stowed away or locked up in the tissue locally for months and very probably for years; that phagocytes may carry tetanus spores from some spore infected locality of the body to a different part of the body where there is dead anaerobic tissue produced by quinine or injuries of various sorts; that stray spore-laden phagocytes getting stranded in dead tissue, germinate there and produce tetanus toxin. Many of the possibilities suggested by Semple should be susceptible of demonstration and it may be well to inquire how far they are borne out by laboratory data. (1) Have tetanus organisms or their toxins been found in the necrotic tissue at the site of a quinine injection in man? (2) To what extent have tetanus spores been found in the feces of human cases of so-called idiopathic tetanus? (3) On account of the high mortality in tetanus, opportunity is offered to examine not only the intestinal contents throughout, but to determine the condition of the mucous membrane as well; have such examinations disclosed injuries or ulcers of the bowels or the presence of tetanus spores in the bowel contents? (4) Can laboratory animals whose gastro-intestinal tracts have been artificially contaminated with tetanus spores be made to contract tetanus by injections of quinine, or can tetanus spores be demonstrated in parts of their bodies other than the lumen of the gut? (5) Have tetanus spores been found in healed wounds of patients who had exhibited no symptoms of tetanus? (6) Have latent tetanus spores been found lying dormant in the healed tetanus wound of a patient after recovery from his attack of tetanus? (7) Has tetanus been produced in animals when the injection of tetanus spores and quinine were separated, not only in place, but by a considerable length of time as well?

On questions 1, 2, 3, 5, and 6, I am unable to submit data, since their elucidation requires the presence of clinical facilities and cases of human tetanus which are not available at this laboratory at the present time.

In regard to question 4, the demonstration of the problem has not yet been attempted in this laboratory.

The hypothesis is made that tetanus in man following an injection of quinine is due to the tetanus spores which have become transported by phagocytes into the site of the quinine injection from a site elsewhere in the body where they had been implanted at some time previous to that of the quinine injection. If this hypothesis be true, one should expect to find it corroborated by a laboratory experiment in which not only the spore site and the quinine site were located in different parts of the body, but in which a considerable interval of time had separated the injections of spores and quinine. The hypothesis does not provide for a simultaneous implantation of spores and injection of quinine; it is assumed that the spores at some time previous gained entrance to the body tissues through external

wounds now healed or through an injured intestinal mucous membrane. In the experiments in animals a separation in respect to time does not seem to have been made between the spore injection and the quinine injection and yet such a separation is assumed to occur in man. Out of Semple's 24 animals in which the site of injection of spores and quinine were the hind leg and the chest respectively, the time of the first quinine injection was one day previous to that of the spore injection in 14 instances, it was the same day as the spore injection in 7, and on the day following the spore injection in 3. The results might have been very different had the injection of quinine been delayed for weeks or months after the spore injection. Such a separation in time of injections is called for by the hypothesis.

HI. MISCELLANEOUS OBSERVATIONS UPON TETANUS.

In the preparation of the data for this bulletin, considerable quantities of media, material, and animals were used; some general observations upon the properties of the tetanus bacillus were made which will now be presented.

INHIBITION OF GERMINATION OF TETANUS SPORES BY CARBOLIC ACID.

Some manufacturers of vaccine virus do not rely upon glycerin alone to reduce the nonspore-bearing organisms but use carbolic acid in addition to the glycerin. The solution used for grinding the vaccine is made as follows:

50 c. c. glycerin.

9 c. c. carbolic acid.

49 c. c. water.

To every gram of the vaccine pulp, 4 c. c. of the carbolized glycerin solution is added. The amount of carbolic acid in each cubic centimeter of the finished virus would therefore be approximately 0.007 c. c. or per cent.

If 1 c. c. of such a virus should contain tetanus spores and should be tested for tetanus by planting the entire cubic centimeter into 6 c. c. of bouillon, the spores if they germinated would be compelled to do so in per cent solution of carbolic acid; if planted into 13 c. c. of bouillon, the strength of the carbolic acid would be reduced to per cent, etc. These percentages of dilution of carbolic acid are based on a thorough mixing of vaccine virus with the entire culture medium. If instead of thoroughly mixing virus and culture medium, the heavy virus is allowed to settle to the bottom of the medium, it is conceivable that the dilution of the carbolic acid might not be greater than per cent. The question arises as to what percentage of carbolic acid will prevent the germination of the tetanus spores.

The attempt was made to germinate tetanus spores in glucose bouillon containing per cent carbolic acid with the result that two out of five tubes thus inoculated showed no growth of tetanus while growth in the other three was delayed 36 and 60 hours. (See Table No. 14.)

TABLE NO. 14.-Inhibiting effect of one-tenth per cent carbolic acid on the germination of tetanus spores.

Ten control tests in which tetanus spores were inoculated into glucose bouillon to which no carbolic acid had been added showed growth of tetanus within 18 hours in all except one, in which growth appeared within 24 hours. Bacillus coli and staphylococcus grew readily in per cent carbolic acid; in 18 hours their nonrestraint would therefore bring about a lengthened period of high acidity preceding the delayed germination of the tetanus spores.

A young culture of tetanus showed growth in glucose bouillon containing 0.1 per cent of carbolic acid.

CULTURE MEDIA FOR TETANUS.

Glucose bouillon, freshly prepared, made from lean beef, not fermented with colon bacilli, containing 1 per cent peptone, and 1 per cent glucose, titrated 1 per cent acid to phenolphthalein, tubed into Smith fermentation tubes and raised to the boiling point just before inoculation in order to expel air, is the medium in most common use in laboratories for testing for tetanus.

Ordinary bouillon prepared in every respect like the above, except that the glucose is omitted, is not generally considered to be the best medium for growing tetanus on account of the nonaddition of glucose. While the presence of glucose in the medium favors the growth of tetanus in pure culture, it hinders the germination of tetanus in mixed culture almost to the degree of absolute prevention provided the contamination consists of many cocci, colon bacilli, Bacillus