nervously tired. Since his sickness X has been in unusually good health. He has had no jaundice and no abdominal pain. In 1906 X began to keep two cows, and since that time cases of typhoid fever have occurred among his milk customers as follows: This number of cases is larger than the total number reported by the Maynard board of health for the whole town during this period. These cases of typhoid fever have been confined to a residence portion of the town about half a mile square, centering about X's house. The region is in the better portion of the town, well elevated, with good hygienic conditions, and not close to the mills or the river. The people are seemingly of moderate means, not foreign born, and of good intelligence. The houses have separate cesspools. There is no town sewer. The water supply is the same all through the town. The ice for the whole town comes from the same source the river. There is a well in the neighborhood used by a large number of people. Several of the typhoid cases did not use this well, and many other uninfected persons did get some of their drinking water there. X did not use this well. The hygienic condition of X's place is quite unsuited for the production of milk. The cesspool is not carefully sealed. The barn is filthy and fly infested. An unguarded privy drains into the cellar, where the manure and a pig are kept. Complaints have been made by the neighbors of the smell from the place. X has kept two cows, which have yielded him about two 82 quart cans daily. He milks the cows and strains the milk himself. Mrs. X washes the cans. The milk is peddled about the neighborhood by X soon after it is milked. It is not iced. No definite conclusions as to the source of the typhoid cases have been drawn. The time that has elapsed since many of the cases occurred has made the obtaining of accurate information difficult. The possibility that X is a carrier has been considered. If he is a carrier it is more than probable that he has infected the milk. The prompt distribution of the milk may account for the small number of cases that have occurred at any one time. Specimens of urine and stool have been obtained for examination. SUBSEQUENT NOTES. Shortly after the above date specimens of feces and urine were obtained from X. Bacteriological examination of this material showed no typhoid bacilli in the feces, but there was an abundant growth of motile bacilli in the urine. The bacilli corresponded in cultural and agglutination characters to typhoid bacilli. A second examination of the man's urine and feces was made. The same condition was again found. X was informed of his condition and forbidden to distribute any more milk. The board of health of Maynard was notified. On November 4 X was given urotropin, with a dosage of 10 grains, t. i. d. At the end of about ten days a specimen of urine was examined, and showed apparently a marked decrease in the number of typhoid bacilli. Owing to complaints of discomfort, the urotropin was then discontinued for a few days. Examination of the urine at the end of this period showed a marked increase in the number of bacilli. Urotropin was then resumed, but with a dosage of 5 grains, t. i. d. Ten days later, December 3, a specimen of urine showed an apparent decrease in the bacteriological content. December 13 the urine showed a moderate number of bacilli. On December 15 the patient was directed to discontinue the use of urotropin and take copper sulphate in one-quarter-grain capsules t. i. d. After two weeks' trial of this medication urinary examination showed no improvement in the condition. At the beginning of the year 1910, four and a half years after his typhoid fever attack, in spite of treatment with urotropin and copper sulphate, this man shows a constant typhoid bacilluria. He has no symptoms of cystitis, and feels better than he did before being sick. A STUDY OF SOME OF THE SPORE-BEARING ANAEROBIC BACTERIA IN MARKET MILK.' By HERBERT R. BROWN, S.Bc.2 The sanitary bearing of bacteria in cows' milk has been the subject of many publications in recent years. Yet the study of individual species, aside from their relation to flavors in the industrial dairy products, has not been pursued with much thoroughness. Notably, the anaërobes have been more or less neglected, although their capacity for the production of toxins and of putrefactive products would lead one to assume that they may be of considerable significance as producers of diseased conditions in the digestive tract. It was therefore suggested by Prof. Theobald Smith, pathologist to the State Board of Health, that I undertake a study of the spore-bearing anaërobes as they may be found in milk offered for sale, especially during the summer season, in Boston. MATERIAL. The material for this research was ordinary market milk purchased at the small stores of the urban and suburban districts, which obtain their milk supplies from different contractors, who, in turn, receive their milk from dairies scattered throughout the State of Massachusetts and the neighboring States. The milk was shipped, in accordance with legal regulations, in refrigerator cars to the contractors, who distributed it to the small dealers in wagons not supplied with cooling arrangements. The small dealers as a rule kept the milk in large supply cans surrounded by chopped ice packed in specially prepared ice boxes. There was, however, a lapse of several hours in some cases between the removal from the cars and the delivery at the suburban stations, and during this period bacterial multiplication could go on to a greater or less extent, according to the temperature of the milk. The samples collected at the. small stores were received in sterile jars and transported to the laboratory in a chamber kept cold by chopped ice and salt. 1 Manuscript completed July, 1909. 2 Assistant in bacteriology, Massachusetts State Board of Health; from the Laboratory of Com. parative Pathology, Harvard Medical School. METHODS. On reaching the laboratory the samples of milk were heated to 80° C. for twenty minutes, to destroy the nonspore-bearing bacteria present, and then inoculated into special tubes to enrich the anaerobes, so that convenient numbers for plating could be obtained. The use of the fermentation tube for the cultivation and biological studies of anaërobes, as suggested by Theobald Smith et al, has given a most satisfactory means of investigating the behavior of many anaërobes in the different culture media. 46 The conditions obtaining in such tubes furnish a favorable environment for the growth of obligate and facultative anaërobes, and the quantity of culture medium necessary to fill the tube is sufficient for the carrying out of the common biochemical tests to demonstrate the presence or absence of specific products. When the anaërobes were sufficiently developed, dilutions of the culture were made and inoculated into blood agar for plating. The blood agar used was prepared by adding to melted agar in tubes of 12 cubic centimeters each, cooled to 40° to 50° C., 1 cubic centimeter of defibrinated horse blood, or blood of some other animal, and a few drops of a 20 per cent. dextrose solution. (Only horse blood was used in these studies.) The plates used were Petri dishes, with specially prepared earthenware covers, which absorb moisture and thus prevent spreading of the surface growths in the condensation water that is often found on the medium when glasscovered dishes are used. After pouring the inoculated blood agar into the Petri dishes, lots of six were placed in Bordet 5 chambers containing pyrogallol, for the absorption of oxygen. The chambers were sealed with wax made of beeswax and vaseline, and were then placed in the incubator at a constant temperature of 37° C. for about four days, when colony development was at its height. Jars other than the Bordet. chamber (which is in reality a desiccator) have been used with pyrogallol, some with exhausted chambers filled with hydrogen, some with illuminating gas, etc., with varying success; because of its simplicity the Bordet chamber was used most extensively. 37 Sterile tissue was obtained from chloroformed guinea pigs, according to the method described by Theobald Smith, by exposing the abdominal viscera with sterile instruments and tearing away convenient sized pieces of the spleen, kidneys and liver, taken in the order named with sterile forceps. The spleen and kidneys were almost without exception found to be sterile, but the liver was occasionally found to contain aërobic and sometimes anaerobic bacteria, the sources of which are uncertain. Incubation of tubes prepared in this way from three to six days was sufficient in most cases to demonstrate infection or sterility. Liver tissue generally undergoes a slight disintegration, causing a deposit to be formed on the walls of the bulb of the fermentation tube, and this may be mistaken for aërobic growth and may necessitate a microscopic examination to prove the absence of bacteria. About 85 per cent. of the tissues used were found to be sterile, and the remaining 15 per cent. of the tubes that showed contamination contained bacteria which were probably traceable to the dust in the air, to the skin when the abdominal viscera were exposed, or to an infection of the liver itself. Sugars. Dextrose, lactose and saccharose were used for the special study of gas formulæ and reactions. The specific sugars were added to sugar-free bouillon without tissue in 1 per cent. quantities just before inoculating, and then steamed in an Arnold sterilizer for fifteen to twenty minutes, to mix broth and sugar and also to drive off any dissolved air that might be present. Inoculation of the tubes so treated generally resulted in rich growths that produced relatively constant and differential gas formulæ and reactions. In some cases, however, it was necessary to pass the anaërobes through a freshening process, consisting of one or two transfers from the stock cultures to fresh tissue tubes before inoculating the prepared sugar tubes, in order to insure growth; or, as was found in one or two instances, even freshened cultures might refuse to grow without tissue, and did so only after pieces of sterile tissue were added. The greater number of anaërobes, however, were found to grow. well in the absence of tissue after passing through the freshening process, that served to increase the vigor of the bacilli. Culture Media. - The culture media used consisted of fermented (sugar-free) and unfermented beef bouillon, bouillon plus tissue, coagulated blood serum, gelatin and milk. The reaction of the beef bouillon was kept at 0.8 to 1.5 per cent, acid to phenolphthalein, that of gelatin. was about the same and the sterile milk was from 1.6 to 1.9 per cent. acid to the same indicator. The milk used for the cultural tests was chiefly a high-grade milk collected under the most cleanly conditions possible. This was chosen because it seldom contained anaërobes and was more easily sterilized at 100° C. than the cheaper market milk, and could be sterilized at lower temperatures when desired. Sterilization was carried out according to the method described by Theobald Smith,1 and at 80° C. in the manner described below. Nonspore-bearing aërobes were frequently found capable of resisting temperatures from 70° to 80° C., but these were devitalized and destroyed by heating to 80° C. on two successive days, and then after the second heating by subjection to a third heating within a few hours of the second treatment. On the third and fourth days the same heat as before was applied, and |