erably old ones-are first washed to remove all the coarser particles of soil, and then solid cylinders are cut out of them with a cork-borer or apple-corer. These cylinders should be of a suitable diameter to fit into the testtubes used for other culture-media, and should be about 5 cm. long. They are then cut longitudinally in an oblique direction with a sharp knife, so that a smooth slanting surface is produced, beginning near one end and extending diagonally to the other end. The pieces of potato thus prepared are next to be washed in running water over night. After washing, each piece is placed in a testtube, the larger end resting on the bottom of the tube, a few drops of water being added to prevent drying, and then sterilized as indicated on page 87. If desired, a small piece of glass rod may be placed in the bottom of the tube to elevate the potato above the few drops of water (Fig. 13).

Dunham's Pepton Solution. - Formula for 1000 c.c.:

FIG. 13.-Potatoculture.

The pepton and sodium chlorid are dissolved by boiling and the mixture filtered. The clear filtrate is then run into test-tubes, each test-tube being filled to a depth of 5 cm., and is to be sterilized as indicated on page 87, after which it is ready for use.

The Adjustment of the Reaction of Culture-media by Titration. Because comparatively small variations in the reaction of culture-media may have a marked effect upon the morphology and mode of growth of bacteria grown upon them, a more exact adjustment of their reaction than is possible with litmus paper is desirable. This is especially

cultivation of the bacteria For ordinary purposes of

important for media used for the of water, of soil, and of the air. cultivation of bacteria, especially of the pathogenic forms, the adjustment of the reaction with litmus paper, as elsewhere described, if carefully done, will be found to be sufficient.

The more exact method of adjusting a reaction is one of titration with phenolphthalein as an indicator. The method is as follows: When the culture-medium, whether it be bouillon, agar-agar, or gelatin, has been neutralized with the aid of litmus paper, made up to the proper volume, and when it is all ready for filtering, as described elsewhere, 5 c.c. of it are transferred by means of a pipette to a 6-inch porcelain evaporating dish; to this 45 c.c. of distilled water are added, and the 50 c.c. of fluid are boiled for three minutes over a flame to expel any carbon dioxid which may be present.

Next, I c.c. of a 0.5 per cent. solution of phenolphthalein in 50 per cent. alcohol is added to the mixture in the dish, and immediately after this enough of a twentieth normal solution of sodium hydroxid is cautiously run into the dish, from a burette, to produce a pink color in the mixture. The judgment of the proper color which indicates that sufficient alkali has been run in requires some practice. The color to be obtained is a bright pink. The appearance of the proper pink color is preceded by a pinkish darkening of the fluid which may deceive the inexperienced.

The quantity of the twentieth normal sodium hydroxid solution required to effect this result is then read off from the burette. The number of cubic centimeters required denotes the percentage by volume of a normal solution of sodium hydroxid which would be required to make the total volume of culture-medium neutral to phenolphthalein. That this is so will be apparent after a simple calculation.

The reaction recommended by the Bacteriological Committee of the American Public Health Association as a standard to which culture-media should be adjusted is such that 1.5 per cent. of a normal solution of sodium hydroxid would be required to be added to the medium to make it

neutral to phenolphthalein. This reaction corresponds closely to a faint alkalinity toward litmus, for the neutral point of phenolphthalein is not identical with that of litmus. The adjustment of the reaction to this standard is effected by adding to the bulk of the culture-medium sufficient normal sodium hydroxid solution or normal hydrochloric acid solution.

For example: If the titration shows that 5 c.c. of the medium requires 1.9 c.c. of the twentieth normal solution of sodium hydroxid to make it neutral to phenolphthalein, then the total mass of the medium will require the addition of 1.9 per cent., or 19 c.c. for a liter, of a normal solution of sodium hydroxid to make it neutral; but the reaction required is such that 1.5 per cent. of a normal solution of sodium hydroxid should be required to make it neutral. Therefore, 0.4 per cent, or 4 c.c. for a liter, of a normal solution of sodium hydroxid should be added to the main mass of the medium.

When the calculated amount of normal solution has been thoroughly mixed with the medium and the latter boiled for a few minutes, the titration should be repeated as above described. If the desired reaction is not found to be present, then further adjustment by addition of the calculated amount of normal acid or alkali solution should be made. It is not to be expected that the first addition to the medium of the calculated amount of normal solution will give exactly the required reaction in every case. This is due to unknown side reactions which take place in the culture-media.

When the reaction has been sufficiently adjusted, the medium is to be filtered and is then ready to be distributed. in test-tubes.

The methods of making the normal and twentieth normal solutions required may be found in standard works on chemistry. If one has not some knowledge of chemistry, he would better have the solutions made by a chemist.

The filling of the test-tubes with the fluid culturemedia described in this section is best effected by means of a funnel of a capacity of about a liter. In this the fluid me

dium is placed, and by means of a pinch-cock the requisite quantity of medium is run into each test-tube. In running the medium into the test-tubes the left hand holds the testtube while the right hand removes the cotton stopper and manipulates the pinch-cock (Fig. 14). Care should be exercised not to allow any of the medium to come in contact with the neck of the test-tube, for it will make the cotton stopper

stick to the walls of the tube. To avoid this, the deliverytube of the apparatus should be inserted some distance into the test-tube in filling.

The quantity of culture-medium run into each test-tube varies according to the form of culture desired and the character of the medium. In the case of liquid media and solid media designed to be used in the form of "stab" cultures the

tubes should be filled to a depth of 5 cm. For "slant" cultures of solid media a depth of about 3 cm. is sufficient, or enough to give a slanting surface from the bottom of the tube to about halfway up the opposite side.

Small Ehrlenmeyer flasks are sometimes used for bouillon cultures. These are of about 100 c.c. capacity, and are filled to a depth of about 1 cm. with the medium. The necks are provided with cotton stoppers, and the whole sterilized and treated as test-tube cultures.

STERILIZATION OF CULTURE-MEDIA.

In general, the sterilization of culture-media is effected by allowing them to remain exposed to the action of live steam in the steam sterilizer for twenty to forty-five minutes on three successive days. The period of exposure to live steam varies somewhat with the kind of culture-medium. A single exposure for the time mentioned is sufficient to destroy all bacteria present in what is called the vegetative or non-resistant form, but it will not kill spores, which represent a stage in the life-history of certain bacteria, in which form the organism is highly resistant to sterilizing agents.

Under favorable conditions, such as are to be found in culture-media at ordinary room-temperature, these spores develop into the vegetative or non-resistant form, which are easily destroyed by heat. Therefore, in order that the culture-medium be made sterile, it is necessary that it be again subjected to the action of steam on the following day for the same length of time, when the vegetative forms of the few surviving spores will have developed, and will be capable of destruction by ordinary exposure to live steam.

As a further precaution a third similar sterilization on the next day is necessary. Therefore, three steam sterilizations, of from twenty minutes to one hour each, on successive days, are required to keep culture-media sterile for an indefinite period.1

1 As has been pointed out by Theobald Smith, this intermittent sterilization at 100° C. may not be sufficient in some cases to kill all the spores, because the condition in the media may not be favorable for their development into vegetative forms, between sterilizations. This seems to be especially true