first place the liquefaction that results through the activity of a peptonizing ferment is to be looked for. This occurs in part only superficially, and is in part funnel-shaped or stocking-shaped at a depth. If liquefaction does not take place, varied and often characteristic peculiarities of growth occur (nailculture, tree-like division, etc.).

CULTIVATION OF ANAEROBIC BACTERIA.

Anaerobic bacteria may be facultative or obligate. The former may develop also in the presence of oxygen, although but sparingly. The latter require special cultural methods. The cultivation of strictly anaerobic bacteria is undertaken either in a room free from air, or in an atmosphere of indifferent gas-as, for instance, hydrogen-or with the employment of substances that absorb oxygen, or, finally, by means of stab-culture in a high layer. The usual culture-media are employed, but with the addition of

Fig. 28.-Fränkel's method of making Fig. 29.-Hesse's method of making anaerobic cultures. anaerobic cultures.

two per cent. of grape-sugar, as all of the anaerobic bacteria thus far known form from this substance gas in abundance (carbon dioxid, hydrogen sulphid, methane, mercaptan, etc.), in this way displacing the oxygen of the air.

Plate-cultures.—According to a method devised by R. Koch, a sheet of mica, sterilized by heat, is placed upon the liquid gelatin spread upon the plate. After solidification has taken place the gelatin is thus rendered air-tight, and beneath the mica the anaerobic colonies undergo development. It is more serviceable in the preparation of anaerobic plates to

employ special culture-dishes, which permit the entrance of hydrogen through two openings in the lid that communicate with a gutter-like excavation of the dish. The lid is fastened to the periphery of the dish by means of vaselin, and it is revolved as soon as the vessel is filled with hydrogen, in order that the openings in the lid and the gutter are no longer opposed to one another, and communication with the outside is cut off (Kamen dish). The hydrogen is generated in a Kipp's apparatus (Fig. 30) that is filled with pure zinc and sulphuric acid, and is freed of hydrogen sulphid and of oxygen, by means of two wash-bottles con

taining an alkaline lead-solution and an alkaline pyrogallicacid solution. The employment of Kamen's plates is attended with certain difficulties; especially is it difficult to drive out all of the oxygen.

In the preparation of anaerobic plates in an atmosphere of hydrogen Botkin's apparatus (Fig. 31) is employed. This consists of a large bell-jar, within which, upon a glass stand, plates are exposed free, without a cover. The bell stands upon a lead cross in a large glass dish. Between the margin of the bell and the stand is a space through which passes a U-shaped rubber tube for the conduction of the

hydrogen gas into the upper portion of the bell. closure is effected with the aid of liquid paraffin.

Perfect The dis

placed air escapes at the bottom through a second. rubber tube, which is removed after the apparatus has been completely filled. Beneath the glass bell a vessel containing an alkaline solution of pyrogallic acid is placed for further security. A disadvantage of Botkin's apparatus consists in the fact that the plates are but incompletely protected against contamination by the air.

Novy's apparatus is to be warmly recommended, both for plate-cultures as well as for test-tube cultures. This consists of a high glass jar upon the

edges of which an air-tight helmet-like cover is placed. The latter is provided above with a revolving, doubly perforated glass stopper, through which the hydrogen gas

At

is introduced and the air is at the same time expelled. the close of the operation the stopper is simply rotated, and