Fractional Sterilization of Tyndall. Granted that so many spores originally exist in the object to be sterilized, it is subjected to 60° C. for four hours, in which time a part at least of those spores have developed into bacteria, and the bacteria destroyed by the further application of the heat. The next day more bacteria will have formed, and four hours' subjection to 60° heat will destroy them, and so at the end of a week, using four hours' application each day, all the spores originally present will have germinated and the bacteria destroyed,

Cotton Plugs or Corks. All the glass vessels (test-tubes, flasks, etc.) must be closed with cotton plugs, the cotton being easily sterilized and preventing the entrance of germs.

Tin-foil may be used to cover the cotton, or caps made of india-rubber.

Test-tubes. New test-tubes are washed with hydrochloric acid and water to neutralize the alkalinity often present in fresh glass. They are then well washed and rubbed with a brush, placed obliquely to drain, and when dry corked with

FIG. 18.

FIG. 19.

Wire-Cage.

Cotton plugged Test-Tubes.

cotton plugs. Then put in the hot-air oven (little wire-cages being used to contain them) for fifteen minutes, after which they are ready to be filled with the nutrient media. (The cotton should fit firmly in the tube and extend a short space beyond it.) Test-tubes without flaring edges are more desirable since the edges can easily be drawn out so as to seal the tube.

Instead of test-tubes, ordinary 3 oz. panel medicine bottles can be used for retaining the nutrient media and cultures.

According to late investigations, the glass tubes become sufficiently sterile in the steam-chest without the preliminary sterilization in the dry oven.

CHAPTER VIII.

NUTRIENT MEDIA.

Of the many different media recommended and used since bacteriology became a science, we can only describe the more important ones now in use. Each investigator changes them according to his taste.

FLUID MEDIA.

Bouillon (according to Löffler). A cooked infusion of beef made slightly alkaline with soda carbonate: 500 grammes of finely-chopped raw lean beef are placed in a wide-mouthed jar and covered with 1 litre of water; this is left standing twelve hours with occasional shaking. It is then strained through cheese cloth, the white meat remaining being pressed until one litre of the blood-red meat-water has been obtained. The meatwater must now be cooked, but before doing this, in order to prevent all the albumen from coagulating, 10 parts of peptone powder and 5 parts of common salt are added to every 1000 parts meat-water. It is next placed in the steam-chest or water-bath for three-quarters of an hour.

Neutralization. The majority of bacteria grow best on a neutral or slightly alkaline soil, and the bouillon, as well as other media, must be carefully neutralized with a sat. sol. of carbonate of soda. Since too much alkalinity is nearly as bad as none at all, the soda must be added drop by drop until red litmus paper commences to turn blue. The bouillon is then cocked another hour, and filtered when cold. The liquid thus obtained must be clearly alkaline, and not clouded by further cooking. If cloudiness occur, the white of an egg and further boiling will clear the same. To make bouillon, beef-extract can be used instead of fresh meat, 2 grammes to 1 litre of water. This is boiled with 5 grammes of salt and 10 of peptone, neutralized as above, and filtered when cold.

Schultz's Method of Neutralization.-A more accurate method of obtaining the required reaction is to use an alcoholic solution (per cent.) of phenolphthalein as an indicator; a few drops of

this are mixed with 10 c.c. of the bouillon, and from a burette a solution of caustic soda 0.4 per cent.is added drop by drop until a faint red color appears. An average is taken from three different samples, and the amount of soda needed for the entire quantity of bouillon is calculated therefrom.

Sterilization of the Bouillon. Erlenmeyer flasks (little conical glass bottles) or test-tubes plugged and properly sterilized are filled one-third full with the bouillon, and placed with their contents in the steam-chest. A tin pail with perforated bottom makes a good container in which they can be lowered in the Koch's oven. They are left in steam of 100° C. one hour for three successive days, after which the tubes and bouillon are ready for use.

Solid Media. The knowledge of bacteria and germs or moulds settling and growing upon slices of potato exposed to the air, led to the use of solid media for the artificial culture of the same. It was also thus learned that each germ tends to form a separate colony and remain isolated.

Potato-Cultures. A ripe potato with a smooth skin is the

best.

Several are brushed and scrubbed with water to get rid of the dirt and the "eyes" are cut out.

FIG. 20.

Moist chamber for potatoes.

Next placed in 1 to 500 solution of bichloride of mercury for hour. Then in the steam-chest for & hour.

In the meantime, a receptable is prepared for them. This is called the moist chamber

The moist chamber consists of two large shallow dishes, one, the larger, as a cover to the other.

These dishes are washed in warm distilled water.

A layer of filter paper moistened with a 15 to 30 drops of 1 to 1000 bichloride is placed in the bottom of the glass dish.

The operator now prepares his own hands, rolling up his coat sleeves and carefully washing his hands, then taking a potato from the steam-oven and holding it between his thumb and index finger in the short axis, he divides the potato in its long axis with a knife that has been passed through the flame. The two halves are kept in contact until they are lowered into the moist chamber, when they of their own weight fall aside, the cut surface uppermost. They are then ready for inoculation.

FIG. 21.

Method of slicing potato. (After Woodhead and Hare.)

Esmarch's Cubes. The potato is first well cleaned and peeled. It is then cut in cubes inch in size.

These are placed, each in a little glass dish or tray and then in steam-chest for hour, after which they are ready for inoculation (the dishes first having been sterilized in hot-air oven).

Test-tube Potatoes. Cones are cut out of the peeled potato and placed in test-tubes, which can then be plugged and easily preserved.