the following method is adopted :--Flame, i.e., set fire to the cotton wool plugs of the two tubes to burn off any dust (bacteria) which may have fallen on to the stopper, then blow out the flame. Hold the tubes together in the left hand between the thumb and first two fingers, take up the platinum wire like a pencil and sterilize it in the flame; with the little finger of the right hand remove the cotton

wool plug from one of the two tubes, keeping them inclined at an angle to avoid spores, &c., dropping in; remove the second plug with the third finger, holding the two plugs in the right hand (see fig. 24). Flame the open mouths of the two tubes and then remove a small portion of the culture from one tube and transfer it to the other,

FIG. 24.-METHOD OF INOCULATING ONE TUBE FROM ANOTHER (viewed from above). Note the method of holding the cotton-wool plugs.

carefully avoiding touching the edge or sides of the tubes in the process; replace the plugs, flame the wire, and then the tube plugs again, label and place the inoculated tubes in the incubator. Exactly the same procedure is adopted if the cultivation is made from any material from which we wish to cultivate bacteria; when the material is fluid the loop is used.

Solid media are inoculated in three ways: (a) "streak," (b) "stab," (c) "shake."

Streak Cultures.-The media is first "sloped," i.e., melted, and the tube laid down at an angle, care being taken that the fluid does not touch the plug; several tubes should be sloped at once, and a folded duster placed over them while they cool, to prevent dimming

The apparatus consists of a copper tank filled with water enclosed in a wooden frame. The temperature is maintained by a gas jet, the supply to which is regulated by a capsule with a definite boiling point. The excursions of the capsule are communicated to a valve which automatically regulates the gas supply. Two incubators are required, one working at 37.5° C., the other at 22° C.

by condensation. When sloped the tubes should be kept twentyfour hours before using. With agar it is a good plan to add a little gum arabic or gelatin to the media to prevent the sloped media slipping to the bottom of the tube. To inoculate the sloped surface

draw the platinum needle or loop, charged with the culture, gently up the surface of the medium and replace the plug. The condensation water in agar tubes should not be poured out, as it has a characteristic appearance with certain organisms.

Stab Cultures.-These, like the latter, may be made on agar or gelatin; the tubes are not sloped. A charged needle is passed to the bottom of the medium and withdrawn. This method is largely used in making cultures of anäerobic bacteria and determining the production of liquefaction in gelatin.

Shake Cultures may be used to determine anäerobiosis, production of gas, &c. The solid medium is melted in a water bath, conveniently fitted with a rack perforated to allow of the tubes standing upright; when thoroughly melted the medium is allowed to cool to 40° C., inoculated as described above, and then well shaken by four or five rapid swings (not up and down). When set the tube is placed in the incubator.

FIG. 26. ENLARGED VIEW OF GAS VALVE IN HEARSON'S BIOLOGICAL INCUBATOR. A, Gas supply; B, tambour; C, gas supply to jet; D. weight on lever to regulate size of flame; P, pin communicating with capsule in the incubator; S, sideway tap.

Liquid Media.-The loop or spatula charged with material should not be directly inoculated into the fluid, but the inside of the tube just above the meniscus is touched with the charged wire; a loopful of the medium is then taken up and mixed with the drop of material so that a complete emulsion is formed. On placing the tube upright and giving it one or two swings the material is diffused through the tube.

Plate Cultivations.-Bacteria rarely exist in nature alone and are invariably associated with other species, it is therefore necessary for the bacteriologist to adopt a method of separating the various

species. This is usually done by means of plates The procedure is the same for gelatin and agar.

Three tubes of agar or gelatin are melted in the water bath and cooled to 40° C., and one tube inoculated with the mixed culture and shaken up. Two loopfuls of this tube are now transferred to a second tube and four or five loops of the second into the third. The plug of each tube is removed and the contents poured into a sterile Petri dish, which has been placed upon the levelling tripod, the glass dish of which has been filled with warm water at 40° C. The mouth of the tubes are flamed before pouring out the contents, and the lip of the tube used to assist the medium to flow over the dish. After the plates have set they are incubated. In summer it is often necessary to put ice into the reservoir water, otherwise the gelatin does not set for hours.

The amount of media placed in the first tube determines the number of loopfuls used for the subsequent dilutions, in fact the whole process can only be satisfactorily learned by practice in the laboratory.

In determining the number of bacteria present in a sample of, say, drinking water, a sterile pipette graduated in tenths of a cubic centimetre is used, and 0.5, 0.3 and 0·1 ccm. of the water added to the various tubes, which are then plated.

In the process of plating the point aimed at is to separate the bacteria from one another in such a manner that when they develop in the nutrient substratum the colonies each one forms are sufficiently separated to observe and make sub-cultivations from, or in the water-dilution plates to count the individual colonies, each colony representing one organism in the original sample, the total number of colonies on the three plates representing the number of bacteria present in a cubic centimetre of the water examined. A sub-culture made from one of these colonies will generally be found to be pure, i.e., will consist of one species of organism alone.

Having obtained cultivations from a plate colony the culture is examined by means of the hanging drop and coverslip preparation stained in the various methods given above, and then sub-cultured into the various test media, coverslip preparations of each being made.

The test media to be used should always include the following:-gelatin stab, streak, shake, plates, agar streak, broth, litmus milk, blood serum, potato, media containing carbo-hydrate, and others containing nitrate.

So far I have only described the cultivations of äerobic organisms, the anaerobic bacteria requiring special methods.

Glucose formate media are especially adapted to the growth of anäerobes, but other media may be used.

The oxygen of the air must be excluded by one of the following methods:

(a) Buchner's Tubes (fig. 27).-A large boiling tube fitted with an india-rubber cork is used, and a little pyrogallic acid placed in the

bottom; some caustic soda solution is then added, the culture tube, previously inoculated, gently dropped in and the rubber cork replaced. This method is adapted for fluid or solid media.

(b) An agar or gelatin stab is made in a tube containing about twice as much of the medium as is used for ordinary purposes, and the top of the stab covered by pouring in a little agar or gelatin which has been melted in another tube.

(c) Hydrogen or other indifferent gas (p. 17) is used to drive out