CASE LXXIV. Dercum, July 27, 1889.-Male, age forty-nine. Cause of death meningitis. Heart weight 11 ounces. Valvular disease, mitral. Hypertrophy slight. Atheroma, root of aorta. Contraction of kidney, slight, cyanotic.

CASE LXXV. Dercum, August 1, 1889.-Female, age fifty-three. Cause of death cerebral apoplexy. Heart normal size. Valvular disease, mitral, slight. Atheroma, aorta, coronaries, and cerebral.

A STUDY OF THE BACILLUS SUBTILIS.

BY J. LEFFINGWELL HATCH, B.S., M.D.

Having had some doubts as to whether or not a non-specific bacterium could set up a septicemia, not necessarily per se, but on account of materies pecans that it might carry, I determined to investigate the subject carefully. It is also interesting to know whether such bacteria could set up an inflammatory process from the mechanical action on the cells, subsequent to their lodgment in the tissues.

It will at once be seen how difficult it would be to determine whether or not, a bacillus was the bearer of any other toxic agent. If a septicemia results from a pure culture, it is rather evidence of the fact that it was due to the bacillus itself; if, however, we take a number of zoöglia and filter out the bacteria, then we have a substance which is the product of the bacteria, and any postinoculatory results would be due to this substance and not directly to the bacteria.

Again, this substance may be the normal secretion of the bacteria, or it may be abnormal or pathological. How are we to determine this? Chemical analysis or spectroscopic analysis are the only agents that can aid us here, and of these we shall have to eliminate chemical analysis on account of the minute quantity of the bacterian product. The second idea, as to whether they can produce some toxic result by setting up an inflammatory process mechanically, by the presence in the tissues, is easier of demonstration; and it is this latter course which I have been contented to follow before I take up the more complex and intri

cate one.

The following experiments were made upon rabbits with pure cultures of the bacillus subtilis, and in order that the nicety of

the technique may not be questioned, I submit my methods in detail, in connection with the results of the inoculations:

The bacillus subtilis (hay bacillus) is considered by Klein (Micro-organisms and Disease) as a non-pathogenic bacillus. It is a widely distributed bacillus, found in nearly all decomposing nitrogenous substances; it is a rod bacillus, reproducing both by spores and fission. The rods are from 0.002 to 0.006 mm. in length, and are about 0.002 mm. thick. They divide best at a temperature of 35° C., requiring about twenty minutes. After division they hang together, forming a large zoöglia. This, in the culture, gives us the characteristic dense pellicle of the bacillus, which rests on the surface of the gelatin, and if shaken to the bottom is immediately replaced by another. One peculiarity of this bacillus is that the spore formation will go on regardless of nutrition, increasing in numbers as rapidly when poorly nourished as when well fed. These spores do not stain in ordinary dyes, which is a feature peculiar to this bacillus. The best source for cultivation is from hay.

Method of cultivation.-To obtain the bacillus subtilis from hay it is necessary to make an infusion. I here give Buchner and Roberts' method for bacillus subtilis, quoted by Dr. Dolly in his "Technology of Bacteria investigation." Digest a quantity of finely chopped hay for an hour, with as little water as possible, at a temperature of 36° C. The liquid is then drained off through a wire sieve and diluted with distilled water until it reaches a specific gravity of 1.004. If the fluid is acid in reaction it must now be neutralized with bicarbonate of soda, and not less than 500 c. c. placed in a flask, which is stoppered with a plug of cotton. The flask and contents are now retained for forty-eight hours at a temperature of 36° C., when the developed bacilli will have formed a dry looking pellicle upon the surface. From this pellicle inoculate a number of tubes of nutrient gelatin, in which media they thrive luxuriantly. By repeated inoculations and attenuation a pure culture is obtained.

If still further precautions as to its purity seem requisite, make several plate cultures from the purest tube culture of the lot. Where zoöglia can be seen microscopically, examine each plate under a microscope, and with a sterilized platinum needle pick out an isolated colony and inoculate another tube of gelatin with

it. Make six or more inoculations so that a choice of cultures may be had, and in this way a very pure culture can be obtained.

My own cultures.-The way in which I prepared my inoculating fluid and the number of cultures it entailed I submit below:

On October 7, 1889, I made an infusion of hay after the method of Buchner and Roberts. From the pellicle which grew upon the surface I subsequently inoculated six test tubes of nutrient gelatin. From those inoculations I obtained dense growths, which, however, were impure. On the 13th I inoculated another series of twelve tubes from the best of those previously inoculated. From this inoculation I obtained a much purer growth. On the 18th I inoculated twenty-four tubes of nutrient gelatin, proceeding in a progressive manner, that is, I inoculated No. 1 from the best of the series of twelve tubes; No. 2 from No. 1, then No. 3 from No. 2, No. 4 from No. 3, No. 5 from No. 4, and so on, until the entire twenty-four were inoculated. From these I obtained excellent growths.

Wishing to obtain as pure a culture as possible I made plate cultures from them. In this way I was enabled to get isolated colonies on the plates, and found the best results from those tubes of the greatest attenuation, e. g.: a No. 24 tube inoculated from a No. 23, which came from a 22, etc., was the purest, and the colonies were isolated to a greater extent, rendering further inoculation more exact. I then studied these plate cultures under a low power, and picked out in this manner, with a sterilized needle, single colonies, with which I inoculated six tubes of nutrient gelatin, using a single colony for every inoculation. The cultures ensuing were exceedingly pure, and it was with these that I made my experiments upon rabbits.

Apparatus necessary for inoculation.-As I pursued the method recommended by Dr. Sternberg, I will describe the bulbs which bear his name. They are made by sealing one end of a piece of heavy glass tubing, heating in a blast flame until the glass becomes soft, and then blowing through the tube from the other end, a bulb about one inch in diameter is formed. The tube is then heated about one and a half inches from the bulb and drawn out into a capillary point, one or two inches in length. Besides these bulbs, some watch crystals, a pair of forceps, and a spearpointed lance are necessary.

Modus operandi of inoculation.—The bulb is to be heated slightly over the flame, and the extremity of the neck, after breaking off the sealed point, is plunged beneath the surface of the liquid. As the air cools the liquid is drawn into the bulb, usually filling it to about one-third of its capacity. The neck of the flask is again sealed up, and the liquid which has been introduced is sterilized by repeatedly boiling the flasks in the water bath. They should then be placed in the incubator for two or three days, and if the contents remain transparent and free from film, they may be set aside as stock bulbs, to be used when required.

To inoculate the liquid in the bulb, the end of the neck is heated to sterilize the exterior, the bulb is gently warmed, and the extremity of the neck nipped off with a pair of sterilized forceps. The open extremity is plunged into the liquid containing the micro-organism, a minute quantity enters the tube and mingles with the fluid in the bulb, without fear of contamination by atmospheric germs.

The extremity of the neck is once more sealed up in the flame of a bunsen burner. A separate bulb for each inoculation is necessary, and the point must be passed through a flame before breaking off and introducing into the body of an animal. The fur or hair is to be cut from the part which is to be the seat of injection; then a spear-pointed lancet is to be passed through the flame of a bunsen burner and thrust into the prepared spot, clear into the connective tissue. The point of the prepared bulb is now passed through the flame, the point rapidly broken off with a pair of sterilized forceps and introduced along the side of the lancet. Heat is now applied to the bulb and the fluid is injected into the cellular tissue with considerable force.

Inoculations.-On the 16th of October, 1889, I inoculated six rabbits in the manner just described with my purest culture of bacillus subtilis. These rabbits, in an isolated room, received the best of care and were well fed with appropriate food. On December 4, 1889, I-inoculated eight other rabbits with bacillus subtilis. On December 21, 1889, I inoculated eighteen more with bacillus subtilis.

The results of these different inoculations can be seen from the accompanying table of experiments.