By this means it is always possible to prepare a medium of "standard reaction."

N

Litmus Method. The hot medium (broth, agar, or gelatin) is gradually neutralized by dropping in testing the reaction on litmus paper.

1

NaOH from a burette and When the medium is neutral

to litmus 4.5 cc. more of NaOH are added to each litre. This method is much quicker, but does not take into account the NaH2PO4, and is therefore uncertain, especially as many weak organic acids do not react to litmus at all well.

Note. In making up the filtrate to the litre when hot the method adopted is to weigh the medium in a tared flask and add the requisite amount by weight, having weighed the whole at the commencement. (a) Agar (ordinary nutrient).-Powdered agar-agar, 2 gms.; nutrient broth, 100 cc.

The agar powder is mixed to a thin cream with some of the broth and added to the bulk. The mixture is then boiled in the steam sterilizer until all the agar is dissolved, neutralized while hot in the manner described for broth, and cooled to 60° C. The white of two eggs beaten up in distilled water are added, and the material boiled in the steamer until the whole of the flocculent precipitate

has fallen, which generally takes three-quarters to one and a quarter hours.

It is now ready for filtering; the ordinary filter papers are too fine for the purpose, and the ones generally used are the thick white "Chardin" filters. The filtering may be carried out in a hot water funnel (fig. 20), or in the steam sterilizer, and is a somewhat tedious process. When filtered the clear filtrate is passed into test tubes and sterilized by the intermittent method.

To avoid filtering the medium may be boiled, after the egg is added, in tall beakers and allowed to cool therein; the flocculent precipitate falls to the bottom and may be removed with a knife when the mass has solidified. The agar made in this way is not so clear as when filtered.

Some bacteriologists boil their agar in the autoclave, but the great objection to this is that the medium tends to turn brown.

(b) Glycerine Agar.-This medium, first introduced for the culture of the tubercle bacillus by Roux and Yersin, is ordinary nutrient agar to which 10 per cent. of glycerine has been added. The preparation is as (a).

(c) Glucose Formate Agar.-This medium, which is largely used in the cultivation of anäerobic organisms, is ordinary nutrient agar to which 2 per cent. of glucose and 0.5 per cent. of sodium formate have been added. Kitasatio, who introduced the method, did so on purely theoretical grounds, the elaboration of proteid taking place in its primary synthesis by the formation of aldehyde (Hueppe).

(d) Agar Streaked with Blood. This medium is used in the cultivation of the pneumococcus, and gonococcus particularly. Fresh human blood may be used, or that of an animal; in either case great care must be exercised in excluding adventitious bacteria. The method generally adopted is to take a rabbit, wash the ear well with lysol and soft soap and shave off the hair. Again wash with lysol and finally with alcohol. The large vein is then punctured, and the escaping blood removed with sterile pipettes and smeared over the surface of slanted agar tubes. The tubes prepared in this way are incubated for twenty-four to forty-eight hours in the hot incubator at 37-5° C., and if no development of colonies takes place are ready for use.

Various other substances are added to agar for special purposes; the basis in all cases is the ordinary nutrient agar (a). The following are some of the varieties :

(e) Iron Agar.-2 per cent. of saccharate or tartrate of iron. (f) Sugar Agar.-- Maltose and lactose, 5 per cent., &c. (g) Gelatin Agar.-Broth, 100 cc.; agar, 1.5 gm.; gelatin, 7.0 gm. Prepared as agar (a).

(h) Gelatin.-Ordinary nutrient gelatin is a medium largely employed. Its composition is: best French gelatin, 10 gm. ; nutrient broth, 100 cc.

The gelatin is dissolved in the broth by heat, neutralized while hot (as above), cooled to 60° C., the white of an egg added, boiled for half an hour, filtered in a hot water funnel and run into sterile tubes, and sterilized by intermittent method.

The sterilization of gelatin in the autoclave generally results in the peptonisation of the gelatin, in which condition it will not set on cooling; 20 per cent. gelatin is also employed at times.

FIG. 21.-POTATO CUTTER.

FIG. 22.-ROUX'S POTATO TUBE, the lower end arranged to catch condensation water.

(i) Glycerine Gelatin.-Gelatin, 10 gm.; glycerine, 4 ccm.; broth, 100 ccm. Prepare as gelatin.

(j) Glycerine may also be added to ordinary broth in the same proportion (4 per cent.).

(k) Potato.-A good sized potato is well washed with a brush and hot water, peeled, and the eyes removed. With a circular potato cutter (fig. 21) cut out cylinders about four inches long, and wash well with water. Divide the cylinders longitudinally so that each has a broad and narrow end. Drop them into sterile Roux's tubes or tubes with small plugs of wool at the bottom and sterilize in the steamer. If the potatoes are acid wash the cut slices in 2 per cent. caustic soda for an hour before placing in tubes. Glycerinated potato is also used. The slices are soaked in 6 per cent. solution of glycerine in water before they are placed in the tubes.

(1) Potato Gelatin.-Peel several potatoes and remove the eyes, weigh out 1 kilo., cut up in mincing machine, and add 1,000 cc. of water. Allow to stand twenty-four hours. Filter. Add 1 per cent. asparagin and 4 per cent. glycerine, 10 per cent. gelatin, and the white of an egg. Boil up, filter, and run into tubes.

Potato water is made in a similar way, but without gelatin.

(m) Neutral Litmus.-Two ounces of commercial litmus are extracted with rectified spirit for thirty days, changing the spirit three times. At the end of this time the litmus is emptied into a flask, and the spirit allowed to evaporate; 600 cc. of filtered water are next added, the litmus dissolved up and filtered, and acidified with pure sulphuric acid. An excess of barium hydrate is then added, and the solution again filtered. Carbon dioxide is then passed through till all the barium is precipitated as carbonate, the solution filtered and sterilized.

Sufficient of this solution to give a good blue colour is added to the various media when required.

(n) Litmus Milk.-500 cc. of milk are run into a funnel and heated in the sterilizer, and allowed to stand for twenty-four hours till the cream has risen. The milk is then drawn off, tinted by addition of neutral litmus, run into tubes and sterilized in the steamer.

(0) Blood Serum.—The blood of an animal is collected in a large sterilized jar at the slaughter house; the first runnings are allowed to escape to avoid contamination from the skin, &c. The jar is then filled and the blood allowed to clot. The serum is pipetted off with a sterile pipette and run into tubes, which are placed in the inspissator in a slanting position, and sterilized by heating to a temperature of 75° C. for half an hour on four or five successive days. Reject any tubes that show growth when incubated after the last sterilization.

(p) Peptone Water (Durham's).-Water 100 cc.; peptone 1 gm.; salt 0.5 gm. Boil for twenty minutes, filter and run into tubes and sterilize in the usual manner. Instead of 1 per cent. more peptone

Peptone water is a useful medium to use in determining the fermentation of carbohydrates. Various amounts may be added, 2 per cent. being an average quantity. The solution may be also coloured with neutral litmus. Glucose, lactose, maltose, starch, &c., may be used.

(q) Beer Wort Gelatin, and Agar.-Beer wort is used instead of broth in making these media; it is best not to neutralize.

(r) Nitrate Media.-0.5 per cent. of potassium or sodium nitrate may be added to test the reducing power of organisms upon nitrates. Broth, gelatin or agar may be used.

(s) Bread is a good medium for moulds. Dry bread is grated and the crumbs placed in small Erlenmeyer flasks and just covered with distilled water. The flasks are sterilized on four succeeding days in the steamer.

(t) Media with Iron Salts.-0·2 per cent. of iron lactate or saccharate may be added to agar, gelatin or broth, for testing the production of sulphuretted hydrogen.

(u) Inosit Free Broth.—Bacillus coli is grown in the beef extract before addition of peptone, &c. After eighteen hours at 37° C. the flask is placed in the steamer for an hour and the contents subsequently used for making agar, gelatin or broth. The organisms use up the muscle sugar (inosit).

(v) Saliva Media.-Saliva is obtained by placing a sterile Woolfe bottle on the draining tube of a saliva ejector whilst the latter is in use for dental operations. The saliva is boiled, 1 per cent. peptone added, filtered and sterilized in the tubes. Gelatin 10 per cent. or agar 2 per cent. may be added for solid media. The saliva should be sterilized as soon after collection as possible; if not treated in four hours it must be rejected. For some operations the fresh saliva is filtered through a Pasteur-Chamberland filter. (Figs. 11 and 30.)

Methods of Cultivating Bacteria.

Inoculation of Culture Tubes.-The operation of planting a substance containing organisms on to nutrient media for the purpose of obtaining a culture is termed inoculation, and is performed with a platinum wire mounted in a glass, or better, aluminium handle. Two wires are required: (a) a straight wire slightly flattened at the end into a spatula; (b) a wire terminating in a loop-two of these, different sizes, are useful (fig. 23). Before and after use the wire is heated to redness in the flame, before use to burn off any adhering organisms that would contaminate the culture, afterwards to remove the organisms taken from the tube and still remaining attached to the wire.

To inoculate one tube from another containing a cultivation