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. 18).

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

The pepton and sodium chlorid are dissolved by boiling and the mixture filtered. The clear filtrate is then run into testtubes, 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 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 medium 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. 19). 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

FIG. 19.-Method of filling test-tubes with culture-medium (Warren).

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.

A freshly prepared culture-medium must be sterilized on the same day that it is prepared, or by the next day it may be found to contain living bacteria, especially if kept over night in a warm room.

For the purpose of sterilization the test-tubes containing the media are to be placed in a round wire basket which fits into the steam sterilizer, thus facilitating the handling of the tubes and also keeping them upright.

If the medium be in a flask ready for running into testtubes, and if it be not convenient to do this the same day, the medium may be preserved as long as desired by inserting a cotton stopper into the mouth of the flask and then sterilizing as above indicated.

The time of each sterilization for bouillon, agar-agar, blood-serum, etc. may be fixed at half an hour; for potatoculture tubes and for litmus-milk, forty-five minutes.

In the case of gelatin, however, the time of exposure to live steam should be shorter, owing to the danger of destroying the solidifying power of the medium by too much heating. Twenty minutes' exposure is sufficient.

Large quantities of culture-media contained in flasks should be sterilized for forty-five minutes to an hour, for obvious reasons.

III. BACTERIOLOGICAL EXAMINATIONS AT

AUTOPSIES.

THE aim of the bacteriological examination at an autopsy is to determine whether bacteria are present or not in the tissues, and, if present, to ascertain not only their species, but also the extent and comparative numbers of their distribution throughout the principal internal organs.

This is accomplished chiefly by means of two methods of examination-viz. the direct examination with the microscope of cover-glass preparations, and the results of cultures made from the tissues. Both of these methods should be employed together, but the culture method is perhaps the most important. A third but less frequent method is the inoculation of animals with pieces of tissue or material taken from the body.

Cover-glass Preparations.-The method of demonstrating the presence of bacteria in pathological material by means of cover-glass preparations depends upon the fact that bacteria have the property of being colored by certain of the aniline dyes, and thus may be more readily seen by the microscope. The cover-glasses are best kept in alcohol, and as required for use wiped dry with a soft cotton cloth. A cover-glass preparation is made as follows: A very small amount of tissue or material to be examined is thinly spread over the surface of a clean thin cover-glass, so as to give a streaked appearance to the surface, but not a definite layer, which is ordinarily too thick for satisfactory examination. The charged cover-glass is then dried by holding it in the fingers over the flame of a Bunsen burner, and when dry it is placed, charged surface uppermost, in the grasp of a pair of Cornet forceps, by means of which it is passed rapidly three times through the flame of a Bunsen burner or alcohol lamp. This "fixes" the material on the glass, and the preparation is then ready for staining by one of the various methods given below. In staining, the cover-glass is held by means of Cornet forceps with the charged side uppermost and level, and the surface is then completely covered

with the staining fluid, which is poured upon it from a dropping-bottle. It may then be heated over the flame of the Bunsen burner, washed in water, and submitted to any further manipulation which may be necessary while still in the grasp of the forceps. When the staining is completed the preparation is next to be prepared for microscopic examination. This is done by placing the cover-glass, with as much water as will adhere to it, charged side downward, on a "slide," and then removing all remaining water, except a thin film of water between the slide and cover-glass, by gentle pressure with several thicknesses of filter-paper. The preparation is then ready for examination with an oil-immersion lens. The presence of this film of water is very essential for a satisfactory examination with the microscope, and its evaporation may be compensated for by a drop of water placed at the edge of the cover-glass. The preparation may also be mounted in balsam after carefully drying it first between filter-paper and then holding it in the fingers over the Bunsen flame, but the examination in water mount is better, because the apparent size of the bacteria is greater in this than when mounted in balsam.

It is of the greatest importance that cover-glass preparations be made from all exudates or acute inflammatory conditions of organs or tissues, so that the results of cultures can be controlled and some idea formed of the number and character of the bacteria present. They are of especial use as enabling one sometimes to recognize the presence of an organism which does not grow in the culture for some reason, and whose presence might escape notice, while their importance in many cases in the identification of the pneumococcus and the bacillus tuberculosis must be apparent. In certain cases it is best to examine the material in its natural state and without drying and staining. This is especially true of suspected infections with actinomyces and the amebæ coli. In these cases the material should be spread over a cover-glass, and this placed, while the material is still moist, charged surface downward, on a "slide," and then examined with various powers of the microscope. If neces