neutral or slightly acid gelatin they grow so as to form isolated colonies along the stab, somewhat similar to the growth of the streptococcus of

Bacillus pyogenes soli, showing the great diversity of size and shape ordinarily met with, and also the irregular staining.

erysipelas; but the colonies in the depth are usually more isolated and larger than near the surface. The gelatin is not liquefied, and the

I have gotten great differences in the rapidity of growth in different batches of gelatin, made with no intentional variation in the preparation, and of apparently the same chemical reaction. A quite rapid growth has been recently obtained in distinctly alkaline gelatin.

growth remains confined to a narrow line, the colonies on plates or in rolled tubes remaining very small. They are lemon-yellow and very finely granular under the lower power of the microscope, and are either perfectly round or have a more or less broken or wavy outline. The growth on potatoes and on blood serum is very weak, and presents nothing characteristic. To the naked eye the growth in all cases is white. In hydrogen tubes the growth is very good. I have rarely succeeded in getting a growth in agar.

The effect upon animals differs with the mode of inoculation. In rats, gray mice, rabbits, and usually in white mice, subcutaneous inoculation of small, or even large, amounts, produces an abscess confined strictly to the seat of inoculation. Injections of from to c.c. of liquid cultures into ear veins of rabbits, produce, in some cases, multiple abscesses, especially in the joints and kidneys. Subcutaneous inoculation produced in one case abscesses of the joints in a white mouse.

The abscesses following subcutaneous inoculation form very quickly, within twenty-four hours, and run a longer or shorter course, from forty-eight hours to eight or ten days, in direct proportion to the amount of the culture introduced. The animals do not seem to suffer any inconvenience, as a rule, and after the abscess is opened suppuration ceases.

The organism is found aggregated in small and large irregular clumps in the pus, many of them lying in the pus-corpuscles. It seems to form metastatic abscesses only under exceptional circumstances, such as when injected directly into the blood. Otherwise the abscess remains strictly confined to the seat of inoculation in rabbits, white rats, and gray mice. My investigations of tetanus, which led to the finding of the above organism, are not yet complete, but I have obtained the organism in pure cultures from different specimens of earth and also from a case of human tetanus; this is perhaps worth a preliminary report.

Dr. Theo. B. Burnett, who was working with me in 1889, succeeded, by Kitasato's method, in isolating from garden earth a bacillus which I could in no way distinguish from the tetanus bacillus in cultures or under the microscope; but which did not produce the disease upon inoculation in rats and mice. Dr. A. T. Bristow, also working under my direction, succeeded in isolating an organism which behaved in the same way. These two gentlemen were unfortunately prevented from continuing their work. They used solid cultures for the most part, and I think the reason we were unable to produce the disease with these cultures is that we did not inoculate sufficient amounts.

The method employed in all cases was that recommended by Kitasato, viz.: An animal (I have used exclusively white rats, and gray and white mice, for this purpose) was inoculated at the root of the tail with several pinches of the forceps of garden earth, and as soon as tetanus developed the wound was opened, and some of the secretion was

smeared upon an oblique agar tube. This tube was placed in the incubator for forty-eight hours. It was then placed in a water-bath at 80° C., for from twenty minutes to three-quarters of an hour. This heated tube was used to inoculate a tube of melted agar, from which the customary dilutions were made as in making plates. The contents of each of the tubes so inoculated were poured into a modified Liborius's appaatus,' shown in the cut (Fig. 3), through a sterilized funnel. The small

FIG. 3.

B

tube B of the apparatus was attached to a hydrogen generator, and hydrogen bubbled through for ten or fifteen minutes, until all the oxygen was driven out. Without stopping the flow of hydrogen the larger tube was quickly sealed off at a, and then the smaller tube at b. These sealed tubes were then put in the incubator, and as soon as colonies appeared the tubes were carefully cut open, and deep stab cultures were made from isolated colonies. I prefer to make deep stab cultures as follows: An ordinary tube of agar or gelatin is inoculated in the usual way, and the melted contents of another tube poured in on top. All contamination is usually avoided by heating the mouth of the tube from which the medium is poured, and by having the medium as hot as possible. In this way I usually obtain pure cultures.

As will be seen from the photograph (Fig. 4), the organism I have obtained does not differ under the microscope from the organism described by Kitasato, and it also agrees with my recollection of the organism in preparations shown me by Nicolaier, who first described

1 Dr. Paul Liborius: "Beiträge zur Kentniss des Sauerstoffbedürfnisses der Bakterien " Zeitschrift für Hygiene, Bd. i. p. 115, 1886.

2 Dr. S. Kitasato: Ibid.

the organism. The behavior in cultures tallies with Kitasato's description. But ordinary inoculations, as usually practised upon animals, I find often unsuccessful-for the reason, I think, that in this way not enough of the material is introduced. I am very often unsuccessful with solid cultures, and when I have succeeded it has been only where large lumps of the culture were introduced. Inoculations from liquid cultures are more satisfactory. In agar cultures, the liquid which separates out of agar, when the latter solidifies, and which sooner or later becomes filled with tetanus bacilli, often acts very promptly, even in small quantities. The age of the culture does not, invariably at least, increase the virulence; for some of the most promptly acting cultures are less than twenty-four hours old. My experiments were confined to white rats, white and gray mice, and rabbits.

I have only once found the organism at the seat of inoculation, or elsewhere, where pure cultures were used, after the death of the animal, and that was deep in the tissues at the seat of inoculation in a mouse that died forty-eight hours after inoculation.

I have had two very singular results, which are worth special notice, as I find no exactly similar cases noted in the literature. One was in a rabbit inoculated with a pure bouillon culture, cultivated in the incu

bator in hydrogen. Three days after inoculation the leg nearest the seat of inoculation became stiff and somewhat extended. A few days later all the symptoms of tetanus set in. The slightest disturbance brought on spasms. The animal was kept in a quiet, dark place, but even the careful introduction of food into the cage brought on violent spasms. This condition lasted two or three days, and then rather suddenly the symptoms abated, except the stiffness in the leg. Except this, the animal was apparently well, and remained so three weeks, when it was found one morning in a spasm; all the other symptoms returned also, and the animal died. The culture with which this animal was inoculated afterward killed a mouse very promptly.

I also have a similar case in a gray mouse which was inoculated with the cloudy water in an agar culture. Two days after inoculation the left leg was perfectly stiff, and the tail was bent over to the left side. The toes of the affected leg were all stretched out and rigid; the animal had no control of the leg or tail. There was also decided pleurothotonos to the left side. These symptoms have now disappeared, and the mouse is at this time alive-ten weeks after inoculation. The subsidence of the symptoms in this case was more gradual than in the case of the rabbit. The culture in this case also killed a rat promptly. Through the courtesy of Professor J. M. Van Cott and Dr. William E. Butler I was able to obtain cultures of the tetanus bacillus from a case of tetanus in a human being. The case was one of a boy who had fallen from a tree and broken his wrist. It was a compound fracture with earth ground into it. Inoculations from the wound and spinal cord produced tetanus in white mice. Cultures were obtained from the mice inoculated from the wound. The cultures do not differ from those obtained by inoculating rats or mice with garden earth.

I am indebted to Dr. C. N. Hoagland for the photograph used in Fig. 4, which he kindly made from one of my preparations. Dr. A. T. Bristow rendered me valuable aid with the photograph used in Fig. 1.