~ \*"? *V s THE UNIVERSITY OF ILLINOIS LIBRARY ii I 41 RON CIRCULATING CHECK FOR UNBOUND CIRCULATING COPY UNIVERSITY OF ILLINOIS Agricultural Experiment Station BULLETIN NO. 161 FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY BY CHAS. F. BRISCOE URBANA, ILLINOIS, NOVEMBER, 1912 LETTER OF TRANSMITTAL University of Illinois, June, 1912. DR. EUGENE DAVENPORT, Director of Hie Agricultural Experiment Station. Sir : I have the honor to transmit and recommend for publication the accompanying paper prepared under my direction by Chas. F. Briscoe, Ph.D., upon the FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY. This is a companion bulletin with No. 149 of the University of Illinois Agricultural Experiment Station issued under date of February, 1911, entitled TUBERCULOSIS OF FARM ANIMALS, 'by Chas. F. Briscoe and W. J. MacNeal. That bulletin was based upon a general study of the disease in bovine ani- mals its characteristics, modes of dissemination and infection, methods of recognition and prevention, and the relation to tuberculosis in man. The import of the present paper is well expressed in the title, with the understanding that the studies were made principally upon the bovine type of the causative organism. Much previous work had been done upon the same subject, as is indicated in the literature herein cited, but important contribu- tions to existent knowledge are made in the results of the experiments em- bodied in this publication, while earlier announcements have been confirmed or disproved. The whole matter is of such tremendous significance that any additions to knowledge and any further dissemination of knowledge upon the subject is of wide and vital importance. The information here presented will be welcomed by specialists, sanitarians, and the general public. T. J. BURRILL, Chief in Botany {including Bacteriology). SUMMARY oi> BULLETIN No. 161 1. There are four recognized types of tubercle bacilli; human, bovine, avian, and a type that infects cold-blooded animals. Only the first two types have any important part in the infection of man. Page 284 2. The beaded appearance of these germs led the early inves- tigators to a belief in spore- formation. This is now known not to occur. The fatty content of the bacilli varies from 10 to 42 percent, which is five times as much as found in any other micro- organism. It appears that this fatty material has little or nothing to do with the duration of their viability. Page 284 3. The tubercle bacillus does not secrete a soluble toxin, but that poisons are formed is well known. It has been shown by various investigators that tubercles can be produced in test animals by the injection of dead cultures. Tubercles thus produced may be mistaken for those produced by living germs. Page 284 4. A clear criterion of death is necessary in reporting results on the duration of life of the tubercle bacillus. This fact many investigators have disregarded. Page 285 5. The tubercle bacillus may be classed with the nonspore- bearing organisms as to viability; but in this class it is one of the most resistant, especially as to drying and to the antagonism of decay organisms in water ~and foul matter. Page 286 6. The action of sunlight upon bacteria was first studied by Downes and Blunt in 1877. Numerous investigators have con- tinued this study to the present day. One of the most important facts brought out is that bacteria when directly exposed to the sun are killed in a few minutes. This is due to the effect of the ultra violet light. The ultra violet rays are now cheaply produced artificially by the mercury vapor lamp. This lamp is destined to play an important part in sterilization and disinfection. Pages 287-291 7. It is shown by the results of all the investigators given in Table 2 that tubercle bacilli when exposed directly to the sun are killed in a few minutes to a few hours. The time .of killing is less at higher altitudes; but it is ten to fifteen times longer in diffuse light. Pages 292-295 8. Tuberculous sputum reduced to dust and inhaled by test animals causes tuberculosis. A much less amount is necessary to produce tuberculosis by inhalation than by ingestion. This, how- 279 ever, must not be taken to indicate that inhalation plays a more important part than ingestion as a cause of tuberculosis. The more important method depends upon the opportunity of infection from each. Investigators do not agree on this question. At pres- ent both inhalation and ingestion should be considered dangerous sources of tuberculous infection. Pages 295-306 9. The Mills-Reincke phenomenon, which has been given a mathematical equivalent by Hazen's theorem, viz., "Where one death from typhoid fever has been avoided by the use of better water, a certain number of deaths, probably two or three, from other causes have been avoided," has been found by Sedgwick and MacNutt to be sound and conservative. Their studies show that tuberculosis has decreased in certain cities of Massachusetts, which decrease is evidently due, in part, to the improvement of their water supply. Pages 307-308 10. It is reported in the literature that tubercle bacilli live for a very long time, several months to more than a year, in water and other material. Pages 309-310 11. In experiments to determine the time that tubercle bacilli live in various conditions the chief difficulty is the "index of death" for these germs. This is true since cultivation of the tubercle bacilli from contaminated material is not feasible, and since the dead germs produce, in test animals, tubercles indistin- guishable by microscopic appearance from those produced by live tubercle bacilli. Pages 311-313 12. Pure cultures of nonspore-bearing organisms and the vege- tative cells of spore-bearing germs when exposed to direct sun- light in thin smears are killed in l /> to 6 minutes ; the human, bovine, and avian types of tubercle bacilli exposed in the same way were killed in i to 4 minutes. Pages 314-317 13. When exposed to desiccation in a dark, well-ventilated place, the nonspore-bearing organisms and the vegetative cells of spore-bearing organisms died in i to 4 days; spores of B. subtilis and B. vulgatus used as controls were not killed in 35 days ; the human and bovine type of tubercle bacilli exposed at the same time and under the same conditions were dead within 4 and 8 days re- spectively. Pages 317-318 14. Pure cultures of bovine tubercle bacilli mixed in cow manure and exposed in a two-inch layer in a pasture field in the sunshine remained alive and virulent for two months. Pages 323-324 280 15. As would be expected, these germs exposed in cow manure retained their virulence longer in the shade than in the sunshine, as shown both by the greater severity of the disease produced in the guinea pigs inoculated with the germs exposed in the shade, than that produced in the guinea pigs inoculated on the same day with the germs exposed in the sunshine, and by the greater length of time that the guinea pigs which were inoculated with the germs exposed in the sunshine remained alive. Pages 325-326 1 6. Tubercle bacilli in the manure of a naturally infected cow exposed in the same manner as the artificially infected manure were dead within two weeks after exposure. Pages 327-334 17. Tubercle bacilli in garden soil and in a dead tuberculous guinea pig buried in garden soil were alive on the 21 3th and the 7 ist days, respectively, and dead on the 23Oth and 99th days, after first exposed. Pages 334-339 1 8. Tubercle bacilli live for more than a year in running water. A watering trough harboring these germs may be a dangerous source of infection to cattle. The better disposition of dead tuber- culous animals is to destroy by burning. Tubercle bacilli in drink- ing water is one of the possible sources of infection for man. In- fection is not prevented by dilution, since clumps containing a great number of these organisms may be inclosed in mucoid ma- terial which prevents their separation and destruction. Pages 340-359 19. Tubercle bacilli in market butter placed in cold storage live for more than ten months, which is a longer time than such butter is usually kept in storage. Pages 359-363 20. General discussion. Pages 364-365 21. References. Page 366 281 FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY BY CHAS. F. BRISCOB, INSTRUCTOR IN BOTANY 1 INTRODUCTION The fact that one-tenth of all deaths in the human family are due to tuberculosis, and that millions of dollars worth of farm animals are lost annually from its ravages, makes any knowledge concerning the fate of tubercle bacilli outside of the animal body of great value. These questions have often come from stock owners : How long is it necessary to keep healthy stock from a field where tuber- culous cattle have been previously allowed to run? How long do tubercle bacilli live in manure, in a watering trough, and in a dead tuberculous animal ? And again, some authors have questioned the reports in the literature that tubercle bacilli remain alive and viru- lent for periods of a year and more outside of the animal body, They have suspected that in such cases the tubercles, found in the test animals after inoculation with such tuberculous material, kept for these long periods, had been produced by dead tubercle bacilli. It has been, in part, the purpose of this bulletin to answer these questions. The first section is devoted to brief notes on the biology of the tubercle bacillus; then a tabular review of the literature is given \vith brief discussions; and lastly an account of the experimental work, followed by a general discussion. The experiments deal with pure cultures of tubercle bacilli, and with those of some other bacteria for purposes of comparison as to their duration of life in sunlight and under desiccation; and with the time -limit of life of tubercle bacilli in cow manure, garden soil, water, butter, and dead tuberculous animals. BIOLOGY OF THE TUBERCLE BACILLUS MORPHOI OGY tu b erc l e bacillus varies in form according to type, method of growth, and age of the individual culture. It is a slightly curved, rod-shaped organism measuring from 0.3 to 0.5 microns in diameter and from 2 to 5 microns in 'Now Professor of Bacteriology in the Mississippi Agricultural Experiment Station, Agricultural College, Mississippi. 283 284 BULLETIN No. 161 [November. length. There are two well-recognized types that infect mammals, the human and the bovine. The latter is shorter and thicker and stains more uniformly. The avian type, found in domestic fowls and birds, and a type that infects the cold-blooded animals, espe- cially fishes and frogs, play almost no part in the infection of man. The tubercle bacillus often presents a beaded appearance. This is more common in the specimens found in old pus and sputum. It is due to a fragmentation of the protoplasm. This peculiar struc- ture of the organism led the earlier scientists, to the belief in spore formation. Koch 74 , in his first paper, Die Aetiology der Tubercn- los, held to this belief, and this idea of spore formation has found place in many publications even to the present day. It is now known, from the relation of these organisms to the action of heat, sun- shine and chemical disinfectants, that they do not form spores They are killed very readily, in thin layers, by the direct rays of the sun, and also at a temperature of 60 C. in fifteen to twenty minutes. In these respects they are like other nonspore-bearing organisms. Their power of resistance to drying and to the WAXY OR FATTY v . , . J SUBSTANCE antagonism of decay organisms appears to be greater than that of other nonspore-bearing or- ganisms, tho less than that of the spore 1 organisms. This power of resistance is no doubt due, in part at least, to the content of waxy or fatty substance found largely in the outer layer of the tubercle bacillus. The presence of this waxy material gives them their well-known character of "acid prqof" power when stained. (When this waxy or fatty substance is extracted with ether, stains are no longer held on treatment with an acid or alcohol.) The bacillus has the largest amount of fatty substance of any known micro-organism. The fat content varies, according to different in- vestigators, from 10 to 42 percent; while in other micro-organisms an alcohol ether-extractive has been found to vary only from 1.7 to i o.i percent. The tubercle bacillus does not secrete a soluble THE 3( T N u S BERCLE toxin > as do B - diphtheria and B. tetam. It has not BACILLUS " been demonstrated that the tubercle bacillus forms a true toxin. Levene 87 proved the absence of tox- albumins from extracts of the bacillus. That poisons are formed is well known, tho their character is not understood. Baldwin 10 thinks that the symptoms and toxemia of tuberculosis are accounted for by the presence of the nucleic acid products in the blood. Koch 73 , Von Prudden and Hodenpyle 155 , Vissman 154 , Straus and Gamaleia 144 , Sternberg 145 , Krompecher 75 , Miller 97 , and Rosenau 121 I9 1 -] FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 283 have produced tuberculosis in test animals (guinea pigs and rab- bits) by injecting dead cultures. These results have been confirmed in the laboratory here. (See experiments page 312). The injection of dead cultures killed by heat or DEA'D CULTURE s^ chemical disinfectants will produce necrosis, abscesses, caseation, emaciation, and death. So closely do these lesions resemble those produced by the living germs that it is difficult to know whether we are dealing with dead or with living cultures. JRosenau 123 suggests that much of the work done upon the duration of life of tubercle bacilli has little value for lack of a clear criterion of death. Lesions from dead cultures, as from live ones, may be characterized by giant cells. Tuberculin cannot be depended upon to distinguish between the lesions pro- duced by dead tubercle bacilli and those produced by live germs. The reactions caused by the injection of 2 cc. of tuberculin under the skin of guinea pigs with lesions produced by dead cultures are similar to those caused by a like injection into guinea pigs with lesions produced, from living cultures, even to the death of the guinea pig. Most investigators have not taken into account the fact that dead tubercle bacilli produce lesions, and in reporting their results have taken for granted, when at autopsy even local lesions have been found, that the tubercle bacilli have been alive and virulent. The only safe method of distinguishing is by inoculating a second- ary healthy guinea pig with a small amount of the tuberculous material from the lesions of the original test animal. If the bac- teria are alive, there develops a generalized tuberculosis, usually severe; if dead, either no lesions at all or only a slight localized effect will occur. Cultivations from this tuberculous material from both the original and the secondary test animal will give further evidence. If the organisms are dead, no growth occurs; if alive, growth is usually evident. This method, tho time consuming, is "ecessary to obtain the most trustworthy information. That the tubercle bacillus does not possess the re- R EL ATI ON OF si stance to external agents as do the spore-bearing QILLI TO OTHER organisms is unquestioned. The spores of Bact. ORGANISMS anthracis have been known to hold their virulence from ten to twelve years (Aiello and Drago 2 ). We have found that the spores of B. subtilis dried on an agar slant and remaining in this state for eight years, gave growth when seeded into broth. It might be expected, however, that tubercle bacilli, from their waxy-fatty content and from their analogy to spores in respect to staining qualities, would be more resistant to 286 BULLETIN No. 161 [\ r orember, external injurious agencies such as heat, drying, light, chemicals, and putrefaction. This, in general, is found not to be the case. Rosenau 123 says, "The tul>ercle bacillus may be classed with the nonspore-bearing organisms so far a"s its viability is concerned." This statement is surely correct with respect to heat and light. The thermal death point of the tubercle bacillus, as determined by the more careful investigators (Herr 50 , 1901 ; Hess 57 , 1901 ; Rus- sell and Hastings 127 , 1904; and Rosenau 121 , 1908), is given at 60 C., with an exposure of from fifteen to twenty minutes. This is practically the same as the thermal death point determined for most other nonspore-bearing organisms. (Sternberg 145 , Smith 130 ). As regards desiccation, and more especially the antagonism offered by decay and foul matter, tubercle bacilli appear rather to take an intermediate position between spore- and nonspore-bearers. Rickards, Slack and Arms 119 found that tubercle bacilli in sputum resisted drying for 88 days; LeNoir and Camus 83 found these organisms alive after 33 days; Kuss 77 found them alive from 20 to 30 days, and killed in 40 to 60 days; Noetel 105 found them yet living after 35 days. Maffucci 90 shows that pure cultures of avian tubercle bacilli dried on silk threads live for 14 days. Kirstein 69 finds pure cultures dried in dust living from 3 to 8 days. In my own experiments they lived 8 days. Other nonspore-bearing organ- isms die in a very short time, as B. ziolaceus, I day ; B. "typhoid, 3 days; and B. coli, 3 days. There are reported in the litera- ture cases where nonspore-bearing organisms have resisted drying, under special conditions, for a very long time. Rosenau 123 finds B. pestis to live over four months on a piece of dry sponge. Sirena and Alessi 135 report that the pneumonia diplococcus, when dried on silk threads kept in a moist room, did not die until after 192 days. More comparative work must be done with cultures of different nonspore-bearers and with tubercle bacilli exposed under the same conditions before definite conclusions can be drawn. In the presence of foul material tubercle bacilli live from a month to a year and more (see Table 7). In my own experi- ments they were always found to live in water for 202 days and as long as 441 days. In this work much care was taken to deter- mine that the tuberculosis produced in the -test animals was by living germs. Careful workers like Jordan, Russell and Zeit 65 have found B. typhosus living in sewage from 3 to 4 days only; Russell and Fuller 126 report from 8 to TO days. It .would be expected that tubercle bacilli protected by the mucoid material, as found in sputum and diseased tissues, in which these germs more frequently occur, and also by their abundance of naturally waxy constituents, 191-} FATE OF TUBERCLE BACILLI OUTSIDE THE AXIMAL BODY 287 would be protected against drying and injuries from the presence of foul material. This conclusion is well borne out by the litera- ture and by the experimental data given in this bulletin. REVIEW AND DISCUSSION OF THE LITERATURE THE ACTION OF SUNLIGHT ON BACTERIA Historical The killing power of sunlight on bacteria was first noted by Downes and Blunt 34 in 1877. They worked with mixed cultures suspended in culture solutions and found that the organisms were killed in a short time, as shown by the solutions remaining clear. Tyndall 150 questioned the results of Downes and Blunt but later published results 151 confirming them. Duclaux 37 (1879) was the first to test the action of sunlight upon pure cultures and thus place the work upon a scientific basis. Arloing 7 (1885) was the first to test the action of light upon pathogenic bacteria (Bact. anthracis}. He was also the first to use the electric light in sujcji experimental work. Feltz 38 (1890) was the first to test the action of light upon tubercle bacilli. The classic works of Buchner 16 and Ward 157 (1892-3) deserve special mention. Buchner was the first to ex- hibit the killing power of light in reproducing characters by the growth of the organisms in that part of the Petri dish protected from the sunlight. The organisms are thickly seeded in a solid medium like agar, and the desired characters cut from black paper are fastened over this Petri dish. The organisms exposed to the sunlight are killed ; where protected by the black paper, they grow, forming the letters. Ward called especial attention to the effect of different rays of the sun, which he separated by the use of screens of colored glass and colored solutions. There early arose the questions : To what is the killing of bac- teria due? and, Are spores more easily killed than the vegetative cells? Arloing 7 (1885) showed that spores of Bact. anthracis in broth were killed in two hours, while to kill the vegetative cells required from twenty-six to thirty hours. Nocard 104 (1885) sug- gested that during the exposure the spores developed into bacilli and the light acted upon the more sensitive vegetative cells. Straus 141 (1886) 'apparently confirmed this suggestion of Nocard by exposing anthrax spores in broth and in distilled water. He found that the spores in the broth were killed in nine hours while those in distilled water were not killed in this time. In 1886 Ar- loing* repeated his experiments, exposing the cultures on ice so that the spores could not develop, and confirmed his earlier results. Roux 125 (1887) finds the explanation in the fact that spores are more readily killed by the action of sunlight in the presence of 288 BULLETIN No. 161 [November. oxygen than when the oxygen is excluded. He thinks that from the nature of the spore constituents more oxidation products are formed in the spore chan in the vegetative cell, and that for this reason the spore is more readily killed. Dieudonne 31 (1894) con- cludes that the formation of hydrogen peroxid by the action of sunlight on the presence of oxygen forms an important part in the killing of bacteria. Kruse 76 (1895) and Richardson 118 (1893) also came to this same conclusion. Thiele and Wolf 147 ( 1906) carried out carefully planned experiments in which their cultures of B. prodigiosus, B. pyocyaneus and B. coli were exposed in the presence of air, oxygen, or hydrogen. They took special precau- tions to confine the gases used, with mercury joints, so as not to allow, the slightest diffusion. They found that the bacteria were killed as readily anaerobically as aerobically, and conclude, there- fore, that the killing of the bacteria by light is not assignable to the indirect influence, of oxidation of water. They exposed their cultures in broth diluted i-ioqo, physiological salt solution and Elbe river water. It can readily be shown that by the exposure of physiological salt solution to sunlight for the length of time the investigators ex- posed their cultures there, is formed some condition sufficient to kill bacteria. The question whether the bactericidal action is due to an injurious chemical formed by the action of the sunlight, or whether it is a direct bactericidal action of the light, is still unset- tled. The facts thus far established indicate that the action is due to both a chemical and a direct bactericidal action, sometimes one and sometimes the other being predominant, depending upon the condition of the experiment. A more important fact, which has been especially emphasized in a recent publication (see Weinzirl, Table 2) is that bacteria when directly exposed to sunlight are killed in a few minutes, in contrast to most of the published results of a few hours to a few days. This is due to the well-known germicidal effect of the ultra violet rays. Since glass is an excel- lent screen for these rays, an exposure of bacteria in glass contain- ers lengthens very greatly the time necessary to kill. Powerful beams of ultra violet rays are now artificially pro- duced with the quartz mercurv vapor lamp. A number of these machines are upon the market. The rays from them are so effec- tive that bacteria directly exposed in thin layers are killed almost instantly. Indeed, so strong are the most powerful of these lamps that the skin accidently exposed is killed and later sloughs off. It is stated that the sterilization of a city water supply can be done economically with these lamps. A test 5 at Marseilles, France, shows that the cost per million gallons of water is only ten dollars. 1913] FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 289 i. e., one cent per 1000 gallons. This indicates a feasible system of supplying a city with a good, potable water from a contaminated source. Years ago it was suggested by Marshall Ward that the dis- infection of cow sheds might well be done with electric lights. Tho this was thought fanatical at the time, it may be feasible with such light as the present powerful mercury vapor lamp produces. Why cannot the "toning up" effect that is to be felt in a room which has been exposed to the sunlight thruout the day be in a way brought about by exposing to one of these powerful mercury vapor lamps, for a few hours in the evening, such rooms as have no access to the sun, at the same time securing the great benefit that comes from disinfection by its powerful beams? For further discussion of this most interesting subject we refer our readers to the recent numbers of the periodicals, more espe- cially the chemical and engineering journals. The following table gives a brief review of the results found in the literature upon the action of sunlight on bacteria. TABLE 1 ACTION OF SUNLIGHT UPON BACTERIA Author Date Conditions Not killed Killed Dowries 1877 Cultures in Pasteur solutions 1879 Tyrothrix filiformis spores Duclaux 36 1885 Tyrothrix scaber from milk: A drop dried in an empty flask In broth culture vegetative cells. . . Kept in the dark 14 days 3 yrs 60 days 15 days Duclaux 36 1885 Micrococci, young cultures veal broth: In dark 40 days In sunshine, July ..... jn dried condition 8. 3 and even 2 days Arloing 6 18S5 Bact. anthracis spores in broth Bact. anthracis vegetative cells in broth 1 to \Yt hrs. 2 hrs. after 2 hrs. 26 to 30 hrs Straus 141 1886 Bact. anthracis spores: 9 hrs 9 hrs. Arloing 8 18S6 Bact. anthracis spores in broth on ice 4 c 5 hrs Dowries" 1886 Bacteria in mixed cultures, diffuse litrht 5 days Roux 133 1887 Bact. anthracis spores: \Vithout air 83 hrs. With air . 29 to 54 hrs. 290 BULLETIN N.o. 161 TABLE 1 Continued [November, Author Date Conditions Not killed Killed Pansini 108 1889 B. violaceus, B. prodigiosus, B. pyo. cyaneus, Bact. anthracis, Msp comma, Murisepticus, and Staph' pyogenes aureus: Exposed to diffuse daylight, devel- opment hindered 24to48hrs. Exposed to sunshine in tube cultures In hanging drop on needle 1 day. y* to2j^ hrs L/aurent 80 1890 Bacillus of Kiel on potato 1 to 3 hrs. 5 hrs Janowsky' 8 ' 1890 B. typhosus in culture media 4 to 10 hrs Buchner 18 Buchner 15 1892 1893 B. typhosus on agar plates B. typhosus, B. pyocananeus, and Msp. comma on plates 1.6 meters under water 1 to \ l / 2 hrs. \y 2 hrs Geisler 80 1892 B. typhosus (almost all killed) 3 to 6 hrs Moment 100 1892 " Asporegeiie anthrax" in phenol broth: Dried and exposed in air 5 to 5j4 hrs Dried and exposed in vacuo 6% hrs. In moist condition in the air 2^ hrs. In moist condition no air 50 hrs Dried anthrax spores in the air 100 hrs. Dried anthrax spores in vacuo 100 hrs. Moist anthrax spores in the air . ... 44 hrs. Ward 167 Frankland and Appleyard 41 1893 1893 Moist anthrax spores in vacuo Bact. anthracis spores on agar Bact. anthracis spores in Thames river water: In diffuse daylight 1 6 mo. 100 hrs. 4 to 10 hrs. In direct sunshine 151 hrs. Bact. anthracis vegetative cells: In unsterilized water 56 hrs. 84 hrs. L/edoux- In sterilized water 84 hrs. L/ebard 81 Ward 167 1893 1893 B. diphtheria in broth culture ... Organisms on agarinPetri dishes with quartz glass covers few days 2 to 6 hrs. Ward and Cartwright 158 1893 Bact. anthracis in unsterilized Thames d'Arsonval 1894 B pyocyaneus on agar and Charrin 9 Gaillard 45 1894 3 to 4 hrs Dieudonne 81 1894 B. coli, B. typhosus, Bact. anthracis, B. prodigiosus, and B. fluorescens By electric arc light 900 candle- power % to 2% hrs. 8 hrs Bact. anthracis spores: In the air 3^ hrs Air excluded ...... 3^ hrs B. tetani spores, air excluded 9 hrs Kruse 76 - 1895 Bact. anthracis spores in hanging drop \y 2 hrs 2 to 5 hrs B. typhosus: In broth air present iy 2 to 7 hrs In broth, air replaced with hydrogen Bact. anthracis diffuse light . . . 7 hrs. (13 da3 T s SO days) 1 Tho the spores were not killed, they were so weakened that one cubic centi- meter did not kill mice. A broth culture of the same in much smaller quantity killed mice. ^ 19 J-] FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY TABLE 1. Continued. 291 Author Date Conditions Not killed Killed Beck and Schultz 1 a 1896 Bacteria short time Gehrke 49 1899 Bacteria: 1 In clear water 2 to 8 hrs Agar cultures 6 hrs. Kedzior 67 Jones 6 * 1899 1900 B. pyocyaneus, B. diphtheria, Msp. metchnikovy, and spores of Bact. anthracis, exposed on gelatin .... B. cartovorus directly exposed 5 min. IX to 3X hrs. 10 min. Mettler 95 1904 Msp. comma, B. typhosus, and B. coli in broth culture few hours Huber 61 Thiele 147 and Wolf 1905 1906 Streptococci and B. diphtheria in broth B. coli, and B. typhosus in diluted broth, Elbe river water, or physi- ological salt solution 2 to 3 hrs. 9 hrs Weinzirl 160 1906 . Exposed directly to the sun in thin smears: B. cholera suis, B. prodigiosus, B. ty- phosus 5 to 10 min B. typhosus (repeated), B. dysenteria, B. prodigiosus (repeated), B. 2 to 5 mill Msp comma 2 to 6 min B. diphtheria 5 min. Pus cocci - ' 5 to 15 min B coli ... /*2 to 2 min B. f riedlJlnder > to 1 min. B. phosphorescens 1 to 2 min. Sar. aurantiaca 90 min. Pink air micrococci 120 min. Neumark 103 1907 Exposed to direct sunlight on agar plate: B anthracis spores 25 to 30 min B anthracis vegetative cells 20 to 25 min B coli 60 min. ' 'Schweinpest bacilli' ' 110 to 120 min. Chicken cholera ...... 8 to 10 min. 240 min. Orsi t06 1907 B. typhosus and Msp. comma in sun- light 8 to 10 hrs B coli in distilled water 6 hrs. 12 hrs. McNaughtarid Korich 34 1910 B. coli: In distilled water in air at to 15C. in diffuse daylight 25 days In strong sunlight, numbers much decreased 6 hrs. In strong sunlight ... 12 hrs. Dried in a desiccator . 11 davs 292 BULLETIN No. 161 [November. THE DURATION OF LIFE OF TUBERCLE BACILLI IN SUNSHINE AND IN DIFFUSE DAYLIGHT INDEX OF Every author consulted, except Weinzirl. used the DEATH animal test to determine when tubercle bacilli were dead. \Veinzirl, using only pure cultures, em- ployed the method of cultivation. No investigator whose work is recorded in Tables 2 and 3 has called attention to the fact that dead cultures produce tuberculosis (for discussion see page 112). Just what error a neglect of attention to this point has introduced into the results given in the following table cannot be definitely known Tho it is certain that dead tubercle bacilli do produce tuberculosis, yet in no case was it found in my own experimental work that localized tuberculosis was produced by dead organisms. In every case the secondary guinea pigs inoculated from cases of localized tuberculosis became tuberculous and usually severely infected. Cul- tures from diseased tissues produced growth, either from the origi- nal or from the secondary inoculated guinea pigs, or from both. It appears, therefore, that the error arising from not taking into account tuberculosis produced by dead cultures is probably slight. TIME REQUIRED P ure cultures of tubercle bacilli when exposed in TO KILL thin layers to the direct sunlight are killed in from a few minutes to a few hours. This has been the result obtained by all investigators. To kill these germs in sputum requires only a slightly longer time. The mucoid mass is a slight protection which increases with the thickness and opacity of the material, but even here tubercle bacilli are killed in a very short time. With two exceptions, that of Feltz 38 and of Mitchell and Crouch 98 (see Table 2), no investigator reports these bacilli surely living after twenty-four hours' exposure to the sun. In these two cases the sputum was exposed on soil, and a small layer of soil would afford good protection from the action of the sunlight. EFFECTOR Experiments definitely planned to test the killing HIGH ALTITUDE power of the sun on tubercle bacilli at different altitudes have been carried out by a Russian, Tres- kinskaja 148 . In our country Mitchell and Crouch 98 have determined the effect of the sun on tuberculous sputum at a height of 1356 meters. Treskinskaja used pure cultures emulsified in one-percent peptone solution, spread in thin layers and exposed directly to the sun. These were killed as follows: At a height of 1560 meters, in three hours ; at 903 meters, in four hours ; at sea level, in four hours. The peptone solution when dried gave a thin protective layer covering these organisms. Treskinskaja thought that this protection would be about equal to that of a thin layer of sputum. 1QI2\ FATE OF TTBERCLE BACILLI OUTSIDE THE ANIMAL Bony 293 The difference in killing power is due to the difference in ultra violet light. These rays are largely absorbed by the atmosphere, especially when laden with moisture. According to Langley, only 39 percent of the ultra violet light reaches the sea level. The higher the altitude, the dryer the atmosphere, the more intense is the light and the greater its killing power. Mitchell and Crouch found that tubercle bacilli in sputum were not killed in 35 hours, but were killed in 45 hours. These experi- ments are not comparable with the experiments of Treskinskaja, done at nearly the same altitude, since the soil and sputum give far more protection than exposure in a thin layer on glass from a one-percent peptone solution. DISCREPANCIES ^ ' s not to ^ e ex P ecte d that tubercle bacilli under IN RESULTS different conditions will be killed in the same length of time. It is indeed difficult to expose different pure cultures under the same conditions. There may be a difference in the age of the culture, in the uniformity of the emulsion, in the manner of exposing, and in the means of deter- mining the time of killing the germs. When exposed on threads of linen or silk some of the germs may be well protected and live for a much longer time than when exposed in a thin layer on a glass slip or on sterilized glazed paper. Then if the material in which they are exposed, as sputum, is of such a nature as to give protection from drying as well as from the rays of the sun, the difference will be still greater. This accounts, partly at least, for the difference in the results of Sawitsky 128 , which showed that tubercle bacilli in sputum on linen threads lived two and one-half months, and the fact reported by Weinzirl 160 , that a pure culture in thin layers exposed directly to the sun was killed in a few ; min- utes. It is not possible to give definite reasons for all the discrep- ancies found in the results shown in Table 2. ; but excepting those of Feltz 38 , whose material mixed in the soil might well give com- plete protection from the sun, it will be seen that tubercle bacilli exposed to the sunlight are killed within a. few minutes to a few hours. DIFFUSE LIGHT It takes ten to fifteen times as long for tubercle bacilli to be killed in diffuse light as in direct sun- light; yet these germs are killed much sooner in diffuse daylight than in the dark, when under the same conditions otherwise, as may be seen by comparing the results of Rickards, Slack and Arms 119 , Twichell 149 , and Ransome and Delepine 114 . Tables 2. and 3, respectively, give in brief the literature upon these subjects. 294 BULLETIN No. 161 [November. TABI,E 2. EKKECT OK SUNSHINE ON TUBERCLE BACILLI Author Date Conditions Not killed Killed Feltz 38 Koch 78 Sawitsky 128 Ransome and Delepine 114 Renzi 117 Migneco 96 Straus 148 Gardiner 47 Ottolenghi 107 Jousset" Mitchell and Crouch 98 Abba and Barelli 1 Annett 4 Cadeac 18 Bang 11 Twitche,!! 149 Didonna 30 Weinzirl 160 Rickards, Slack and Arms 119 148 Treskinskaja 1890 1890 1891 1894 1894 1895 1895 1898 1899 1900 1900 1900 1901 1902 1903 1905 1905 1905 19U6 1906 1909 1910 Sputum and soil mixed and exposed to direct sunlight, tested by gui- 137 days 2% mo. 6 hrs. 10 to 15 hrs. 1 to 12 hrs. 24 hrs. 1 hr. 35 hrs. 1 4.76 percent 2 to 24 hrs. 24 hrs. 1 hr. 2 to 8 hrs. 2 6 hrs. after 137 days after2>mo. few min. to few hrs. 2y 2 mo. 45 days 4 days (12# hrs. sun) 24 to 30 hrs. 2 hrs. 40 hrs. 24 to 30 hrs. 9 hrs. 6 hrs. 4 to 14^ hrs. 5 to 7 hrs. 1 hr. 45 hrs. 6 days 48 hrs. 24 hrs. 48 hrs. 6 min. 7 hrs. 2 to 10 min. 3 hrs. 4 hrs. 4 hrs. Exposed to changing climatic condi- In pure culture Sputum on stretched linen Sputum exposed to light Pure culture on glazed paper Sputa mixed with 10 parts water, ex- posed at 28 C Sputum on stretched linen and woolen threads Culture in a glass container, in broth In a thick layer of dried sputum In sputum on woolen cloth Sputum the thickness of spitting, on Sputum exposed to direct sun Tuberculous sputum placed upon Tested 105 specimens of sputa taken at random; 5, or 4. 76 percent con- Tuberculous sputum size of spitting. Tuberculous sputum on a board Tuberculous sputum on glass Tubercle bacilli on agar, exposed to a 30-ampere lamp at 30 cm. distance Tuberculous sputum, direct rays Exposed culture to sun 2 to 8 hours; then inoculated guinea pigs in order to immunize them. Pro- duced only a local abcess Pure culture on paper slips or glass Sputum exposed in sunshine Summer months, direct sun: Pure culture tubercle bacilli, height 1560 m Pure culture tubercle bacilli. 903 m Pure culture tubercle bacilli, sea level ... Virulence diminished after 20 hours. 2 It is not certain whether the organisms were alive or dead, since dead organ- isms can produce tubercles. igi2\ FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 295 TABLE 3 ACTION OK DIFFUSE LIGHT UPON TUBERCLE BACILLI Author Date Conditions Not killed Killed Galtier 46 1889 Tuberculous organs: Dried at 30 38 days 30 days Dried at room temperature (some) 30 (usually) af- Sawitsky 128 1892 Sputum on stretched linen days ter 30 days 2% mo. Ransome and Delepine 1 ^ * 1894 Tubercle bacilli on dried paper 2 days lyucibelli 89 1899 Sputum dried on glass 18 days Fluid sputum in reagent glass 4 mo Jousset 66 1900 4 to 7 hrs 1902 8 to 14 days Mixed with dust 8 days On threads 10 days On coarse cloth 20 days Hill 80 1903 Sputum in reagent glass in glass cup- 16 days Cadeac 1 8 1905 2 to 6 days 4 to 10 days Twitchell 149 1905 Tuberculous sputum in paraffined bot- tles 124 days 175 days Sortnani 1 38 1906 Dry sputum in a room 1 mo Rickards, Slack and Arms 119 1908 Dry sputum in a room 1 mo Weinzirl 159 1908 Fine emulsion of tubercle bacilli dried on paper slips (average of ten trials) .'. 2 8 days 4.4 days EFFECT OF DESICCATION UPON TUBERCLE BACILLI IN SPUTUM AND OTHER MATERIAL The difference in the length of time that different bacteria withstand desiccation is very great. B. cartovorus is killed in a few minutes to a few hours (Jones 64 ) ; B. tuberculosis may be killed in a few days ( 14 days, Maffucci 90 ) to several days (88 days, Rickards 119 ); and spores of Bad. anthracis live twelve years (reported above). The difference depends upon the kind of substance on which they are exposed and upon the difference in the kind and form of organism exposed. Harding and Prucha 54 have shown that Bact. cauipcstre remains alive much longer when dried on cabbage se^d than when dried on glass covers; on glass it was dead at the end of ten days; on the seed it remained alive for thirteen months. This difference is no doubt largely due to the difference in the hygroscopic moisture retained by these substances. The kind and form of the organism exposed to drying has even more to do with its capability of living. The spore form lives very much longer than the vegetative form. 296 BULLETIN No. 161 [Xorembcr, It appears that tubercle bacilli, especially in sputum and other mucoid material, withstand desiccation better than other nonspore- formers. The difference is not great and there are many apparent exceptions where other nonspore-bearers live for a very long time, like the one given aboVe after Harding and Prucha 54 . Diplococcus pneumonia has been reported as living for 192 days when dried on silk (Sirena and Alessi 135 ). The important fact which has been so thoroly established in recent years is that tuberculous sputum reduced to dust causes tuberculosis when experimental animals (guinea pigs, rabbits, cats, dogs and calves) are made to breathe such dust-laden air. How great the danger is to man and cattle to breathe dried tuberculous material is yet a disputed question. In 1882 Koch, in his work on "Die Aetiologie der Tuberculose", pointed out that dried tuber- culous sputum is one of the most important factors in causing tuberculosis. It was not until 1905 when the work of Calmette and Guerin 21 was published that ingestion was brought forward as a very important factor in producing tuberculosis. It was sug- gested by the advocates of this theory that practically all tubercu- losis is produced by ingestion that even inspired material is swal- lowed and what apparently is inhalation tuberculosis is actually tuberculosis by ingestion. This conclusion will not hold in the light of the most recent experiments, especially those carried out at Breslau and collectively published by Fliigge 40 (1908), "Ver- breitungsweise and Bekampfung der Tuberknlose". It is shown here that in all animals with which experiments have been made it required a hundred to a thousand times more tubercle bacilli to produce tuberculosis by feeding than it does by inhalation. So the thought that the swallowing of a part of the few tubercle bacilli necessary to produce tuberculosis by inhalation is the cause of in- gestion tuberculosis is wholly precluded. A complete discussion of this most interesting subject cannot be given here. The data in the two following tables show the fact mentioned above, that it requires many times more germs to pro- duce'tubercnlosis by feeding than by inhalation ; also another most important fact, that severe tuberculosis may be produced by either method of infection. It must not be understood that the facts exhibited in these two tables in any way indicate which is the more frequent method of infection. For what matter if it does require one thousand times more tubercle bacilli to produce tuber- culosis by feeding than by inhalation, who knows whether we are, on the average, taking in one thousand times more of these germs in the food than in the breath ? On one hand, Calmette 19 is found declaring that "In the ordi- nary daily life, the infection of the digestive organs is predomi- FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 297 Remarks erms inhaled in moist spray thru a tracheotomy tube, erms inhaled as above. All became severely tubercu- lous. 11 except 3 of the 65 guinea pigs became tuberculous; two that had received 40 and one of the three that had received 20 individual germs remained healthy. he guineapigs in each series were killed 1, 12, 24, 72 and 144 hours after inhalation. 11 became tuberculous. ses the Findel tower ap- paratus for inhalation in the three series, nly doses of 50,000 germs surely positive in the last series. he dose was given with the Buchner spray apparatus in the open air; so only a part was inhaled. nimals inhaled tuberculous sputum dried 12 to 13 days on a carpet; beaten and swept for 20 minutes, ame as above except dried for 6 days and beaten and swept for 12 minutes, uinea pigs exposed during the time of the above two series and also the time of removing the first and plac- ing the second series of test animals. 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H to w 45 "a 0) cd a 'O a r- n a 0) l~ < "a! a 1 rt o to to n J8 *D 3 S w < K M 6 - n M # - 298 BULLETIN No. 161 [November, Remarks uckled mothers whose teats were injected with pure culture of tubercle bacilli, ame as above, ed thru a stomach tube, he human type of tubercle bacilli appeared not to injure the goats. In June they were given 200 mg. of bovine type. 11 severely tuberculous: lung involvement. 11 became tuberculous. ix Breton heifers aged 8 to 10 months. 11 became tuberculous. hree were fed at three in- tervals during 40 days; other two at one feeding, wo control guinea pigs in- oculated with 19,000 indi- vidual tubercle bacilli be- came severely tuberculous. o tuberculosis in any ani- mal, nly slight evidence of tu- berculosis in one that was fed 50 mg. ot the slightest evidence of tuberculosis in any animal. nlyone calf was completely negative. The presence of the disease was found 21 times in the abdominal or- gans, but only 3 times in the lungs. 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H rO * vd FATK OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 299 nant"; and on the other hand Chausse' j: says, "Tuberculosis in adult bovine is due to inhalation in 98 percent of the cases, if not more; in calves in about 90 percent, ingestion and congenital in- fection being responsible for the remainder." Dr. Ravenel 115 , a strong believer in ingestion as one of the important causes of tuberculosis says, "One must take an impartial view of the whole problem, and be willing to agree that both channels of infection are open. In animals, however, the alimentary tract seems to be a more common port of entry". At present, at least, both ways of infection should be consid- ered very dangerous and every precaution taken to guard against them. A brief summary of the literature concerning the length of time that tubercle bacilli live in dried tuberculous material and concerning the infectiousness of such material is given in Table 6. TABI.E 6 EFFECT OK DESICCATION OF TUBERCLE BACILLI IN SPUTUM AND OTHER MATERIAL Author Date Conditions Not killed Killed Villemin 153 1869 Dried tuberculous sputum several hrs. Koch 74 1882 8 wks. Cochez 2 * Malassez arid Viffnal 91 1883 1883 Tubercle bacilli incased in sputum Dried sputum killed a dog, by inhala- tion Alternate drying and moistening of tuberculous sputum 8 times. ...... 3 wks 12 days Schill and Fischer 1 * 9 1884 Tuberculous sputum with spores, dried on glass 126 days 179 days Tuberculous sputum without spores, 186 days 226 days DeThoma 89 1886 10 mo. Sormani 137 1886 Dried tuberculous sputum 2 mo. virulence Galtier 46 1887 Tuberculous sputum dried on linen. . . Dried tuberculous material 6 mo. 20 to 38 days deceased thereafter no virulence after 4 mo. Cadeac and Malet 17 1888 Pieces of dried tuberculous lung ex- posed on paper in laboratory 43 days 102 days Dried tuberculous lung allowed to de- 76 days 80 days Same repeated 150 days after 150 De Souza 28 1888 Inhalation of dried tuberculous mate- rial caused tuberculosis in 12 of 14 guinea pigs. Time of drying not days Galtier 46 1888 Inoculation of tuberculous material dried at 30 C., or by breathing dried tuberculous material 15, 30 and Same material dried at room temper- ature 38 days 30 days after 30 days 300 BULLETIN No. 161 TABLE 6 Continued \Xorcinbcr. Author Date Conditions Not killed Killed Cornet" Feltz 38 Sawitsky 128 Stone 140 Koch 72 Maffucci 80 Marpmann 92 Ransome and Delepine 114 Hance 83 Kirchner 68 1889 1890 1891 1891 1892 1892 1893 1894 1895 1895 1 Dust samples examined for tubercle bacilli were found positive: In hospitals 17.6 percent 17.6 43.6 " over 7 mo. about 140 days 7 to 9 mo. 2% mo. 2}4 mo. 3 yrs. 2 to 8 wks. 14 days 2 mo. (?) (?) 19 days 19 days 45 days 4.9 percent 25 percent 2 mo. (?) 19 days all killed In insane asylums In dwelling 1 houses )ried tuberculous sputum in road dust: Exposed to weather. Exposed to sun > Dried sputum in rooms Tuberculous sputum dried under or- dinary conditions and exposed in the dark in living" rooms Same exposed to sunlight Dried tuberculous sputum, only de- Guinea pigs were inoculated with tuberculous sputum dried for 2 weeks, for 4 weeks, and for 8 weeks. In each case tuberculosis developed as with fresh material. 3 ure culture of avian tubercle bacill dried on silk threads, inoculated into a hen abdominally Same repeated. Hen not tuberculous abdominally; one tubercle in the Tubercle bacilli frequently found in street dust by microscopic test . . . Tuberculous sputum exposed in watch glasses: To air and light 4 days; then 15 To air and light 8 days; then 11 days in the dark In closed cupboard In dark air shaft, produced slight tuberculosis in a rabbit The author, under the direction of Dr. Trudeau of the Adirondack Cottage Sanitarium, examined 81 samples of dust from the differ- cottages. Four guinea pigs dice too early to make the test for tu bercle bacilli; of those remaining 5, or 4.9 percent, became tubercu- lous. These were from a group of 10 guinea pigs inoculated with the dust from one small cottage. The author later inoculated 9 guinea pigs with dust from a New York hospital, 5 of which died in 3 days, and 1 of the 4 remaining became tuberculous Fourteen guinea pigs were inoculated with 7 samples of dust from rooms in homes of consumptives; 9 guinea pigs died early and 5 re- main^d healthy. In a second ser- FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY TABLE 6 Continued 301 Author Date Conditions Not killed Killed Kirchner es (continued) Cornet 2 Laschtsch- enko 78 Neisser 102 Beninde rs 1893 1898 1898 1899 Cornet 26 Sticker 139 Jones* 1899 1899 1900 Peterson 1 Heymanu 59 1900 1901 ies three rooms were investigated, from which 8 guinea pigs were in- oculated; one died early and one of the other 7 became tuberculous. Tuberculous sputum placed on a car- pet and allowed to dry naturally produced tuberculosis in 35 out of 36 guinea pigs which were placed in a room at a distance of 134 to 300 cm. from this carpet Tuberculous sputum dried in thick layers on paper and cloth was not virulent after 9 months Tuberculous sputum rubbed up with dust and aspirated at a velocity of 3 to 5 cm. per second will pro- duce tuberculosis, when inocu- lated into guinea pigs, in a large majority of cases Handkerchiefs that were used by con- sumptives from 2 to 12 hours were dried for 24 hours, and then rubbed up in a box. The dust from these was aspirated and injected into two guinea pigs for each t'est. In 12 tests 8 were positive and 4 nega- tive Dried sputum in one case lost viru- lence in 3 months; another time remained virulent from 6 to 8 months Tested 29 samples of sputum dried from 2 to 21 days, by inoculating guinea pigs with dried sputum dust and by causing them to inhale the dried sputum dust. Tuberculosis was produced in 27 out of 29 tests. The author inoculated guinea pigs interperitoneally with the mucus from the nostrils of 131 healthy persons. Three of these guinea pigs died of tuberculosis on the 8th, 14th and 59th days after in- oculation. No tubercle bacilli could be found b} r stains of the nasal mucus' One of 8 guinea pigs became tuber- culous after inhaling sputum, dried for 2 months; while sputum dried for 3 months gave one posi- tive in 6 The aspirated air from sputum, dried 6 days and injected into guinea pigs, produced tuberculosis 14 percent 9 mo. 8 positive 8 mo. Positive 27 of 29 tests 4 negative 2 mo. 3 mo. 6 days 'It is probable that these three guinea pigs, or at least the first two, died of spontaneous tuberculosis. 302 BULLETIN No. 161 TABLE 6 Continued. [November, Author Date Conditions Not killed killed Heymann 51 * (continued) Gotschlich" Hill 60 1903 1903 Noetel 105 1904 Cadeac 18 Kirstein 69 1905 1905 Dust from rooms of consumptives: Samples taken on a brush No. of No. of Percent samples positive positive Private rooms 59 5 8.5 Hospitals 61 5 8.2 Samples taken on a moist sponge Private rooms 57 9 15.78 Hospitals 62 25 40.3 Total sa'ples 239 44 18.4 Hospitals 123 30 24.3 Pri'te homes 116 14 12.0 No tubercle bacilli were found in 119 samples of dust taken from 15 places where consumptives were in the habit of visiting. Samples of tuberculous sputum from 10 rooms, using 496 swabs, were .taken by rubbing them on carpet, furniture, etc. In 8 of the 10 cases the sputum was proven to contain tubercle bacilli from stained preparations; the other two were not tested. Tubercle bacilli dried on glass rods were dead after 16 days. Clothing that had been and that was being worn daily by consumptives was enclosed in a large box of three cubic meters content. The dust was beaten and shaken out and allowed to settle; then it was collected and thoroly rubbed up in 5 cc. of broth. The original material, and dilutions of .1 and .01 were respectively injected into guinea pigs: Coat and vest worn daily Jacket and hose worn daily New jacket, hose and old vest with no evidence of contaminated sputum Old coat Coat, hose and plush vest, none worn for three weeks Wool jacket and old hose, not worn for five weeks Tuberculous sputum exposed on glass to air and light Tuberculous sputum, in thick layers dried on a marble slab Tubercle bacilli from pure culture or from tuberculous sputum were sprayed upon collected dust, and then after different lengths of time tested for virulence by inoc- ulation of guinea pigs. Dust from : Positive Positive Negative Positive Positive Positive 2, 4 and 6dys 4,6andlOdys. 14 days 1<)I2\ FATE OF TI.T.ERCI.E BACILLI OUTSIDE THE ANIMAL BODY 303 TABLK 6. Continued Author Kirstein 1 "' 1 ' (continued) Park and Williams 110 Twitchell 1 * 9 Sormani 138 LeNoir and Camus 88 Rodet and 120 Delanoe KShlisch 71 Datt- 1905 1905 1906 1907 1907 1908 Conditions Books and papers Sputum dust Cloth ravelings Street dust Dried tuberculous sputum gradually loses its virulence, but still infective Tuberculous sputum: In paraffined bottles in dark moist box In paraffined bottles in diffuse light In paraffined bottles in thermostat. In cotton-stoppered bottles in dark moist box In cotton-stoppered bottles in dark closet On ice Tuberculous sputum: On handkerchiefs. On towels On carpets In sand in moist light place In sand in dry light place In open bottles, out doors, winter . . Dried tuberculous excretion at 35 C. Ivarge quantities of air were aspirated from tuberculous hospitals and the dust injected under the skin of guinea pigs. From 4 tests aspi- rating respectively 270, 2000, 20000 and 53000 liters, in no case were tubercle bacilli found Eleven samples of dust from local offices and private dwellings were inoculated into guinea pigs, with wholly negative results Fresh tuberculous sputum or a pure culture of tubercle bacilli was inti- mately mixed with dust obtained from dwellings and factories, and the number of tubercle bacilli that a guinea pig inhales in a given time was determined. It was found that tuberculosis was pro- duced in the test animals when as low as 50 tubercle bacilli were in- haled, and that the inhalation of 270 or more of these germs al- ways produced tuberculosis. An- other series of experiments were made with dust from 15 dwellings where consumptives were living. The rooms were small and poorly kept. F/ighteen guinea pigs breathed for some time in dense clouds of this dust. None of these guinea pigs became tuberculous. 8 days 4 days 5 days 3 days 2 to 3 mo. 170 days 124 days 33 days 157 days 100 days 102 days 70 days 70 39 123 30 110 15 days Not killed Killed 14 days 7 days 10 days 8 days 188 days 175 days 100 days 172 days 141 days 153 days 110 days 110 70 148 70 132 all 11 samples 304 BULLETIN No. 161 TABLE 6 Continued [November, Author Date Conditions Not killed Killed Kuss 1 1908 Tuberculous sputum dried in thin layers in the dark, free to the air: Virulence wholly preserved Viruletice diminished Virulence much decreased, yet a large dose produced a tuberculous guinea pig Virulence entirely lost Tuberculous sputum, dried in thin layer, in diffuse light of a room rapidly decreased in virulence: Noticeable decrease Marked decrease Very noticeable decrease Not entirely lost Entirely lost Two guinea pigs were exposed to the dust made by shaking of hand- kerchiefs on which tuberculous sputum had been dried for 25 days. Neither became tuberculous Six guinea pigs were exposed to the dust from a plank containing sputum that had been drying for 17 days. Remained healthy Experiments in a box of 125-liter con- tent: Tuberculous sputum dried for 6 days in the dark was powdered and 1 to 2 grams suspended in the air of this box. Guinea pigs with their heads projecting into this box were made to breathe this dust from 30 to 60 minutes. In all cases the guinea pigs became tubercu- lous Experiments in a room of 30 cubic meters: Series i 125 cc. of tuberculous sputum were dried on a carpet from 11 to 13 days. Nine guinea pigs were swung 60 to 90 inches above this carpet. Then the car- pet was beaten and swept for 20 minutes. Four of the 9 guinea pigs became tuberculous. Also the dust obtained by the aspira- tion of 60 liters of air, at a point 60 inches above this carpet, was inoculated into a guinea pig, which became severely tubercu- lous Series 2. 20 cc. of tuberculous spu- tum were dried on another carpet for 6 days. Six guinea pigs were suspended above this carpet, which was beaten and swept for 12 minutes. Two of the 6 guinea pigs became tuberculous. Also 12 to 14 days 18 days 20 to 30 days 3 days 7 days 10 days 15 days 40 to 50 days 20 days 25 days 15 days 6 days 11 to 13 I9I-] FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY TABLE f> Continued 305 Author Date Conditions Not kilk-d Killed Kuss 77 (continued) LeNoir and Camus 83 1908 1908 L/eNoir and Camus 86 1908 dust obtained by the aspiration of 71 liters of air produced a tuber- culous guinea pig Series 3 Six of the 9 guinea pigs exposed to the beatings and sweepings of the two carpets of Series 1 and 2, described above, became tuberculous Tuberculous sputum rich in tubercle bacilli was mixed with dust a part of which had been sterilized, another part not sterilized, and each mixed sample was dried in a large open flask and kept in the laboratory for 33 days. Each sam- ple was inoculated into a series of 5 guinea pigs: Series i. Dust not sterilized. Three of the 5 guinea pigs became tu- berculous only in the lymphatic glands near the point of inocula- tion 1 Series 2 Dust sterilized. Three of the 5 guinea pigs died of acute in- fection; the other 2 had tubercu- lous lymph glands near the point of inoculation, and one of the two had a tubercle in one lung 1 Again, the author examined dust from a tuberculous hospital: Series 3. The dust was collected from the cornice near the ceiling, wash-board, window-sill, window- seat, bed railings and floor. All was thoroly mixed, divided into two lots of .6 gram each, and placed in two flasks. One flask was protected from the light; the other was exposed to diffused light for 5 days and to sunlight 3 days. Each sample was inocu- ulated into a series of 5 guinea pigs. In each case only one of the 5 test animals became tuber- culous. Series 4. Four grams of dust were collected and treated as in Series 1. In this series 2 of the guinea pigs died of acute infection and 3 remained healthy. Series 5. Dust was collected and treated as in Series 1. 0.7 grams of the dust was placed in each of two flasks. The first was exposed 6 days 6 to 13 days 33 days 33 days J The tuberculosis may have been produced from dead tubercle bacilli. 306 BULLETIN No. 161 TABLE . Continued \ November, Author x j Date Conditions Not killed Kilk-d. L n uds n sxpos < i o cloi ar tl ed + + + f? + Excelh it ii Contarr Excelle Modera No gro Con tat :nt growth < i ination nt growth te growth wth ination mould ads ? le horizon i < f) O |i SSvo3?o -s a V s 2 c 6 3 a g>8 !s VO VO VO ^O iO - 'S * rO l^ rO 00 >H 00 00 00 00 00 \O O bo ^* ^ ,b S 00 O rO rO O w^ ~* ^ o.a rH CO VO I> 00 rH M c tc E (LI .5 O iO O tO Q >O 00 O W a 3 IO rH r-t ^ ^ O r-t rH M CO 1C 00 w i o rH c*5. O j d 5 "C 3 aj ^O i rO O rO vo rH 00 Li ni 3 M rO ) tfi l-O ON *0 ^ IO rH C-l -" 00 (kj O -- I* 55 ! b tc he "S ^j > Q 3 3 3 5 + +++++; : : : : CU rt &42 .2 ++ ++ +-I- ++ + : : : : : : 4 ^ M . HH O Q^D 1>CJ C^IJ "ti -*-* "3 to O D 1> V fl 8 o 33 33 33 ^CC ^5 SS SS 2-9 rt 11 11 11 r w i cti cti . , rt rt rt ^^ ^^ ,. |_j (UDiDiUDfUOO. ^ CflflClGfl..X3 OO OO OO 3S JH *o "O .fl g-s ^-g "g-s "g-g -g-g -g-g -g-g -g-g * k ^ fc ^ k ^ '^ ^ ^ ^ s *S *S *S ^S *S 'S *S *s --~ f to ^ C ri VO VO O O vo *O t'* 1 s * fO rO ON O\ vo 10 t'* O\ "^ ^ ^ .^ -^ ^t" O VO iO ^ -^ "^ vo to vo c*) H OO OO OO OO OO OO OO IH rt f inocu- lation II II II II II II II II ONON voiO vovo O\O\ VOVO OOOO ONON vovo f t^ t> t> i^- oooo oooo oooo oooo 1 3 /) d to O O t^- 1^ vovo i^--> sl y y ; ! + + + ; ! y \^ w 3f diseaset f s r'- to 60 ,0 4> .0 4> & o 2- 1 * S M P w 3 rH Sf TVT M w to o> i Oi P .be C > I -\ p *1 r T + ~t r 3 rt ftl II II II 1 1 II . Jl a '3- bo i % a n r ~l r +~r + T -\ r M I o w t-l Result of acroscopic v v v ^ "be t tn to "tn 'to 'to rrt 'to 'to ^^^OOO"da SS .2.2.2333 rta-33 a 33 COCO (Oo OO OO O ^0^0 ^0^! V*!- _ rH V(U H Uiu 1-|3 33 >2 'Sf-( ^^ Mn-t- 1 -4-<-t-> ^- 0)5 5"^ -*-*-*- 1 3 jra +** a*3 *3"3 aa ^ X3 ^>rj 'O'O ^" Crfl 'O'O *** 4) 0>0> 33 30> 0)0) a *vH.2 O)O> 0)HH hHhH V->"-4-> -tJ*J -- NN "*O * NN -MMH MHMH OO OF BOVINB ' S rii*i5*!jaaa c^aJaa DD Hlooo^^soo gg 00 W a *0 4) X -*-< rt o> O O * VO "bo 6 MH OO OO OO O O OO OO OO rH rH o 5 0) "rt Q- inocu lation ON CTs lAJ) lA'O C^ 0\ VO vi 0000 Oi Os >O yt Mf-1 rHrH N M rH-t rHrH Md rHrH tlti 0000 oi,00 00 ob C^C^ 00 rHrH J, ^ rH rH rH rH g H 6- bed ELfc t^ 00 ^ VO rO ^J" C^ rO l>" 00 CO ON O rH CO ON iO M") O ^O 1^* t^ 00 00 O O d C$ ^O *O t^ I s * !>!>. tI>. t>-t>- t> t~ 0000 0000 0000 0000 3 C w l* O to 0) f-> to rt &^ OO l^I> VOO rH rH ONON rHrH rOfOrHrH rH rH rH rH 326 BULLETIN No. 161 [November, the 8 1st, 1 23d and I7ist days of the exposure to sunshine showed the tubercle bacilli to be dead or at least not sufficiently virulent to produce tuberculosis when the centrifuge sediment was injected sub- cutaneously into guinea pigs. It thus appears from these tests that pure cultures of bovine tubercle bacilli, when mixed with cow manure and exposed in an open place in a pasture field, remained alive in this instance for ap- proximately two months. Exposed in the Shade. The part of the artificially infected manure exposed in a place protected from sunshine was tested on the same days and in the same manner as was the part exposed in the sunshine. These results are given in Table 14. The parts tested on the 7th, i6th, 3ist and 49th days were shown at each testing to contain virulent tubercle bacilli. The infection of the guinea pigs in the first two tests was severe. The guinea pigs in- oculated with samples taken on the 3ist and 49th days of the exposure were infected with tuberculosis but not so severely as the guinea pigs inoculated on the 7th and i6th days. However, they were much more severely infected than the guinea pigs in- oculated with the sample taken from the part exposed in the sun- shine and tested upon the same days. The three samples taken on the 8ist, I23d and I7ist days were shown not to contain viru- lent tubercle bacilli. Three of the guinea pigs inoculated with the first two samples remained healthy until killed on the 49th and 55th days respectively after inoculation of the sample. One of the two guinea pigs inoculated with the sample taken on the 8ist day died five days later with an acute infection. The two guinea pigs inoculated with the sample taken on the I7ist day died of acute infection with no evidence of tuberculosis. From these tests it appears that tubercle bacilli mixed with cow manure remain virulent to guinea pigs for 49 days after ex- posure in a place protected from the sun. All tests made later than this date showed these organisms to have lost their virulence. Virulence was retained longer in the shade than in the sunshine, as shown by the production of more severe tuberculosis in the guinea pigs inoculated from samples exposed in the shade than that produced in the guinea pigs inoculated on the same days with sam- ples exposed in the sunshine. FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 327 FROM NATUR- The tests of the samples of manure from the ALLY INFECTED tuberculous cow, both the one exposed in the MANURE , , , . j xi i shade and the one exposed in the sunshine, at no time after exposure produced tuberculous guinea pigs. The re- sults are tabulated in Tables 15 and 16. The sample exposed in the sunshine was tested on the I3th, 34th, 63d, and io5th days after first exposure. All the guinea pigs save one, which died of acute infection in four days, remained healthy until killed and examined 49 and 52 days after inoculation. The sample exposed in the place protected from sunlight was tested on the same days as the one exposed in sunshine, with one exception. No test was made on the I3th day of exposure from this part of the infected manure since there were not a sufficient number of guinea pigs available at that time. This sample was omitted because it was thought that the tubercle bacilli in the part protected from the sun would be the least likely to die. It was indeed unexpected that these bacilli would be dead in either of these two sampled at this time. It was a hot time in August. During the exposure from August 1 6 to 29 there was an average temperature of 72.32 F. and a rainfall of 2.32 inches, having six clear, one wholly cloudy, and seven partly cloudy days. A number of showers occurred, making it an excellent time for the growth of decay organisms found in the manure. The killing of the tubercle bacilli in so short a time was no doubt due partly to the antagonism of the decay organisms and partly to the weakened virulence of these germs. Slight virulence was shown by the producing of only localized tuberculosis in one of the two control guinea pigs inoculated with a sample of the fresh manure. More experimental data upon this subject is very desirable. 328 BULLETIN No. 161 [November, ' O p (A 3s + 3 "O o 2 s 0) /! 3 *" CL .Ho ^! y + ^ to . . CO 5 <4H - 6 o ~ -i o C 1 * jo 4, : : : : : : : : 3 w M D ^ H p o *J -1 rt %% . > a a) a; to tt u c r( ; too ,-c ........ * a B M ^8,55 00 * 10 0> -J ( i O 3 V* J3 + : : : : : : : i% ho -4 o (X Rl > a H 3 bo i H o 3 o i in JACII,!,! IN MANUI Result of o 'a o at c * "M! S CC rt X2 | ^ aj r s S H c 5^ >% o "-M Crt >,>> >^ >,>^ >^[^ ^^ a 55 5 5-g 55 55 H -r o w B M &. w Macro ^3 rtrt .5ni rtpj rtrt rt "3 g I-H Oil) 4> 4^4, 0)4, 4,4, o^ K- 13 ^K WK WK ffiffi s s 8 1 11 o ^ '3 3 >* s 3 H ^ H hJ to S-i O V *o x 4-> 4 *O *O _<'O 'O'O 'O'O *O*O 4, 4, ^1> 4>D DO DO O o a 0) c S S Q w Sw 22 SS K p I since inocu- lation O CO -*00 MN O\O\ CNON 00 -V VO iO IO tf * TJ- * TH Q 2 5 *rl 3jd vi ^o o\c\ o\o> oooo ONO\ T-I fH rlN rHrH >-i>H NM 00 00 0000 O\ O\ OO rH rH H H rH r-( 5 <$' 00 CT\ * >O * 1C VO t^ M CO 1^ l^ 00 00 r-l iH MM iO 10 l^ l^ 1^- t^ 00 00 00 00 00 00 ' c 1) 1* 3* d" t o >> * nJ o -o CH O O rOfO ** rOr<5 >OUJ HtH t^rO VO'O OO H fH 1912} FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 32 p : : : : : : Jrl j^ o 58 u . bco iH & 1 ( O &S5 00 ; I ) j, v o -i-> Is + : :::::: 1* o bo ( ->5 w 0. oj 4.2 t> x "^ p> L Cri^>% >, >, >^>^ >, >, rt ^>j3^ x j: XJ3 XJ3 OF BOVINE; TUBER Macros ^'s'rtrt rtrt rtrt rtrt g^HDl* OD 1) ft *O 'O 'O'C *O*O 'O'O D 1> OJCJ /5 O O >OiO iH M HH O OO OO OO Q vo r-( O 4> -4-J rt P inocu- lation O VO i^ON 0000 <^ON T T -TV -Tj; "7 00 00 C\O> OO r-irH -( tH ?-( l-l W a Or-l -HO 1> t^ r-t< tOfO lO'O I> 1> 0000 0000 0000 1) 1 ( l = 5 CO O to UJ P-.- ; tfl Rj a* O O ** r^rO UM/J CO c*) >O VO O O I 1 rH 330 BULLETIN No. 161 > [November, Series 2 PREPARATION A second series of experiments exposing cow ma- AND EXPOSURE nltre artificially infected with a pure culture of OF SAMPLES , . , , t . ATA, bovine tubercle bacilli was made, ine prepara- tion and exposure of these samples were made in the same man- ner as in Series I (see pages 319 and 320). The places of exposure were within a few feet from the places where the samples were ex- posed in the first series. WEATHER Table 17 gives the weather conditions during the CONDITIONS time of exposure of this series. The same plan is followed as in Table 12 giving the weather conditions in the first series. These conditions were just the re- verse of those in the first series, the first series beginning in the hot month of August, the second in the cooler and more moist month of March. No especially unusual weather conditions oc- curred during this period. Manure 'Exposed in the Sunshine. The results of RESULTS exposing this infected manure in the sunshine are given in Table 18. The tubercle bacilli were still alive at the third test after 45 days of exposure. Tho the tuber- culosis produced in each of the two guinea pigs from this test was only slight, as shown when these guinea pigs were killed and ex- amined 53 days later, Guinea Pig 973, inoculated with the dis- eased tissues from these two, produced severe generalized tuber- culosis. Typical cultures were obtained from the diseased tissues of both the original and the secondary inoculated guinea pigs. The samples taken after this date produced no tuberculosis in the six guinea pigs inoculated. Manure Exposed in the Shade. The results of exposing the sample in the shade are given in Table 19. Here we find that the tubercle bacilli were alive for 73 days, while in the sample ex- posed in the sun they were dead at this testing. Also the test made on the 45th day shows more severe tuberculosis in the guinea pigs inoculated from the sample kept in the shade than from the sample in the sun. i. As shown by the results in both series of ex- CONCLUSIONS periments, a pure culture of bovine tubercle bacilli mixed with cow manure and exposed in the sun- shine in a pasture, remains alive and virulent for approximately two months. 2. The virulence of the tubercle bacilli in cow manure was re- tained in the samples protected from the sunshine longer than in those exposed in the sun, as shown both by the increased length 1912} FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 331 0> t- 3 O iH ON 'f -l H 2 * j O iO * rO * 00 i SI'S ^s r*5 O\ 00 l^ * iO CO fO rt VO t> t^ n O M * CJ ro T- 10 o O W n .e CO C 3 | S H c a g* D * S e N rt * i/> to t>. 00 X Q W 5 < S 1 7 "a P *J S ^ 3 01 *" - 4-1 > S H "^ rH O >O * Ot^ W W 0> a o'S* 3 *- cfl * p | " a H -M S tfl a -4-1 co S 1 o Q ^a bc'S- B| -(-> rt O\ ro rove 00 M vo t>- t- O\ O\ O rH W G / i C CO - "o c c iO iO t- rO C3 CN Oi C^ O O> O / E g- s CO iO t> t^ 00 rS V i2 -5 K 3 <4-i J3 M VO vo ro ON M - 3 '2 ^ JQ ^ fe X? JS lr< i C t-t O N M 1 1 . 3 & i CO ^ ^O ^O vO vo P j j 60 C. 5 M K*l >^ s Q ri t rt o 3 oo o vo rj * * fc ( 5 cu "y o x = i r J ^ d ; CO O * f) rH t> I-H T-t C-l rO * 9 C, ^ J W ^ "C 1 tc 1 K_ X bo n a ^^^is T ~ ( P x W * M <4H ro M ^ *j OJ O t^. Ct) CU t" 332 BULLETIN No. 161 [November, ' 4> 3 + + : : : : : CO d 2" W fc i 3 u 11 fcH O + + : ': : : : M co 9 r fc in 71 o a. -*-> 4) *-> D M Q O S " j3 4) j3 4) * ' 4) CO 4) to '. ' o o W O ' 5f 2 S : : : : ~ " <* : : : P? to W 0) 4-" 3 2 + + 1 + + B be ( J -^ P. , S rt 2 '"* i i i i i i ' \ 1 *g 1 1 + TT ~r + : : d be 1 RCI,E BACILLI IN periment 2) Result of o 'a o 4) 4) 4) W - '- 4> 4) 4) CO CO CO ^ CO tO CO .2* "0*3 's'S Kt^ >.>> >> >> DO OO to 01 J3J3J3XJJ3 w ^ p W I O oq o w Macros .3,0,2,0^49 rtrt rtrt rt rt 3 VH VH r\ *cJ 'O *o tu 4> 4> a c a 4) 4> 4> M ta "o V TJ ^-S -S-S -8 S W -g-g -2 "S O fc 3 4) o ^M MM SH M Ld't^d ' ^ "^ Ld Ld M 1 &l inocu- lation iO *O *O ^-O iO ^* O\ *O \O ^O fO T 00* rH W MH O -4-> rt Q inocu- lation vrfi iii T 7 r i H ii i j, t* t^ fO CO tO CO rH rH t^ t^ *> 00 C^O^rOcOrr^r ujx/j \o 1 d II rO ^^ rH C^ 10 vo ^^ 10 ^^ *O ON VO rH rH C^ d ^ ^)" ^O 1 O t^ t^* 00 rH O\ ON ON ON ON ON ON OA ON ON O\ o rH i < 1* B O cfl > tn rt 1* OO rfrj- 10 iO fOrO QQ O vc rHrH 't *4- l^t> OO rl O rH rH rH rH 1912} FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 333 -3S 3 3 CO VV +J CO ++ + : 6.2 fd kH O 4) f^ CO cfl . llation of d Macro- scopic lip : "i"s * : 1-1 S : ^" ^ r h o O bp O M ri c^ I>t^ CJ C\ C7\ O i-t I \ u *3 + + ++ + : ' > . i guinea p 6.2 i + ++ ++ + jeriment 2) Result of o o 1-1 U U ^ > > t2 <2 CO CO CO 'S ' "2 Q 'S 'co 'S o o "rt "-S ^ 3 -Ij 1> 1> O O 3 33 42 X! PV-H ^ >-> >^ >-> cpo oo 33 <^ ^Ci^ J3 ,C t * is ) f 1 1 1 4 ) Macros . O O ,Q *^ '^ Cw tO ro Cy Cw rt - 44 4)4) ~ CU4) CU 4) |2 54 NN B3 W W v ^ 15 22 *"* ^5 N K N QJ il) * rj rt nJ I 5 s| ag aa sa aa a a 4 4 ( S" 3 ^ ri O i S H o 'J a ss ss KS ss e K 5 1 "o 3 C 4) ^^ IL 31 HH ^v vt v v !>!> tOrO tOfO rHr-t tt- 1> 00 \ 4 M ^ S3 ri C<1 N fO rO * 4- VOXO vo v> vo 00 J 3 3 4 oil rO"* rO^ rO^t voi> vor- O t> C\O\ O^ O\ O\ O} O\ O\ O\ON O\ O r-( S 4) 55 rt rt O -o OO ^" "^" lO'O fOrO OO O VO r-t r-t ^f'^- t-l^- OO M VO 334 BULLETIN No. 161 [November, of time that they remained alive and by the severity of the disease produced in the guinea pigs inoculated upon the same days from each of these samples. This difference, however, was doubtless due mainly to the difference in drying. 3. Tubercle bacilli in the manure of a naturally infected cow were dead within two weeks after exposure. More experimental re- sults are necessary before trustworthy conclusions can be drawn. 4. Danger of infecting farm animals from tuberculous ma- nure is indicated. i DURATION OF LIFE OF BOVINE BACILLI IN GARDEN SOIL DIIDDOCC Some investigators state that tubercle bacilli will KUKrUot .,,.,, , . , , OF TEST live in soil and in dead carcasses buried in the soil from two to three years. If this be true, ma- nure from tuberculous cattle when put upon cultivated fields which are later used to pasture stock may be a source of infection for farm animals, especially hogs. For this and other reasons it was thought advisable to test the length of time a pure culture of bovine tubercle bacilli will live in garden soil. The culture of bovine tubercle bacilli was of the CULTURE same strain as that used in the experiment forarti- fically infecting the cow manure. About four milli- grams of pure culture were obtained from a four-weeks' growth on the surface of two large tubes of glycerine agar. The organisms were carefully removed from the glycerine agar and emulsified as described under "Cultures and Emulsions," page 315. CA .. DI c The sample of soil with which the tubercle bacilli oAlir*LC . . ...... OF SOIL emulsion was mixed was obtained from a garden plot that had been in cultivation only two years. Previous to that time, this plot of ground had been in sod for at least fifteen years. The part from which the soil sample was ob- tained had been well manured with horse manure the first year it was under cultivation. The second year no manure was added but the ground was well stirred and made into a lettuce bed. A suf- ficient amount of this soil was obtained to fill a ^4 -inch mesh wire basket having the dimensions of 4x5x6' inches. This required 1700 grams. This pulverized soil was placed in a large pan and the emulsion of tubercle bacilli sprinkled over it. These were thoroly mixed by being constantly stirred for some time. The amount of emulsion was sufficient to make the soil quite wet and stickv. FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 335 PLACE OF g rams ot this infected soil were removed to EXPOSURE be tested for tubercle bacilli as a control. The re- maining soil was put into the wire basket and covered with a wire gauze. It was buried in another garden that had been under cultivation for one year and had received no ma- nure or fertilizer of any kind. The place where it was buried had been previously well pulverized. The basket with the infected soil was buried six inches under the surface of the ground. TESTS OF Samples of this infected soil were tested for tuber- SAMPLES cle bacilli on the first day of exposure and on the 7th, 1 6th, 34th, 55th, and thereafter about once a month for 352 days. Ten grams of soil were removed by dig- ging down beside the mouth of the basket and with a sterile pota- to knife making an opening to the center of the basket. After the removal of the sample, the opening was filled by pressing. the soil in around this opening with a potato knife. The wire gauze was placed over the mouth of the basket and the garden earth filled in over it to a depth of six inches. The soil sample taken to the lab- oratory was thoroly shaken with 200 cc. of 0.8 percent salt solu- tion in a 300 cc. flask. After standing ten minutes until the coarser sediment had fallen to the bottom, 40 cc. were removed and placed in two sterile centrifuge tubes and centrifuged for five minutes. The supernatant liquid was drawn off and put into two other centrifuge tubes and centrifuged for thirty minutes at 2000 revolutions per minute. The supernatant liquid was drawn off and discarded. The last 5 cc. of the liquor and sediment were thoroly mixed and in- jected subcutaneously into guinea pigs, in graded doses, giving one i l /2 cc., another i cc., and for the first test a third received y 2 cc. RESULTS OF ^ e resu l ts considering the length of time that THE TEST tubercle bacilli live in the soil are recorded in Table 20. The guinea pigs inoculated from samples taken on the day of exposure, and on the 7th, i6th and 34th day after exposure showed in each case, when killed and ex- amined, severe generalized tuberculosis. Microscopical and cul- tural tests showed the germs from the diseased tissues to be char- acteristic of active, living tubercle bacilli. Tests after this time indi- cated a weakening in virulence, but slight tuberculosis was produced in the test animals from material taken on the 21 3th day of expos- ure. Microscopic, cultural, and guinea-pig tests from the diseased tis- sues of the original guinea pig showed the tubercle bacilli in the soil sample to be active and virulent. Five tests were made after this date. In every case the guinea pigs, when killed and examined, were found to be healthy. The two testings after the last one in which tubercle bacilli were found, viz., the ones made on the 336 BULLETIN No. 161 [November, ' 0) "r; u 3 3 3 + + + + * O-2 * o 6 H ^ &'< S " + . . + + + 8 *8 ; o "o P H Is ! I T 4) "2 ,0 t) a w 2- -M 0) -t-> U -4J D -> V T O rt S .y 1^0 M ^5 tC Co c 4> tfl O 1 S 4J C 4) O O O =. 4) O V tfl 3 O . 2 vo vo t> 3 J3 ^'afc 8 oo oo 00 R 5 j 3 4> 4 -*j ! j 3^ o5 +++ ++ ++ ++ + + + d -BsuinS 1 KTT T*TTT~I Micro- scopic +++ + -h ++ ++ + + + + jo ;insa^ vrr 'cr^T-wr'a-cr n T *ErkTTAr\rr af\ x TTTTCTVT Macroscopic Tub. only in R.S. inguinal, slight Tub. only in R.S. inguinal, slight Generalized tuberculosis, severe Generalized tuberculosis, severe Generalized tuberculosis, severe Generalized tuberculosis, severe Generalized tuberculosis, moderate Generalized tuberculosis, severe Generalized tuberculosis, severe Tub. at point of inoculation and in R.S. inguinal Generalized tuberculosis, not se- vere Generalized tuberculosis Tuberculosis only at the point of inoculation ^ SH ox V +J irt flj *o *d *o JH ^ 'd'O 4) to -a D V T) -d O O\ f) CO * M M * * VO vO 1> ro fO 1C IT) % c?5 000 O O O O O o O o 3 a iJ o-2 -M O -(-1 rt C nJ Q.^^H $& -*> 33 otot 1 1 i-H rH rH r-1 a^oi, ^^ C5 M 0000 c^ ck iH -l .^ d. 10-20-] 10-20-] rh S?0 0*S,J5 *r\n vo XO iO i/) T-l M 10 o i> i> 1> 00 ^o *c t^ l^ o I> oo oo t> t^ 00 Oi TH T-l oo oo M ro 00 c<5 ro 00 ( v a, ^ ? 815 rt g-0 C * O- i> i> vc ^o r-1 rH fO rO 1C iO O 00 00 FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 337 TABL.E 20 Continued. + + 'The right superior inguinal enlarged in the third and fourth week and then became normal again. + + Acute in- fection 2 ~ - C i> 0> CO rQ 41 3 4- QJ C 4> i + + + + + + : : : : : : : : Acute infection Generalized tuberculosis Slightly tuberculous Acute infection Acute infection Acute infection Acute infection Tub. in R.S. inguinal and spleen Healthv 1 " cdcd rn . - cdc^ ~ HH HHH4 ffiffi pHMH HH 13*0 *O*O f O *Od; 1> C i3 g S fifl 03 gg gg gg gg g C4 C^ d *O C^ *O CO T< t 1 ^ !> fO tO iO '-O TH 00 rO 32 Jl r^ dlfJl 4 TH TH CO tO C^ r-l N n M ri M . f rH i l TH TH i ii ii ii ii i "t tOtO fOrO r-lCO^O I> TH I> t- 00 00 TH TH tO to OO TH TH OQ 0000 (S rO tO iO i/) O^ O\ THTH OrO VOO C>^ THTH to THrH TH TH THTH C-1CS C^C^ C^d C1M COtO tO 338 BULLETIN No. 161 [November, 2301!], and on the 261 st days of the exposure, produced in the third and fourth weeks an enlargement in the right superior in- guinals of each guinea pig inoculated, which then became normal again. Tho it cannot be surely known, it may be that the number of live tubercle bacilli was so small as not to cause the disease, but of sufficient number to produce an enlargement of the glands. Three tests made later than these two showed no such enlargement of these glands. BOVINE TUBERCULOSIS IN A DEAD ANIMAL, crtnorc re Besides the exposure of the pure culture of bovine bUUKL/t \Jr * _ r MATERIAL tubercle bacilli in garden soil, a guinea pig that had died of bovine tuberculosis was exposed in this same soil. This guinea pig was extensively tuberculous. It was placed in a flower pot and covered with a screen of wire hav- ing a mesh fine enough to exclude earthworms. The screen was pressed down close over the guinea pig, which allowed the fine gar- den earth to come into immediate contract with the dead body. The abdominal and thoracic cavities had previously been opened in making an examination of the guinea pig just before placing it in the garden soil. METHOD OF ^ ^ e time of the first test, made the 7ist day of TESTING the exposure, the tissues of the guinea pig were so decayed that the flesh and the skin were easily torn. A part of the tuberculous lung and of the right superior in- guinal lymphatic was removed for the test. At the next test, on the 99th day, most of the soft tissues had been carried away by small ants. On the i33d day only the bones, hair and some ten- dons remained. A few pieces of bones and a bunch of hair were obtained for this test. The results of these tests are given in Table 21. RESULTS Live, active tubercle bacilli were found on the 7 ist day; after this date no tuberculosis was pro- duced in any of the guinea pigs inoculated. No doubt if the small ants had not molested the soft tissues, tuberculosis would have been produced in the test animals at later dates. While no final conclusion can be drawn, it is evident that these germs live a suffici- ent time in dead tuberculosis animals to be dangerous to stock. FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 339 -3 P ^ 3 !0 * J + o P-- 0) ** O. 8 s8 Stfl + llation of d Macro- scopic s P t- > V ffl CJJ o o be o C CD '. 1 rH ri 0) 5s + + Micro- scopic + ++ Macroscopic 4) 0) 0? 0) 0)0) CO 10 tfl CO 5 tfl g cfl tfl to o T-l T-H 1 ^J P PP >>>>,>: J3 J3 J3 J2 ^,Q^ rtrt rt rt.rt -M 55 aw ffi w^ *O *O *O o 0) 0) 0) .- N N N ^i ^ nl ^ n3 ra re ra U V- V- 0) 0) 0) ~ a a 0> 0) 0) O 00 0) M ^ 'l s 0) 0) 0) 0) 0) i$ ''& >3 kp Q A KM KM it W 3 S b" .2 2*3 2*1 Q ^.5 J2 rH \O-3- rHrH rO 00 N iO 00 t"^ VO vO ^J* CO o 3 a D 0,0 n ri ^ 3 f*. O\ ON f<5 rH rH rH rH rH rH 1 O VO | II >O t- 00 00 ll O\ O^i O\ ON ON O^ O O rH rH rH rt ^ O -o rH rH ON ON cO d Cl t> pN ON to vo vc rH rH rH 340 BULLETIN No. 161 [November, DURATION OF LIFE OF TUBERCLE BACILLI IN WATER PURPOSE AND ^ ne P ur P ose f these tests was primarily to deter- PLAN OF TESTS mine how long bovine tubercle bacilli will live in a watering tank such as is found on the average stock farm in Illinois. In connection with this work it was desired to test the length of time human tubercle bacilli will live in drink- ing water. With this in view experiments were planned and car- ried out with tubercle bacilli from the following sources : 1. Pure cultures, Series i. 2. Pure cultures, Series 2. 3. The diseased tissues of a tuberculous guinea pig. 4. Tuberculous sputum. Series i. Pure Cultures CULTURES The bovine culture was the same as that used in the cow manure which was exposed to weather conditions. The human culture was taken from the tuberculous glands of the neck of a patient from the Burnham hospital and iso- lated in this laboratory. The tissues were received May 25, 1910. A pure culture was obtained by inoculating a guinea pig and cultur- ing the diseased tissues of the guinea pig. This culture, which is typical for the human type, has since been kepi upon glycerine agar. SAMPLES OF The sam pl e f water use d to make the emulsion WATER for exposing the bovine tubercle bacilli was ob- tained from the large watering tank used to water the dairy cattle of this station. This tank is 2^2x4x10 feet, and is supplied with running water. A considerable amount of spiro- gyra and various kinds of smaller green algae was floating in the tank. Diatoms were also abundant. A green scum covered a large portion of the surface of the water. The sample was ob- tained in a 500 cc., sterile, cotton-stoppered flask. The water in the tank was slightly agitated with a stick about as much as it was thought the cattle Wtf'n* stir the water by drinking. The mouth of the flask* was pi 1 iQ'ed under about six inches and then allowed to fill. The sampl -vas at once taken to the laboratory and the emulsion prepared aat same afternoon. The spirogyra died soon after the emulsion was made, but the diatoms and the smaller green algae, as well as numerous bacteria, were present at the conclusion of the experiment December 5, 1911, 586 days after the experiment started. The sample of water used to make the emul- sion with the pure culture of human tubercle bacilli was taken from the tap water of the bacteriological laboratory. There were at the 19*-'] FATE OF TUHERCLE BACILLI OUTSIDE THE ANIMAL BODY 341 time some algae and bacteria present in this water, a part of which were still living at the last test made. PREPARATION emulsions both of the bovine and of the OF EMULSIONS human cultures of tubercle bacilli were prepared in the same way. About two milligrams of a four weeks' culture grown upon glycerine agar were emulsified in the manner described under ''Cultures and Emulsions," page 315. The emulsion of bovine tubercle bacilli was diluted to 350 cc. with the tank water obtained for this purpose; 250 cc. were used to expose in the running water, and 100 cc. to expose in standing water. In a similar way a 250 cc. emulsion of human tubercle bacilli was made. EXPOSURE OF SAMPLE For exposing the emulsions in running water two 6-inch flower pots were obtained. The small opening in the bottom was stopped with a cork and this was sealed over with boiler paint. A test of the two flower pots thus prepared showed that they held water. After immersing them for twenty-four hours to within two inches of the top, no water rose on the inside, tho it became very moist. The two emulsions were now poured into these two flower pots and FIG. 1. THE VESSELS AND THE Poor, OF RUNNING WATER IN WHICH THE TUBERCLE BACII^I WERE EXPOSED. The intake is at A; the outlet at B. The submerged vase C has been placed upon the shelf with other vessels so it can be seen. The flower pot D, which contained the tuberculous guinea pig, is placed upon a brick to bring it into view. The two unglazed cylinders and one of the flower pots show the effect of freezing. 342 - BULLETIN No. 161 [November, each flower pot placed in a gallon glass jar containing water and immersed about three inches, so that the level of the emulsion of tubercle bacilli on the inside was the same as the level of the water on the outside. The water in the glass jars around the outside of the flower pots was kept continually running. While this procedure does not give the same condition as water running directly into the emulsion of bacteria, it allows to some extent the circulation of the water inside the porous flower pot with running water on the out- side of these vessels. Another part of the emulsion of bovine tu- bercle bacilli was kept in a cotton-stoppered glass bottle. This bottle was partly immersed in one of the glass jars of running water. The water could in no way circulate in this bottle, but it was kept at approximately the same temperature as the emulsions in the flower pots. The samples were kept in the laboratory until April 15, 1911, 250 days after the emulsions were first made. It was then found neither convenient nor desirable to keep them there longer. A pool was prepared in the courtyard of the Agricultural Building, to which they were transferred. The pool was made by sinking a large tile three feet in diameter and filling in the bottom with concrete. Constantly running water was maintained to a depth of twenty inches the year round. It did not freeze any time during the winter of 1911-12. A shelf of slate was placed four inches under the surface of the water on which the vessels containing the tubercle bacilli were set. .The pool was screened against flies and other insects. (See Figs, i and 2.) FIG. 2. THE POOL SCREENED AGAINST FLIES. i<)i2\ FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 343 -reo-riMf TUP Nineteen tests from these samples were made for TESTING Tnt , A . SAMPLES the presence and for the virulence of tubercle bacilli. These tests were made on the 9th, 23d, and 44th days, and thereafter about once a month until the last test, which was made December 5, 1911, 586 days from the begin- ning. The sample of the bovine tubercle bacilli kppt in the cotton- stoppered bottle was tested for only 202 days, since the sample was exhausted at this time. This sample was not transferred to the pool. Each of the three samples was tested upon the same days and in the same manner. The water in the two flower pots was thoroly agitated by giving it a circular motion. With a sterile glass rod the sides and bottom of the flower pot were scraped so as to loosen adhering sediment that might contain tubercle bacilli. The water in the bottle containing the bovine tubercle bacilli was thoroly shaken before the test sample was taken. It was not convenient nor thought necessary to rub the inside of this bottle, as was done in the case of the flower pots. With a sterile Pasteur bulb pipette from each of the three containers approximately 5 cc. of this in- fected water were removed and placed in a sterile centrifuge tube and centrifuged for thirty minutes at a high speed. Four cc. of the supernatant liquid were removed and discarded. One or two drops of the sediment were placed upon a glass slide and a micro- scopic preparation made and stained for tubercle bacilli. The remaining sediment and liquid in the centrifuge tubes, about i cc. in quantity, was thoroly mixed and injected subcutaneously into a guinea pig. The same precautions were taken with these tests as in the case of the tuberculous manure to guard against mistaking tubercles produced by dead tubercle bacilli for those produced by living, virulent ones. Microscopic preparations and cultures were always prepared to test the diseased tissues of the infected guinea pig for tubercle bacilli. In a part of the cases, however, the tuber- culosis was so extensive that it was not thought necessary to make inoculations of diseased tissue into another guinea pig. RESULTS OF ^ e resu ^ ts f the tests to determine the length of THE TESTS time tubercle bacilli live in water are recorded in Tables 22, 23 and 24. With the first four tests of each of the three samples containing tubercle bacilli the guinea pigs became extensively and severely tuberculous, as will be shown by an examination of the three tables. Further tests of the dis- eased tissue from all these guinea pigs showed the tubercle bacilli to be living- and virulent. The fifth test, made after 126 days, showed at least an apparent weakening of the virulence of these germs. 344 BULLETIN No. 161 4> /: tn J, 35 + + i ! iseased ti 1! + ; : -3 MH O a .2 Macro- scopic S S s : - S -S S 3 5 jj ~ v .2 _4> .2 " '-g j* 3030 t34) c I-H oil i 8 ;. i I r. 4) -> bo 1 i a> + + + 1 - + + + guinea p 1 Micro- scopic + + + + ++ * + + Result of croscopic 4) 4)4)4)4)4) 4) S3 *3 IH bhkiUWi VH O O 8! ifl 09 10 10 00 01 "3 *3 ^-^-. ^ 00 09 tCtflEwCOT) tn OO "3 '3 '3 *3 '3 '3 "3 5 c c o .2 *3 p e P "3^ "3" o *o "o O OOOOO O fli j_. d - - i-i s-i U.J -^ -^ (U ^ ^ C^ ^ CJ 1 V ^9 Q C3 3 " a a ti *rt Trt*rt*r1 t Cl T r1 TrtS'^^ D o a -o -a o Tj-a -o -3 -j 4) 4)4)l>Ji> ^^^^ s a a *9 M s --i .-. .- rt -*^ H ** r^J B ^ ^ ^\ ^\ ^\ M WWW^W W QQ :H 3.1 O-M E .2 O\ -i n to 10 M n -* M -t fsj If) 1Q t!) ^p t^ ^ Cl Cl MH O OOOOO OOrHrH O "rt inocu lation *i i i i j. j. i- i i T I/) ON^OOO rHrHCNM iH MrHNrHr-C fOrO 00 00 ON O M N n N CS rH rH rH rH rH < bod t>- OrHVOrHC^ MrOtOTh t* O rH tO V0 VO 1^* t^* C^ C" . 1> C^ 00 X 0000 00 00 00 00 ?" ' < v >. '1$ * rl 5 15 ^^ l - ^ 00 S rH rH r-( r-l r-> rH FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 345 VH "T" "T" ~~t~ a a rt + + + + O C he 2 .04, ^4, -d ^4, o S 3 Wi 3 l- 2 3^ *Tj *-> i> --'0) g -t- 1 D 4) C C D C D 4-> C D H) > > > ^ d 3 4) 4) 4j D rt to tn at tn C *5 r ^ ~ "S g U3 tfl ! P I W ^c E _C ^ .n ..H .11 hC ^ M " .2 ^ - - X "g^ HH O C 1 = * 3 3 o 3 t || S | | pi | > -t- , > "rt 41 1 ^_) o C C > " C attemp seeded ^_i 4_l +* -4-* *O -4- 1 -r- 1 ^ -4. rn EB BE fli W 3 < 9 < S V C^ 1> 1) *4H ^ 4) .O ^5 ^H *iH '*^ ^- C CJ rQ TH ^ ho )-, U( V-. U. 3 0) t-i C G C G ^ 1 2 he 3 D D I 1/5 TJ- rH * >o M 00 D ^ rH III 1 II 1 ,!< 2 4. 3 rt ^ M r> 33 3 3 vi S 3 3 rH rH ^ rH |l rH rH i-i O ^"* C^ rH l/) t** O rO rO ^ *O 00 ^ C^ JO S s S d-S "I ON ON (^ ^ O\ O\ OS O^ O o o rH 3 tn 1 o r) o 8 rH rH CO 00 l> rH O rH t^ U5 VO - > c t ^ Cl M C3 Cl O <~O ^- Tj- ^ U5 1/3 28 + < 346 BULLETIN No. 161 [November, issue 3^ o2 + + : : + w WATER iseased t Micro- scopic + +:+ + N RUNNING u S-l C _0 J2 g Macro- scopic ^2 $S : : - it" 4J D -MO) -MO) (/) > . > . > p CD gtn a)* 3 ^ o o o " M Q W O C M ;. be 6 ^ua ri >o "i- : : i> o VO CO MM oo oo co ; ; o\ o\ 3 & tO d> 4-1 he ^ < ^ s Is -4- + + + + : :+ + - *I,OWER P guinea p i otdoos -OJOTTAT + + + + ++++- + + W ! fc H M HT o +> 3 .c J5 ij Jj > > > > hchc > >-. o NJ N NN NNQ q. w fc rt rt cti cd rtcti rt cd n "w M o M to D H)l>fl>4)DC 3 s ccnsGccB^ D D4>4>4>4>4>4) ^ O O O O OO OO H O W to <<-. O. ' C M *O 'O'O'O'O'O'C .'O 4J (U 4) 4) 4)t> D'O D'O h4 A o 1- V o W WWMWwM^M fc 2 M I \ i C since inocu- lation o ONtHronr-ii^o-^-io ^f 'J-xOiOr^fOiOiOfOfs) M ^ , O OOOOOOO^itH & q fO M ^ z ^ &* C o *5 (4 r . i ^ i i^..ii 4 f co ot c^ i cU MC!| n N i-l I rH rH rH W 3 B r 6-1 :i 'O OOO\"*-t^OOOOON^O !> CO O CO >O OVOOt-t--OO M <* 1 MM "t * 00 00 1 tf 1 I O." 1912} FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 3>4", I ' + ': I ! : 1 Not enough material for cultures. * 4- '. * . . "3 v a v > > > V 4) .2 f 1> 1> ' K ^ O . . ^ tn ** to co cc ^g in <_) t/i tnt/i 3 o ^oyox-CJ^-cc* D 4 "'4J4)D' I^HI-H' ' *^H -O-S^^^wSSoS 2 o V f> V V I **-' .2 v .2 .2 .2 H 1 | "H 7 2 g - < ^ u) vo I in N C* ro rO -riO t^-OOOOON ^-(^i o o r- n * ^ sG\^OOOOO'~< o o ? 1 < M 00 (X in -i- 1-- I e-i C4 r i i s i s s ii 348 BULLETIN No. 161 [November, 1 4) . ..... "3 Wl + 1 3 i '/} Vj -M o 6.2 4) ^ Q. ' i * Ml 2 ~T" a) kH O 4) tf A tn , -5 M *o I'l i-t 4) r-f 5 - S W c ** D Ij < S ' : c > c ^* *+J ^ f " * r/> M Q C O 'g,^ oo oo 5\ O\ ! 55 < (/} M <&> 4 *4 4) 'a . M .2 ^ o j- -)- Jr )- -| [- | |- O '3 J W ho w <*H O 4) 4) 4) 4) 4) 4) .T ^J b W . g Vi WiJ-i ,.. fj 1 4)4)4)4) 4)4) tfltfl -y. 3 ^ t-' ^ K* ^t* 4)4^ W pq U) ^^ 'to 'S 55 o w o d 55 H > a o o "3 "3 "3 "3 ..'S's "3"" y o u y (flto oy > M s 3 !) V-i t-c )-i VH *S'S '-'J-i "-!J3 V H O 0) PQ 2 y _3 3 3 3 13 "3 33 *3 -J ' &t o w rt 8 _. )- J-i 4) iu Q 3 T O T OT3'Q difli'D _ . ~ *\ 4) 4> 4> 4>^3J3 4>4) fiT< j .2 .2 .2 .2 33 .2 .2 " -S **< >-> o M ^> cd R) RJ n) ^^ ^^ rt co -4-) i . fl r"^ M C .C C wi-i 3;: 4) o 4)4>4)4> 4)4) o^ aces sa Jij; ^>QJD4) 4)4) ^t*^ ja 55 c5 O O cb c5 O o M *o V rr-t tt t-> a 4) 4) 4) 4J 4)4) 4)4) T O T O 4) *O Ui T3 txJ Li M ^ tsii^ v v QQ Mfi CTJ E 4) c< W i-f " i i C 90UTS inocu- lation VO ON d CO Mf^ rHrH ^*'1- 00 t^ !- 13 M-t OOOO OO OO r-l rH r-l rH -0 ^ O i- ! C 1 '+3 rH M -H CJ rHr-( COcO T J fNM i 3 3 r oo oo o\ ON t> 00 rH co uivjS I>1> ONC^ OO Ri H^ 1> 1> 00 00 OOOO OOOO OOOO ONON tfl a 1 i : o 'd' t 4) P-, tfl rt o\ to ^^ u^ vo vo t-* t^ ^5 o c^ c^ M * 1> cNcM M-* OOOO OO rHrH rHrH rHrH CNN H - c i 3 & i<)i2\ FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 349 The tuberculosis produced in the two guinea pigs inoculated at the fifth testing from the sample of standing water contained in the cotton-stoppered bottle, was neither extensive nor severe. Those from the running water had a higher virulence than the tubercle bacilli in the standing water, but the tuberculosis produced was not so severe as that from the former samples. It should be noted that the time of killing these guinea pigs was much earlier, and it may be for this reason alone that the virulence appeared to be weakening. This fact was noted at the time of killing the four guinea pigs inoculated with the bovine tubercle-bacilli-in- fected waters, and so the two guinea pigs inoculated with water infected with the tubercle bacilli of the human type were not killed at this time. Thirteen days later one of these t\vo guinea pigs died with generalized tuberculosis. Unfortunately the death of this guinea pig was not noted until a short time afterwards, and it could not be determined whether tuberculosis was the only cause of death. The other of the two guinea pigs inoculated with the sample of water containing the human type was killed 72 days later, and the examination showed severe generalized tuberculosis. Later, on the I47th day, we find severe generalized tuberculosis produced from each of the three samples. After this date only two more tests were made from the sample in the cotton-stoppered bottle, one on the iSoth and the last one on the 2O2d day of ex- posure. These two tests show the lessening of the virulence of the germs : for from the former no tubercles could be noted 24 days later, when the, two inoculated guinea pigs died of acute infection; from the latter, one guinea pig died too early to make the test; the other, killed 78 days after inoculation, showed only a doubtful sign Of tuberculosis. However, an inoculation of an emulsion of the tissues from the point of inoculation and the right superior inguinal glands produced, in a secondary guinea pig, gen- eralized tuberculosis. From the diseased tissues of this guinea pig an active culture was obtained. Thus no end point was reached from the sample of standing water with the bovine tubercle bacilli in the cotton-stoppered bottle. The other two samples, kept in porous flower pots which were transferred to the pool in the court- yard, remained alive for 441 days, but were dead on the 47Oth day. From the emulsion of the bovine type severe tuberculosis was produced in the guinea pigs even in the last test in which these germs were found alive. From the emulsion of the human type only localized tuberculosis was produced at the last test. The sec- ondary guinea pig inoculated from the diseased tissues of this last test animal showed severe generalized tuberculosis, and cultures from the diseased tissues of both the original and the secondary test animal showed these germs to be active. No test made later than this showed any indication of live tubercle bacilli. Five such negative tests were made. 350 BULLETIN No. 161 [November, Scries 2. Pure Cultures Before the close of the first series it was recognized that these experiments were of such importance that a repetition of the same was advisable. PREPARATION ^ ie cu l tures use d and the preparation of the OF EMULSIONS emulsions were similar to that in Series I. Emulsion of the Human type. The sample of water used for the emulsion of the human type was obtained from the pool June 15, 1911. With the sample was obtained also a considerable amount of floating green and yellow sediment scraped from the inside of the large tiling enclosing this pool of water. A microscopic examination showed very abundant algae anabena, diatoms, desmids, scenedesmus, and confervoideae; also several kinds of bacteria. Among the animalcule there were vorticellae, amoeba, a few paramoecium, water-eels and rotifers. There were thoroly shaken 450 cc. of this algal water to which was added an emulsion of approximately 5 ing. of human tubercle bacilli. This sample was divided into two equal portions: one portion was placed in an unglazed earthen, cylindrical jar eight inches tall and three inches in diameter; the other portion was placed in a small, unglazed vase which was stopped tightly with a one-hole rubber stopper in which was inserted a small glass tube that reached above the surface of the water and allowed the escape of any gases that might accumulate in the vase. This vase was placed on the bottom of the courtyard pool twenty inches below the surface of the water. The other sample in the 8-inch jar was placed on a slate shelf four inches below the surface of the water. This jar was open to the sunlight but the direct sun could not reach the surface of the emulsion inside. During the winter the part of this jar projecting above the surface of the water; was crumbled by freezing. (See Fig. i.) Emulsion of the Bovine type. The emulsion of the bovine type was made in a way similar to that of the human type. Five milligrams of these organisms from a young culture on glycerine egg were removed and rubbed up thoroly in a sample of 225 cc. of water obtained from the drinking, trough at the dairy cattle barns, and thoroly mixed. A microscopical examination showed the following: (a) vegetation spirogyra, oscillaria (large and small species, the small species being very abundant), a few dia- toms, abundant protococcus, desmids, scenedesmus and anabena ; (b) animalcule amoeba, paramoecia, stylontia and vinegar eels. This sample was placed along with the emulsion of the human type in a similar cylindrical jar on the slate shelf below the sur- face of the water in the pool. FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY JS1 TESTS >c The results of the tests of the bovine and the r 1-1 ill- human types exposed in the open, unglazed, cylin- drical jars are given in Tables 25 and 26. The last test in each, made 259 days since first exposed, showed only local tuberculosis in each of the -four guinea pigs inoculated. Tho there was not time to determine by cultures and by secondary in- oculations whether these organisms were living, it is likely that they were. The end point in this series was not reached. The tuberculosis produced in the test animals from all other samples, except those dying with acute infection, was severe and general- ized. A typical autopsy is shown in the case of Guinea Pig 1124 (Fig. 3), which was inoculated with tubercle bacilli of the human type after exposure for 212 days in running water. The results of the tests from the other part of the emulsion of human tubercle bacilli placed in the submerged vase are reported in Table 27. The last test, made on the 25gth day, gave gener- alized tuberculosis in the two guinea pigs inoculated. N FIG. 3. GENERALIZED TUBERCULOSIS IN GUINEA PIG 1124 INOCULATED WITH HUMAN TUBERCLE BACILLI AFTER BEING EXPOSED IN RUNNING WATER FOR 212 DAYS. 352 BULLETIN No. 161 [November, J. * 4) 3 3 "i 1 I ~t~ to ^^ ^j 1 o 6.2 4) ^ a. .2 o + 4- + + + CO - o V <4 t/} - c i <4H 1 -~ C *O " o o ^o X^ 3 3 ^ to 1 1 e _ ft > = -4-> *-> -M u, .2 3 bo . . 4) to 4 4 rt o fH^H C C C J2 1 +2 ^ CO ^ B3 4> D 4) 3 ~ O O OH ) ' j o o c bo 6 rH rO ^O CM VO CO VO 00 ri ro O O O rH rH 5 r-H rH rH ) tn 4) i 4) 4-" ^ s ~i '*"" ~i ~T~ "i "T" ~r be I ^ -4J i 'ft 4 n) 6.2 r (U + i i i i [ i [ r .2 3 o* ~p ~t~ i ~r "r i i 1 5 '3 be 1 I ^ ai 4H j o 4> en 4) 4) 4) -* ^-H ) *^** 3 4) 52 4) 4J 4> G S3 \ o CD 0) D.2 "3 CO 4> M jj 4>33 to 'to o 'S to '3 to (o be bo - <2 u - ^ - .S .5 4 -M s J 4) 1 1 1 1 8 1 8 1 | j ^ 3 J > Wi o o 0"2 S'OXlo^O w^r.c3w a J5 ^ 5 4) 10 C 4/ 4) 4) ^ ^ O - rr E Q 34) 4)^' 1 ^' 3 ^"^"3 ; i rt S a'3 ^3'd^5'O rt4) 4)4)4) .2 ^ *S ^ .2 i .2 ass t 4 I *"O ^c^^r^v o>^"c3 3 C a Q o W 5 i > 4) 4) o O O < O O O J i i X ^ -a -o-o -o -c -o < 4) c *- 4 s s .2 .2 s s .2 s .2^3 I O o g g * . ^ .3 * 9 s g g i n o ?.= 3.2 -2 VO^- COCOrHClOOO rHOO\ n ' ) i/5Tj-vOrf OOOOO i j (. 5 * C rt 3 ij 4 <4H o 4) 3 S J O II 1 1 1 J 1 1 I 1 -4-1 V Ti\ r !T 5 j. i j. Vii OVO t C Ok O ( CT rHcOcO 0- ll 00 ^H rH 00 rH VO ^* ON 'O VO 1^* 00 O\ rH IO ^O t > * O^ ^5 M ^O *C ON^ OOOOOrH rHrHrH 1 9 - ~ / *s >. Oco cooOrH-^-riro ria\ON * to rt rH ^f 00 ^ C^ ^O t'* rH 1C IO B O-d rH rH TH n n n c ft 1 igi2\ FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 353 m w ij ^^ o J7 W O g ^^ fe 0> ^ B a-c P 0) to &? O S S H K B S I 8 w a ' 0) 0) /} 31 + +- + I *C o.H V M .2 o" ~T~ I ~1~ " I 1 rt a o x (=S 03 o MH o 2-2 & & & V & v S o +-> 8- rt ItH O A to > 3 3 . f>. CCjjD {^^Hgiy J i-* O C) (5 O P y S M . be o *O 00 M CO *O O O rH >-l r-t rH rH r-t r-t rH r-t M > > V CO'^MtO QCOtf)^ K .2*2.2"3 cc^'3'3.2 oo O o S .2 .3 3 2 " ^ J2 'a *3w'3* ) r!o ~^ O *^ ^ ^ *^ r-* O CO tO u v* 3 !* *+~t MH C33^, *T2 *ri M 2 D-Md)^ QC^"^ ^ ^ * v v . $ ="3 'JSO'JJC 'Scc*^ r^H orhwO ^CJC3i> C C 3 C U V 04^ C? C5 i r- j S Illl IIII AJ> !l l/30000r-l ^ 23 ^ A T r-tr-t -^ C rf : 3-^ :^rt rHMMrH ^A^^^ 1 ^ COCO rH rH rH rH d-1 il ^^OO OOOr-trHrH rHrH : i i ^ O to i 0) >i OcOcOOO ^ 00 N N CO Cl C\ O\ 5 - il to rj rH-*J-CO C-IC-lvOVOt^-rH If) \f) c a i cu" 354 BULLETIN No. 161 [November, 4) rH 0-2 (0 <. 4 H -d V H 4 i g O i o> *^i /} ac 9 s d c H ; 5 3 s *o a _0 Macro scopic 3 O O 0) ' *~^ . H p Q 3 be 6 to c O'2,5 rH T 4 1 ! 1 T i 3 ** 3 1 ' be C >3 3 .^ 3 P. I S \ 2 if 4- -f + + + s be 1 H >5 03 * * r? > 7 to 1 V 5 D to a i; -M ; 3 O IH o ^ -d g -g - -d -d JJ rt D O nj RJ j N IH o; Ui DO; 1 < g O ^ g O ^ o t> 4 O *d u 4; H O 4) 13 g s s g s g g ; > i 5 * since inocu- lation VO d O 00 f > * ON O^ M * n * rO 00 00 i 3 , O * C-l rO M ON ON S g j to rt rH rH fl fS CJ Cl c Q P< FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 355 The Diseased Tissues of a Tuberculous Guinea Pig In order to test the length of time^that tubercle bacilli will live when exposed in running water, Guinea Pig 918 was selected. This guinea pig, inoculated with tubercle bacilli of the human type, was killed 47 days after inoculation and found to be severely tuberculous. The autopsy showed the liver and the spleen to be much enlarged and thickly set with yellow masses of tubercles; the lungs and lymphatics were also extensively infected. Micro- scopical and cultural tests showed the tubercle bacilli to be char- acteristic and active. MANNEROF EXPOSURE The dead tuberculous guinea pig was placed in a flower pot having the opening in the bottom closed. A half brick was placed upon the guinea pig to keep it it from floating up when placed in the running water of the pool. The flower pot was set on the bottom of the pool, which was twenty inches deep. RESULTS ^^ e resu lts are given in Table 28. All the tests OF TESTS made from the decaying and putrefying tissues of this guinea pig with one exception produced FIG. 4. GENERALIZED TUBERCULOSIS IN GUINEA PIG 1126 INOCULATED WITH TUBERCU- LOUS SPUTUM AFTER BEING EXPOSED IN RUNNING WATER FOR 187 DAYS. 356 BULLETIN No. 161 [November. Result of g-uinea pig- test 1 1) i ** tfl 4 " > + + + + + I 6. 4) >H n, 3 - "* ^H O D S tn + + + + + + llation of d Macro- scopic .. > 0) rQ C - U 3 JH *-* 4) -4-* 4) C 4) CO) t) 10 4) M 4) | 5 4) H c 4) 4) (0 & di & a) 3 i- 3 2 <-* 4) <-> 4) C 4> C 4) ft) 10 4) to +J D . |> fl 4) 4) CO Gen. tub severe o o he o t 1 p<" 00 00 rH rH O Sro vC ^ 00 00 O OOO TH rH TH TH TH rH rH rH rH t* + + + + + 4 -f + + + o.H + + + + + + + 3| + + Macroscopic Generalized tuberculosis, severe Generalized tuberculosis, severe Generalized tuberculosis, severe Healthy YTatiltVlir 4) 4) 4) 4) D 4) J5 V- _'__ t, Ui r> 4) 4) 4) 4) 4) to 4) .^4 > >t>> >,> 4) 4)04> 13 * 'S to tototo c a jj t/T to'to'ifl to^to.5 'to 3 'to 'S 'to ' *S 3 '3 4, 5, 5 E o .2 o o o o ** , 'O *! .2 .2 - ^ tJ'3'3'3 *3 0*33 H^rJ OrJ^Jr- 1 J GO OOO i 5 ^25-2 5 ** 5 9 4)4)D o -g "g -g -g 1 S Soo N "^NNN N .S ^ *<' <5 J< W, V^W,^ 1* & S 4) 4)0)4) 4)?4)4) c cca sjiflrQ .t^'^- rl ^*-OO^J- rOrOVC Tj-l>-l^. t QV ^ rHOVOlO ^ rHTJ-Tf f<) "o p c 4) -M 073 t C rt III III 1.1,11 1 IJ ''J, C-l rH rH C^ O\ tO rH 00' 00 rH t>- ^ XO I^ 1 rH rH 00 rHrH r-lrHMf-JrHrHrH < 1 TH tO . /) > I/J VO'O1> 000000^ O rHC\r-( f^rOf*) rH rH rH *tl rHIOlC OOXO rl^Tj-O l> OrHd ^OlOVC "E. "^ S S | rt rt OTJ CO ^ O O O 0*i ^ ^ d C7\ ^ r*3 t^ 00 rH ^ ^t r^ r-t rHrHrHrH Cl C^f^fO f)f<3rr 1912} FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 357 tuberculosis, save those that died too early with acute infection. The two guinea pigs used in the third test, made 99 days af- ter exposure, remained healthy. Probably only non-tuberculous tissue was obtained for this test. Later tests taken from broken- down tissues showed virulent tubercle bacilli. The tests were positive up to the 32ist day; after this date the test animals all died with acute infection, the last one, however, not too early to have shown tubercle bacilli. On the 229th day and later no structure of tissue could be determined except the hair and bones, and the bones were almost as fragile as garden earth. Sediment from the bottom of the flower pot was obtained for the samples. Tuberculous Sputum To determine the length of time that tubercle bacilli from tuberculous sputum will live in run- ning water, a sample was obtained from an ad- vanced case of tuberculosis. A stained smear of this sputum showed numerous bacilli. A very small sample was inoculated into a guinea pig the first day of exposure. This guinea pig died in three days of acute infection. RESULTS The results are given in Table 29. Tuberculosis has been produced in the test animals up to the last test, 232 days since exposure. However, only local tubercu- losis was produced at this last test and there has not been sufficient time to determine whether the organisms are alive or dead. The test made just previous to the last one, 187 days after exposing the sputum in, water, produced severe generalized tuberculosis in Guinea Pig No. 1126 (see Fig. 4). SOURCE OF SAMPLE TABLE 29. TUBERCLE BACILLI IN WATER Kind and source of the organisms Not killed Killed Human tubercle bacilli: a. Pure culture in flower pot, Series 1 441 days 470 days b. Pure culture in 8-inch cylinder. Series 2 c Pure culture in submerged va*e 259 days 259 days d In tuberculous guinea pig 321 days 381 days g JQ sputum 232 days Bovine tubercle bacilli: 441 days 470 days b. Pure culture in 8-inch cylinder, Series 1 c. Pure culture in cotton-stoooered bottle .. 259 days 202 davs 358 BULLETIN No. 161 [November, O! ssue ' D 52 + + : iseased ti Micro- scopic + + + o <*H a .^ -4-> Macro- scopic - a) a> -^ p C Bfc o M ^-> > 50 c i> c > 2' a 1} tfl 4; 1C O O f-i : O c hH ,b 5P tf-as * b * VO CO VO O O rH rH rH rH 10 he 1 i -*-> + + + + + guinea p i jyiicro- scopic + 4- + ' + + + Result of croscopic ,. > > > v .3 ~ V D u tfl (fl 93 (CM c .2 s .2" c .3 .2" a * 2o g 111/1 I. 3 g H ^ 'So'3'3 tj JT no 5 ODOO D N o3 222 ^s 1 I' 1 11 ^ w D c c c c 0! 0) 0) 0) o o o o HH O a S J *> Id V a *o *d 'O _, _. _. 'O Oa) D u'O'O'U "Ou 4> ^- 1 ' ri rr inocu- lation rOOO VO Mt^-^-rH t^C\ ro to ^Hrj-vo -^fOO HH o V ~- a- lation OOO H |Sr * Ifi rt H-I rH T-l T-( r-t t-( 1 iH II i i M i m. .m t^ GO O\ O rH H r-l rH rH a- 30 o ifc VOtO Ch - M rt 8* O O\ ro ON t^ vo t- ri M to VO O\C-1>OOO roro rH rH rH M n I9i-] FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 359 A summary of the results showing the length of time that tubercle bacilli from various materials -live in water is given in Table 29. Conclusions 1. Tubercle bacilli live for more than a year (441 days) in running water. 2. The length of time that human and bovine tubercle bacilli live in water is practically the same. 3. These organisms live in sputum exposed in water for more than 232 days. 4. They live in the tissues of a dead guinea pig exposed in water for more than 321 days but are dead in 381 days. 5. A watering trough harboring tubercle bacilli may become a dangerous source of infection to cattle. 6. A dead tuberculous animal in a stream on your neighbor's farm may be a means of infecting your stock. 7. The better disposition of dead tuberculous animals is to destroy by burning. 8. Tubercle bacilli in drinking water is one of the possible sources of infection for man. 9. Infection is not prevented by dilution, since clumps contain- ing a great number of these organisms may be inclosed in mucoid material which prevents their separation and destruction. DURATION OF LIFE OF BOVINE TUBERCLE BACILLI IN BUTTER INTRODUCTORY Considerable attention is at present being directed STATEMENT to the presence of tubercle bacilli in foods, more especially milk, butter and cheese. It has been determined by Sedgwick and Winslow and by Park that typhoid bacilli frozen in water die very rapidly. After an hour's freezing 30 to 60 percent were destroyed, and in two weeks 99 percent were killed. The remaining one percent lived for a number of weeks. Tubercle bacilli in butter kept at a temperature below freezing are not killed in this way, as determined by Mohler, Washburn and Rogers in 1909. In order to obtain further information upon this subject the following experiments were planned. Butter was mixed with an emulsion of a pure culture of bovine tubercle bacilli and placad in small vials which were stored in the three following places: 360 BULLETIN No. 161 [November, 1. The cold storage of the Monarch Refrigerating Company, Chicago, 111., at 10 C. below freezing. 2. The University of Illinois Dairy storeroom at 4 C. (above freezing). 3. The basement of a dwelling in Urbana, Illinois, kept at an average of approximately 20 C. PREPARATION ^ pound of fresh butter was obtained from the OF SAMPLES creamery of the University of Illinois directly from the moulding board. It was salted as usual for the market, one ounce of dry salt to one pound of butter. After the butter is mixed, pressed and drained it has a salt content of two to three percent. It was not chilled, but at once taken to the bacterioligocal laboratory and mixed with the emulsion of tubercle bacilli. An emulsion of 3 mg. of bovine tubercle bacilli in 100 c.c. of 0.8 percent salt solution was made, and the butter melted at 35 C. was thoroly shaken with this emulsion. From about 10 to 15 cc. of this emulsion were put into small sterile glass vials and stopped with sterile cork stoppers. Thirty such samples were prepared, ten of which were stored in each of the three places mentioned above. TESTING THE ^ e sam pl es were tested when prepared and at SAMPLES varying intervals afterward on the same day from each of the places stored. They were brought to the laboratory, melted at a temperature of about 38 C., and two guinea pigs were each injected subcutaneously with i cc. of the melted butter from each sample. The samples kept at Chicago at 10 C. below freezing were always in excellent condition; those kept in the basement of the dwelling became very rancid and slightly mouldy; and those kept in the University Dairy storage showed slight moulding in part of the bottles. RESULTS The results of the tests are given in Tables 31, 32 33. No end point was reached. Generalized tu- berculosis was produced in the test animals from each of the three samples taken on the 274th day. It was noted that the tuberculo- sis produced by the samples kept at the lower temperature was more severe. This was probably due to the killing out of other organisms that at higher temperatures acted antagonistically to the tubercle bacilli. 1912} FATE OF TUBERCLE BACILLI OUTSIDE TriE ANIMAL BODY 361 i D ' ' 3 3 S o -S : : : : : : : : : : :+ \\ : : : : I 4-1 8" ;/: 3 .& ^ : : : : : : i : i :+ '. : : : ': f. 5 IIH o .2 : : : : : : : : : 3 : : : : .: : $3 .0 5 a rt : : : : : : : : : ; 2 g : : : : : . . . . 5 . . .0,0 jd : : : : : : : : :H [J : ' ' : y 600 . . : ". ; ; . : : : c^ CO KH "8*3 . . . . . . . . . . ,-( . . . . . . 0) o> l-i be *H < i a> 3s +++:::++:::+ :+ ++ : : guinea p ' k oidoos -OJOITAT ++ ++ -H- ++ ++ ++ ++ ++ ++ o UD VO 4^4) V Q) OD D C^ C^D "3 0) K DO) a; 0)0) 4) CJ O D nV .2 D OJ OJ >> >> >> >> >>.s> -M > >> 0)0) 0)0) 0)0) 0)0) 0)0) Tx 0) 0) 0)0) COE/J tCcO ( tOtO MO) cQ 09 JJifl tS* ** "S 'S tc~!C UU -*--*-> X3 X! J2 ,0 XI XI ^ X3 42 X) >C .0 ? X! J2 X) ^ Tj 33 33 3-3 33 33 03 5 s 33 aid; ri E 'O'O 'O'O 'O'O 'O'C 'O'C x 1 ^ i-t "O "0*0 "3 *2 4)D 4)1) O4) 0)0) 0)0) v o) hn a> a> nJ 'O'd 'd'O 'd'O 'O'd _-(_( _i'O -H--I '--i "^ 'O'd S^ SS S^ SS ^^ ^^ ^ ^S SS s T3 MW MM MM* SM SS Sg SS Sg g^ i i C since inocu- lation 0000 fOOO VOIO -i tH OOrO O\O OO OOM OO rOfO cOfO >OiO f3c<5 fOrO Tf 'i-^t fOrj- c<5cO o - Z\ fc i a J > 3^ opcp 1010 o\ I T f* J J II '-"'-" 'H H II II 0000 II O^ O> OO i-i i-i || || rOrO toiO OOOO H iH rH rH M rl r-( T-l H rH ol J5d Ifc OOON OOO\ ^O vot^ OOO^ IOVD O\O OOO\ NrO ^-H <-OrO * 'O VOVO GOCX) HrH fSfO Tf TJ- t^I> OO OO OO OC rH r-( r-(r-( rHr-l T-(^H c 1 ( a t h s *& , tn a ^ OO t^l> -**1- ONO\ N rO OOOO TC^t lOiO * -f rnr-i roro 1010 O\O\ MM ^O^O OO l>l^ T-tr-l THrH MM MM 362 BULLETIN No. 161 [November V ^ 4) 3 /: 3 3 4-J o B-g a .2 o . : : : ++ : : : : : V r. s : : . . . . : : . . ^ . . . . . : d O go O O o .2 '-M ,28 ^ ce . . S3 1 5 bed ; i i oo m t a!4 . . . . rH T-H PQ ^ C 11 ++ +: :: ++ ' : ++ !+ ++ : H H 3 o 3 W guinea p '! '? scopic ++ ++ ++ ++ + ++ ++ +4- + o 4)4) 4)1) 4)4) 4)1) D Ul> g <0 0>1) BERCI, "3 4) rn 95 CO t093 93cfi 931 aj D3 ^JtC 0)[fl 'Jj y~ ^T Q P Ttc o)T 0T> aT;*' to y^ o* * 00 TH M ^D TH ^h */^ TH TH ^t~ ^J" 1/3 *O *O *O O^ ^ TH TH rO rO i/3 /} X^" t^* OO OO OO OO OO irH THTH THrH THrH rH rH rH rH rH rH 4 1 I (1 V [' tfl ri rHrH rHrH MM MM 1 FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 363 V rH " 3 1 3 a u5 *-> d 2-s. : : ; : . _i i ' : j i_ .2 o o : . : . : : > r . T r to p o o So. :::.::::-. *# 55 .2 5 . . . . . . . . . . rtr 1 v & o . ted '. '. '. : : . : : : . ^ ^ o o hH a * . . . . . . . . rH iH TH TH tfl o be < y ++:;;; -t+ ;;++. ++ i i guinea p 1 ivucro- scopic ++ ++ ++ , + ++ ++ ! ++ ++ o D DD **^ U3 ^ " lH 0)0) ^(U ^O> 0)0) ^^ OJO> 'c^ 22 ui? NS: NN NNI Ns3 ,2^ t^tsi -S.5 q;0) o>o! U2*^ U^^J ^ ^H "T^^H S3 ^3^ a> o Cfl CC rfrt rtcd ricti rtrt oo rtrt" ^j ^ o? 0) '_ "_ 0>0) 0>0? 0)0> 0>OJ 5o) 0>0> o^ OC? Uw cc en cc cc xjxi cc ^^ C5O OO OO OO ^Jn OO d 0)0) 0)0) 0)0) 0)0) *^ *^ 0) 0) *^ *^ 0) 0) 0) 0) * 1 ^H r-H ^H * < ^H ^^ f* 0) 0) ^^ ^-< 0) 0) ^^ ^^ i ^^ 22 22 22 22 SS.gg SS gg gg C ?.= 5 ^ 00 00 rO rO *-O iO ^ i^ ^O ^ O O ^ C'l C^ f^l O O ro rO cO f O iO vo ro CO C^ C^ ^ ^t" rO rO ^t* ^" fO rO o . v : tt il 2 r3 II II II I 1 A J. II II II II OCOO O>O O\ O\ 'oo 00 00 ^r^T O^ &\ THTH o\ O\ 1 i r i n T 1 TH TH TH r-i i i ii OOX II O\& OO THTH II II roco IOUJ WOO THTH THTH dCl THTH TH TH d' yj Q 00^ OO OO OO OO OO THTH THTH THTH T-TH " c u b o >> TH TH CO rO 10 io O O ri M O vC O O t~- l> 364 BULLETIN No. 161 [November, GENERAL DISCUSSION A summary of the results of the experimental work is given in Table 34. It is seen from this table that tubercle bacilli in pure culture, spread in thin layers on sterile glazed paper slips and ex- posed to the direct rays of the sun, are killed in a very short time (i to 4 minutes). In this respect tubercle bacilli may be classed with other nonspore-bearing organisms. When exposed to desic- cation, pure cultures of these germs in thin layers are found to be dead in a few days. In sputum and other foul material they appear to live longer than the other nonspore-bearers. They are known to live long enough to be blown around so that the inhalation of dried sputum dust causes tuberculosis in test animals. Just how frequently people are infected by breathing dried tuberculous ma- terial has been discussed (see page 270). That sunlight plays an important part in the disinfection of this dried tuberculous dust is evident. Also that our homes, factories, and places of business should have an abundance of window space, located so as to admit the light, is another timely lesson. Tubercle bacilli in cow manure lived 73 days when a pure cul- ture mixed in a sample of manure was exposed to weather condi- tions in a pasture field in the shade, and as long as 49 days when exposed in the sunshine. The sample from a tuberculous cow was dead at the first test, made 13 days after exposure. We were dis- appointed in not being able to repeat these experiments. It would be advisable to repeat this work, especially with naturally infected manure from several tuberculous cows that were known to be expelling tubercle bacilli per rectum. Keep pigs from three to four months old in a pasture with such tuberculous cows, and allow the pigs to feed upon the cow dung. Feed other pigs by mixing with their feed tuberculous manure which had remained in this pasture for varying intervals of time. An examination of the internal or- gans of these shoats would give valuable information both as to the infectiveness of the cow manure and to the length of time that tubercle bacilli remain alive in such manure. This would indicate, much more completely than our experiments, the length of time that stock should be kept from a field in which tuberculous cattle had been pastured. When tubercle bacilli, either in manure or in dead tuberculous animals, become mixed in the soil, the danger may be still greater, depending upon the opportunity for hogs to take this material along with their food. The bacilli live longer under these condi- tions, but the opportunity of being taken is usually less. The danger of man becoming infected with tuberculosis from drinking water has been discussed (see page 307). Just how likely FATE OF TUBERCLE BACILLI OUTSIDE THE ANIMAL BODY 365 TABLE 34. SUMMARY ox RESULTS Organisms exposed to direct sunlight Name of organism B. subtilis, spores B. mesentericus vulgatus, spores. B. subtilis, vegetative cells B. mesenteric, vegetative cells... B. prodigiosus B. diphtheria B. coli B. ty phosus B. violaceus . . Not killed 180 min. Yz min. 6 min. 2 min. 1 min. 2 min. Killed 1 min. % min. after 6 min: 3 min. 2 min. 3 min. Tubercle Bacilli Exposed to Direct Sunlight Name of culture Not killed Killed B. tuberculosis, human. B. tuberculosis, bovine B. tuberculosis, avian.. 1 min. 1 min. 2 min. 2 min. 2 min. 4 min. Bovine Tubercle Bacilli in Cow Manure Kind and source of material exposed Not killed Killed Bovine tubercle bacilli, pure culture: Exposed in cow manure, in sunshine, Series 1. Exposed in cow manure, in sunshine, Series 2. Exposed in cow manure, in shade, Series 1 . . . . Exposed in cow manure, in shade, Series 2 49 days 45 days 49 days 73 days 81 days 73 days 81 days 100 days Bovine Tubercle Bacilli from a Naturally Tuberculous Cow Tuberculous manure from this cow, in sunshine. Tuberculous manure from this cow, in shade .... 13 days 43 days Bovine Tubercle Bacilli in Garden Soil Bovine tubercle bacilli, pure culture 213 days 230 days Bovine tubercle bacilli, in tissue of a dead guinea pig 77 days 91 days Tubercle Bacilli in Water Human tubercle bacilli: Pure culture, in flower pot, Series 1 441 days 470 days Pure culture, in 8-inch cylinder 258 days Pure culture, in an unglazed vase, submerged. 259 days In the tissues of a tuberculous guinea pig 321 days 381 days In tuberculous sputum 232 days Bovine tubercle bacilli: Pure culture in flower pot, Series 1 441 days 470 days Pure culture, in an 8-inch cylinder \ 259 days Pure culture, in a cotton-stoppered bottle j 202 days Bovine Tubercle Bacilli in Market Butter Temperature stored Not killed Killed 10 C. below zero , . . . 274 days 4 C. above zero 274 days 20 C. above zero 274 days 366 BULLETIN No. 161 [November. it is that cattle and other farm animals are infected with tuberculo- sis from the presence of these germs in water is not surely known. The common watering tank may become a source of infection. Here the tubercle bacilli live among the algae and in the decaying organic matter for more than a year. Calmette points out that constant and repeated infections are the most dangerous. Cattle would thus be subjected when a watering trough was infected. Another source of danger to man is in the use of phosphates made by grinding up dead tuberculous animals (which is done rather extensively in the United States), this fertilizer often being used in vegetable gardening. One can easily conceive how a small piece of tuberculous tissue containing many dozens of tuberculous germs could be made to adhere to an onion or a radish, especially in a slightly bruised place, and be carried directly to the consumer. That these germs would remain alive and virulent during such a circuit there is no question. It is seen that tubercle bacilli in butter kept at 10 C. below zero retain their virulence longer than when kept at the higher temper- ature. This temperature of 10 C. apparently has no injurious effect on these germs, while the antagonism of other organisms is largely prevented. Butter can be kept in cold storage for months in an excellent condition, but this in no way lessens the danger from tubercle bacilli that were originally introduced into the butter. All such dairy products should be tested by govern- ment officials not only for quality but also for the presence of tu- bercle bacilli. REFERENCES 1. Abba e Barelli, Sulla resist, del bac. tub. negli sputi sopra di- parimenti e dentro le biancherie, Rivista d'igiene e sauita publica 12: 115, 1901. Cited by Treskinskaja. 2. Aiello und Drago, Cited by Sobernheim. G., in Kolle-Wasser- mann Handbuch der Pathogenen Mikroorganismen 2: 28. 3. 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