The experiments given below refer to the active vegetative state of the bacterium in nutrient liquids, as experiments had failed to reveal any other more resistant state. The cultures were, as a rule, but one or two days old. Previous experiments having shown that older cultures are less resistant to heat than recent ones, it was assumed that the vitality is gradually reduced as the culture grows older. All the tubes about which there seemed the slightest suspicion of impurity were examined microscopically and often on gelatine plates. In all cases the last of a series of inoculated tubes which became turbid was carefully examined. This served as a check upon tubes exposed for a shorter period of time to the action of the disinfectant. The percentage of the solution used indicates the ratio by weight in grams of chemically pure substances to grams of distilled water. Mercuric chloride was found destructive to the bacterium when diluted in the proportion of 1:75000 (.001 1-3 per cent.). Several drops of a culture were mixed with about 1e of a .1 per cent. solution, and tubes inoculated from this at the end of 2, 4, 6, 8, and 10 minutes. Tubes remain sterile. To show that the antiseptic effect of the liquid transferred with the platinum loop was nil, one of these tubes was inoculated again from another culture. This tube was turbid on the following day. Five tubes treated in the same way with .05 per cent. solution. All remain sterile. Five tubes inoculated from a culture exposed for the same periods of time to a .01 per cent. solution. All remain clear. Five tubes treated as before, using a .005 per cent. solution. Permanently clear. Five tubes treated as before, using a .002 per cent. solution. All tubes clear excepting the one inoculated after 6 minutes' exposure. Five tubes inoculated at the end of 5, 10, 15, 20, 25, and 30 minutes after exposure to a .0001 per cent. (1: 100000). Tubes inoculated after 5 and 10 minutes turbid next day. On the second day all but the one inoculated after 30 minutes turbid and containing pure cultures of the bacterium. The limit of disinfection for this period of time must therefore lie between 1:50000 and 1: 100000; hence 5 tubes were inoculated as above, using a solution of 1:75000, at the end of 7, 10, 15, 20, 25, and 30 minutes. All tubes remained clear. Carbolic acid destroys the bacterium in solutions containing from 1 to 11 per cent. of the acid by weight. Five tubes inoculated after treating the bacteria from a liquid culture with a 1 per cent. solution for 5, 10, 15, 20, and 25 minutes. All turbid on the following day. The two last tubes were also examined on gelatine plates and the cultures found pure. With a 2 per cent. solution, five tubes inoculated after 10, 15, 20, 25, and 30 minutes remained sterile. The same result with a 14 per cent. solution. With a 1 per cent., tubes inoculated at the end of 7, 10, 15, 20, 25, and 30 minutes remained clear, excepting the first, which contained bacillus subtilis. Passing to a per cent. solution, tubes inoculated at the same intervals became turbid with the bacterium sown. With a per cent. solution the result was the same. Passing back to a 1 per cent. solution, tubes inoculated at the same intervals remained sterile. There seems to be an incompatibility between the first and last series. If we examine the others, however, we must conclude that the limit of disinfection lies between 1 and 1 per cent. Iodine water was prepared by shaking up some iodine in distilled water, which assumed an amber tint. This solution destroyed the bacterium in 15 minutes, as the following experiment shows: Six tubes were inoculated with bacteria after they had been exposed to the action of the iodine water for 7, 10, 15, 20, 26, and 31 minutes. On the following day the first tube became turbid; on the second the 10-minute tube was turbid and found to be a pure culture of the bacterium sown. The other tubes remained sterile. One of them, inoculated later, showed its capacity for sustaining growth by becoming promptly turbid. Permanganate of potash.-A series of experiments with this sub. stance, conducted in the manner described above, showed that the bacterium is killed by 15 minutes' exposure to .02 per cent. solution tion (1:5000). In order to obtain this result a 5 per cent. solution was tried first. Tubes inoculated after an exposure of the virus for 7, 10, 15, 20, 25, and 31 minutes remained permanently clear. One of these tubes, subsequently inoculated with the unaffected virus, was turbid next day. Two and a half per cent., 1 per cent.,per cent., † per cent., per cent., and per cent. solutions were tried in the same way. The six tubes used for each solution remained sterile. Finally a per cent. (1:5000) was used. Tubes were inoculated after an exposure of the virus for 2, 4, 6, 10, 15, 20, 25, and 30 minutes. On the following day the four first tubes were turbid; the fifth and seventh remained sterile; the sixth and eighth contained a fine bacillus. These two tubes, as was found later, belonged to a lot which, through some carelessness, had not been properly sterilized, and the majority became turbid before use. Mercuric iodide was found to destroy the bacterium in solution of 1:1000000 in 10 minutes. Two grams of potassium iodide and 1 gram of mercuric iodide were dissolved in 100% of distilled water, making a 1 per cent. solution of the disinfectant in a 2 per cent. solution of potassium iodide. This solution, diluted with sterile distilled water so as to make .1 per cent., killed the bacterium of hog-cholera táken from liquid cultures in less than 5 minutes; .01 per cent. (1:10000), .002 per cent. (2:100000), .001 per cent. (1:100000), and .0005 per cent. (5: 1000000) destroyed the germ within 2 minutes. When the solution was diluted so as to make .0002 per cent. (2: 1000000) and .0001 per cent. (1: 1000000) it was found that with both solutions tubes inoculated with the bacterium after a exposure of 2 and 5 minutes were opalescent, the bacterium introduced having multiplied, while the remaining tubes (10 to 30 minutes) were sterile. These two solutions, therefore, were still powerful enough to kill the germ in 10 minutes. The dilution had been carried so far as to make them practically equivalent in disinfectant power. Sulphate of copper.-This disinfectant, which seems to be more effective than most other metallic salts, was tried in solutions containing 2 per cent., per cent., per cent. Both the 2 per cent. and per cent. solutions destroyed the germ within 5 minutes. Tubes inoculated with the bacterium after an exposure to the per cent. solution for 5, 10, and 15 minutes became turbid; those inoculated after an exposure of 20, 25, and 30 minutes remained clear. The disinfectant power for short periods of time may be said to lie between and per cent. In this, as in other tests, one or two drops of the culture were added to 5 of the disinfectant. A slight flocculent precipitate formed each time. Of hydrochloric acid a .2 per cent. solution of the acid, made by adding 4.2cc of chemically pure acid (containing about 40 per cent. HCl) to 95.8ce of water, destroyed the germ in less than 5 minutes. Chloride of zinc.-A 10 per cent. solution of this salt failed to destroy the vitality of the bacterium in 10 minutes; 20ce of Squibbs' chloride of zinc, containing 50 per cent. of the salt, were added to 80% of sterile distilled water to make a 10 per cent. solution. A drop from a culture five days old was mixed with 5cc of this solution, from which mixture tubes were inoculated at the end of 5, 10, 15, 25, and 30 minutes. The two first tubes became clouded. Sulphuric acid.-A .05 per cent. solution (1: 2000) was fatal to the bacterium of hog-cholera in less than 10 minutes. Without going into detail, it is sufficient to say that the results were reached as indicated above. Tubes containing sterile beef broth were inoculated at the end of 5, 10, 15, 20, 25, and 30 minutes with bacteria exposed to 4 per cent. and per cent. No development. Those inoculated with a per cent. became clouded, each being a pure culture of the bacterium inoculated. When per cent. was tried, only the 5-minute tube became clouded. The solution (by weight) was made from sulphuric acid containing 96 per cent. of the acid (specific gravity 1.838). It must be remembered that the foregoing tests were made upon bacteria in an active vegetative state. It is probable that in the dried condition it would have taken solutions of the same strength somewhat longer time to destroy their vitality. To briefly summarize the results, placing the least effective substance first, we obtain the following table: Chloride of zinc in a 10 per cent. solution destroyed the bacterium in liquid cultures in 10 to 15 minutes. Carbolic acid, 1 to 14 per cent. (1:100), in 5 minutes. Iodine water in 15 minutes. Hydrochloric acid, per cent. (1:500), in less than 5 minutes. (Only a .2 per cent. solution of this acid tried.) Sulphate of copper, per cent. (1 : 1000), in 15 to 20 minutes. The above table would no doubt be somewhat changed by mixing virus embedded in large quantities of organic matter with the disinfectant solutions. It gives, however, a good working basis for experiments on a large scale, and it throws out at once the use of chloride of zinc and perhaps carbolic acid. In order to determine how much stronger solutions than those above given would be required to destroy the dried bacteria, the following experiment was carried out: Spleen pulp containing large numbers of bacteria was rubbed on sterile coverglasses so as to make a thin film, and allowed to dry for 2 days under a plugged funnel. A solution of mercuric chloride, 1:50000, was poured upon the coverglasses, and one was removed after 11, 2, 3, 5, 7, 10, 15, 17, and 20 minutes, washed in about 100 of sterile water, and dropped into tubes containing beef infusion. After remaining in the incubator for a day it was found that the virus exposed to the disinfectant for 14, 2, 7, 10 and 20 minutes had been destroyed, the tubes remaining permanently clear. The others contained pure cultures of the hog-cholera bacterium. The bacterium may be thus killed by solutions of mercuric chloride, which do not destroy spores. Koch found that anthrax spores may remain in solutions of 1: 50000 for over 60 minutes without losing their capacity for germinating. That all of the germs were not destroyed in the above experiment does not weaken the conclusion. They were undoubtedly incrusted with blood and cellular elements, so that the disinfectant could not exert its full power directly upon them. Koch, on the other hand, used spores from cultures only. The experiment demonstrates the absence of any resistant spore state. in the tissues of the animal, but points out the necessity of considerably increasing the strength of disinfectant solutions in endeavoring to destroy the bacteria in nature, inasmuch as we have to deal with other things besides the germs themselves, which neutralize much of the disinfecting power. Is there any resistant spore state in the life history of the bacterium of hog-cholera. Stained in dilute solutions of aniline colors the bacterium from the tissues of animals which have succumbed to the disease stains in such a way as to leave the impression that it contains an endospore. A narrow band of stained substance bounds an oval pale body, which is but slightly tinged. It appears that a rather resistant envelope prevents the coloring matter from passing readily and quickly into the interior of the bacterium. If a drop from a recent liquid culture be suspended from the lower surface of a cover-glass and examined in a glass cell with a homoge neous immersion objective and small diaphragm, the following appearances are worthy of record: The bacteria in the center of the drop of culture fluid are in very active motion and quite small. If the periphery of the drop be examined there will be found a dense layer of bacteria caught there by the slow desiccation and consequent contraction of the drop. These, some of which are still moving slowly, are larger than the forms in the center of the drop. As the drying proceeds and the film of water becomes thin the bacteria appear to be made up of a distinct dark border surrounding an almost transparent body. In most forms there is a slightly thicker border at the ends than at the sides of the short rod-like bodies. When stained slightly this border takes the stain well, while the body of the rod remains pale. The fact that the structural and color pictures correspond is strong evidence that the microbe possesses a rather dense membrane, which in optical section is seen as a narrow dark border. The form and size of the bacteria under consideration depend upon the culture medium and upon the age of the culture. The appearance which they present in the animal tissues is very closely simulated in liquid media, more especially beef infusion with peptone. When grown on gelatine or potato the appearance just described cannot as a rule be made out, as the bacteria are apt to be smaller. The foregoing facts incline us to believe that we have no true spore state to deal with in this microbe, but perhaps a membrane, which is more or less resistant, according to circumstances, and which is more. resistant in the animal tissues than in cultures. Microscopical characters, however, are now and then misleading, unless we interpret them by physiological experiments. Judging from what has hitherto been considered properties of bacterial spores, the microbe of hog-cholera cannot lay any claim to the production of true endogenous spores. Their absence is determined by results of experiments recorded in the preceding pages: 1. The thermal death-point of the bacterium at 58° C. An exposure to this temperature for 15 to 20 minutes destroys not only the vitality of cultures of all ages, but also the dried germ in the tissues of the infected animal. A momentary exposure to boiling water is equally efficacious. The bacteria are destroyed by disinfectants in solutions which are incapable of destroying spores. 3. They are killed by simple drying far more quickly than are spores; at the same time their resistance to drying is much greater than might be expected under the circumstances. In the experiments recorded some dried bacteria in spleen pulp were killed in less than a month; others resisted forty-nine days. We may put the limit, which is very much less for dried cultures, between one and two months. It is this continued vitality in the dried. state that suggests the existence of a membrane which is more rcsistant than that possessed by the great majority of bacteria in their vegetative state. This difference between bacteria in the vegetative and the spore state is illustrated by the anthrax bacillus. In cultures the bacilli are killed by drying in five or six days; the spores, under the same condition, retain their virulence for years. All the facts brought out by the study of this bacterium lead to the conclusion that a distinct spore state, so called, does not appear either within the animal body or in nature. *There is no reason why the bacterium in the body of animals may not be in an arthro-sporous state, according to the classification of de Bary. The name is of little account as long as we define the properties belonging to a given state. Observations on the pathogenic properties of the bacterium of hogcholera. In addition to the foregoing experiments on the general biological characters of the bacterium of hog-cholera, a few additional observations were made upon its pathogenic activity, with a view to determine more precisely the mode of infection. Growth in vacuo.-It seemed desirable to learn the extent to which the bacterium was capable of multiplying with a minimum supply of oxygen. The following simple experiment was tried: An elongated glass bulb of about 15cc capacity, terminating in a narrow tube about 10cm long, and containing about 5% of beef-infusion peptone, was inoculated from a pure culture. The air was then exhausted by an air-pump for fifteen minutes, while the bulb was kept immersed in a water bath at a temperature of 38° C. It was finally sealed in the flame and placed in the incubator. The results of three separate experiments were practically the same. In the bulbs the culture liquid was turbid on the following day. This turbidity increased but slightly, and within three or four days growth had evidently ceased. Four other microbes, two of which were found in the exudates of hog cholera bacillus luteus described in the Second Annual Report of the Bureau, a micrococcus, bacillus subtilis, and a microbe producing septicemia in rabbits, were treated in the same way. None of the tubes became turbid. When, after three or four days, the bulbs were opened and filtered and air allowed to enter, the liquids became turbid within twenty-four hours, the characteristic pigment of the bacillus luteus appearing a few days later. The bacterium of hog-cholera has therefore the power of multiplying in what is practically a vacuum, but this power is limited. The other microbes failed to show any signs of growth whatever. They were purely aerobic. The bacterium of hog-cholera may therefore be regarded as holding a place midway between those microbes which seem to thrive better without air-anaerobic-and those that fail to grow without it. That no spores were found in these bulbs may be inferred from the following experiment: One of the tubes kept sealed for a month was opened and a number of culture tubes inoculated therefrom. They were then exposed to a temperature of 58° C. for 15, 20, 25, and 30 minutes. All remained sterile. One, inoculated without being subsequently heated, was turbid with the specific bacterium next day, indicating that it was still alive in the bulb when the latter was opened. MODES OF INFECTION. (a) By way of the digestive tract.-In at least 90 per cent. of swine a very severe form of hog-cholera may be induced by feeding to them the viscera of animals which have died of the disease. The lesions produced are exceedingly severe. The mucous membrane of the large intestine is extensively ulcerated or completely necrosed. In animals which have contracted the disease in the ordinary way in infected pens the ulceration of the large intestine, at times very severe, usually stops abruptly at the ileo-cæcal valve. When this is slit up, the mucosa belonging to the small intestine up to the free border of the valve is in the great majority of cases normal, while the mucosa of that surface of the valve facing the cæcum may be extensively ulcerated. In many animals fed with infectious matter the ulceration involves the entire ileum. This is well illustrated by the following cases: January 8, 1886.-Pig No. 165 was fed with the viscera of two pigs which had died of hog-cholera. It was found dead January 26, after manifesting no marked symp |