bacteria as important for the cholera-process, whilst everything else gave the impression that it was something secondary; for the bacteria first described always advanced beyond the others, and gave the impression that they smoothed the way for the other bacilli. Surface of mucous membrane laid bare. smaller than tubercle-bacilli. One scarcely, forms a correct idea of the thickness, length and breadth of bacteria by giving their dimensions in numbers. I therefore prefer to compare the dimensions of bacteria with other objects. As the tubercle-bacilli are known to everybody, I compare the cholera bacteria with them. The latter are about half or two thirds as long as the former, but much more bulky, thicker, and slightly curved. The curve is generally not more marked than that of a comma, but sometimes it is larger, becoming semicircular, as in the adjoining figures (2 and 3). In other cases the curve is doubled, one comma is attached to another, but in an opposite di Fig. 1.-Section of the Mucous Membrane of a Cholera-intestine. An utricular gland (a) is diagonally cut through. In its interior (6) and between the epithelium and basementmembrane (c) numerous comma-bacilli. 600 magnifying power. With regard to the contents of the intestines, at first no clear conception could be formed, as the only cases which came before us for examination were not suitable. Also in these the contents of the intestines were putrid and bloody. There was an enormous quantity of various bacteria in these contents, so that there was no possibility of attending to the real cholera bacilli. Not till I had dissected a couple of acute and uncomplicated cases, in which no hemorrhage had as yet set in, and in which the contents of the intestines had not turned to putrid decomposition, did I recognize that, the purer and fresher the case, the more did a special kind of bacteria prevail in the contents of the intestine, and it was soon clear that these were the same bacteria which I had seen in the mucous membrane. I investigated them in every way in order to establish their special peculiarities, and am able to give the following information regarding them. These bacteria, which I have called comma-bacilli, on account of their shape, are Fig. 2.-Object-glass preparation from the con tents of a Cholera-intestine. Core of the Necrotised Epithelia (a). Semicircular Comma-bacillus (). Specially characteristic grouping of Comma-bacilli (c) 600 magnifying power. rection, so that it forms the shape of S. I think that in both cases two individual ones after being divided have remained stuck together, and give the appearance of a more marked curve. But in artificial cultivations another remarkable form of development of the comma-bacillus is to be observed also. The comma-bacilli frequently grow in threads of longer or shorter length. In one of the preparations, shown in the following diagram, several of these forms can be seen (fig. 4). But they do not form straight threads like other bacilli, for instance, anthrax bacilli, or, as it appears in the picture, simple wavy threads, but very long tender spirals, which as far as their length and the rest of their appearance is concerned, bear the closest resemblance to the spirochætæ of relapsing fever. I could not distinguish one from the other if they were side by side. I am inclined to the view that the commabacillus is not a genuine bacillus, but that it is a transition form between the bacillus and spirillum. Perhaps we have to deal with a genuine spirillum, of which we have do. Hence the milk looks quite unchanged. But if you examine it under the microscope, it teems with comma-bacilli. They grow very quickly, and in great numbers in the serum of the blood and in food-gelatine. This gelatine can serve for facilitating and securing the discovery of the comma-bacillus; for the colonies of the bacilli assume, in the gelatine a most characteristic and definite form, which so far as my experience goes, no other kind of bacteria assumes in like manner. The colony looks, when it is very young, like a very pale and tiny drop (fig. 4), which is however not quite circular, the shape generally assumed by these colonies in gelatine; but it has a more or less irregularly bordered, hollowed out, in parts also rough and jagged, shape. It also has a granular appearance at an early period, and is not of such regular character as other colonies of bacteria. Fig. 3.-Object-glass preparation. Cholera-dejecta on Damp Linen (two days old). Great multiplication of Comma-bacilli, amongst which some S-shaped (a). 600 magnifying power. fragment before us. It is seen in other spirilla that very short specimens do not form the complete thread of a screw, but consist of a short staff, which is more or less less curved. The comma bacilli can be cultivated in meat broth. They grow in this liquid very quickly, and in great numbers; and this property can be utilized for studying their other qualities, by examining with a strong magnifying power, a drop of the meat broth cultivation. It is then seen easily that the comma-bacilli move in a very lively manner. When they are collected together at the edge of the drop, and are moving about amongst one another like a swarm of dancing midges, and those long spiral threads also appear moving in an animated manner, so that the whole affords a strange and extremely characteristic picture. But the comma-bacilli also grow in other liquids, and especially speedily and in great abundance in milk. They do not curdle the milk, and do not precipitate the casein, which many other bacteria raised in milk Fig. 4.-Object-glass preparation. From the edge of a drop of Meat-broth, with pure-culture of Comma-bacilli. Long screw-shaped Threads (a). 600 magnifying power. When the colony becomes somewhat larger, this granulation becomes more and more evident. At last it looks like a heap of strongly refracting granules. I might best compare the appearance of such a colony with that of a little heap of pieces of glass. As they grow, the gelatine liquefies in the immediate neighborhood of the bacteria colony, which sinks down deeper into the mass of gelatine. A funnel-shaped cavity is thus formed in the gelatine, in the midst of which the colony is seen as a whitish point (as seen in the diagram). This appearance is also quite peculiar; it is seen, at least in this manner, in very few other varieties of bacteria, and never so marked as with the comma-bacilli. The sinking of the colonies can best be observed when carrying out an artificial cultivation. A suitable colony is selected on the gelatine plate under moderate magnifying power; touched. by a platinum wire previously heated; the bacilli are transferred by the wire to the wire in a test tube of gelatine, and this is lower temperatures. Experiments have been made on this point which show that they can grow very well at 17° Cent. tho' more slowly. Below 17° Cent. the growth is very small, and seems to cease below 16°. In this point the comma bacilli remarkably resemble anthrax bacilli, which have also this minimum temperature as the limit of their growth power. Once I made an experiment to test the influence of lower temperatures on comma bacilli, and to see if they were not, at a very low temperature, not only hindered in their development, but also if they can not possibly be killed. For this purpose an artifical cultivation was exposed for an hour to a temperature of 10° Cent. below zero; during this time it was completely frozen. When part of it was put into the gelatine, there was not the least difference visible in the development or growth, so that they bear frost very well. closed with sterilized wadding. A cultivation of this kind then grows in the same manner as the colony on the gelatine plate. I am in possession of a numerous collection of artificial cultivations of bacteria made in this manner, but I have never seen in their cases such changes as the comma-bacilli cause after being transferred to gelatine. Here also, as soon as the cultivation begins to develop, you see a little funnel at the place where inoculation took place (see the diagram). By degrees the gelatine liquefies in the neighborhood of this point of inoculation; then the little colony is plainly seen, extending more and more; but a deep spot, sunken in, always remains, which, in the partly liquefied gelatine, looks as if an air bubble were hovering over the colony. It almost gives the impression that the bacilli not only caused a liquefaction of the gelatine, but also a speedy evaporation of the liquid thus formed. We know a number of other bacteria, which, in the same manner, liquefy the gelatine in test tubes, starting from the point of inoculation. But in these cases there is never such a cavity, nor this bubble-like hollow space. The lique faction of the gelatine, starting from a single isolated colony, the best way of observ-self of this very simply by laying a piece of ing it in a layer of gelatine, which is spread out on the glass-plate, never spreads very wide. The dimension of the liquefied district of a colony may be estimated at one millimetre. Other kinds of bacteria can liquefy the gelatine to a much greater extent, so that a colony attains a size of one centimetre in diameter, and more. In the cultivations of comma-bacilli in test tubes, the liquefaction of the gelatine extends very slowly, and continues in such a manner that after about a week the whole contents of the test tube have become liquid. Unimportant as these qualities seem in themselves, special weight is to be laid on them, because they serve to distinguish commabacilli. Comma-bacilli can also be cultivated in Ceylon moss (agar agar) to which meat broth and pepton are added. This agar agar jelly is not liquefied. They can also be raised on boiled potatoes-a fact which is very important for certain questions. On potatoes they grow exactly like the bacteria of glanders, which forms a thin pulpy coat of a brown color on the potatoes. Comma bacilli flourish best at temperatures between 30° and 40° Cent. (86° to 104° Fahr.), but they are not susceptible to It is not the same with the withdrawal of air and oxygen. They cease to grow, when deprived of air, and accordingly belong, if the division into aerobic and anaerobic bacteria be held as good, to the aerobic class. Anyone can convince him talc or mica over the glass plate when the artificial cultivation has been placed on it in liquid gelatine, and when the gelatine is beginning to stiffen; the talc or mica must be as thin as possible, and must cover at least one-third of the gelatine surface in the middle. The piece of mica, owing to its elasticity, adheres completely to the portion of the gelatine covered, and thus shuts off the air. Then, as soon as the development of the colonies follows, it is seen that the development only takes place where the gelatine is not covered, and only a trifle, about two millimetres, under the mica plate, up to which point the mica has been able to force its way. But under the mica plate itself, nothing grows. Extremely small colonies, invisible to the naked eye, do, it is true appear, which probably owe their origin to the oxygen existing in the gelatine, but they do not increase in size afterward. An experiment was made in another manner. Little glasses containing food gelatine which had been inoculated with comma bacilli, were placed under an air pump, and others prepared in a similar manner were kept outside the air pump. It was seen that those inside the air pump did not grow, but only those outside it. But when those that had been in the air pump were placed again in the air, they began to grow. Te same occurs when the cultivations were brought into an atmosphere of carbonic acid. While the cultivations that have been kept for comparison outside the carbonic acid atmosphere grow in the usual manner, those that are in a stream of carbolic acid remain undeveloped. But in this case also they do not die, for, after having been for some time in the carbonic acid atmosphere, they begin to grow immediately after they have come out of it. On the whole, comma bacilli, as I have repeatedly observed, grow extremely rapidly. Their vegetation very speedily reaches a maximum at which it only remains stationary for a short time, then diminishing again very speedily. The comma bacilli when wasting away, lose their shape, they appear at one time shriveled, at another time swollen, and in this state they are not at all or only slightly susceptible to color. The peculiar conditions of the vegetation of comma bacilla can best be observed by bringing substances which are rich in comma bacilli, but also contain other bacteria, e.g., the contents of a cholera-intestine, or cholera-dejecta in contact with moist earth, or by spreading the substance out on linen and keeping the surface in a damp condition. Comma bacilli then increase in a very short time, e.g., in an extraordinary manner in twenty-four hours. Other bacteria that exist with them are at first stifled by the comma bacilli, a natural pure culture is formed, and on examining with the microscope the mass that is taken from the surface of the damp earth or linen, preparations can be obtained which show almost exclusively comma bacilli. Such a preparation is the following, which comes from the damp linen of a cholera patient, polluted with his dejecta. (Fig. 5.) But this luxuriant growth of comma bacilli does not last long. After two or three days they begin to. die off, and other bacteria then increase. The conditions become the same as in the intestine itself; there also a rapid multiplication takes place but when the real vegetation period, which lasts only a short time, is over, and especially when exudation of blood takes place into the intestine, the comma bacilli disappear, and the other bacilli, especially putrefaction bacteria, commence to develop in Fig. 7.--Natural Size of the Colonies as they ap pear on the Gelatine-Plate. But so far sufficient experiments have not been made on this point; this is only a supposition which I make, supported by my experiments with other bacteria cultivations. This point is a matter of importance, because it is not a matter of indifference whether the comma bacilli, when they come into a sink, find a good or very bad soil for reproduction. In the first case they would multiply, and would have to be destroyed by methods of disinfection; but in the latter case they would die off, and there would be no necessity for disinfection. I am inclined to hold the latter view, as borne out by all the experience I have so far had: strained only as long as the carbonic acid was allowed to operate. The same holds for the statement which I now lay before you. The comma-bacilli flourish best in liquids Iodine is known to have been characterwhich do not contain too small a quantity ized by Davaine as a very intense poison of nutritive matters. Several experiments for bacteria, and, under certain circumhave been made on this point. Dilutions stances, correctly so. Davaine made his of meat-broth with an alkaline reaction experiments by diluting, to a very great were prepared, and a quantity of comma- extent, a liquid containing anthrax-bacilli, bacilli were placed in them. In one of e. g., anthrax-blood, to such an extent, that these experiments, the meat broth, after a he finally had nothing but pure water, in five-fold dilution, proved to be no longer a which very few anthrax-bacilli were susnutritive solution. Of course, these experi-pended. He added some iodine to this ments must be repeated, and be made in a more extensive manner, in order to find a definite and fixed limit; but, in any case, it can be seen from these results that one must not dilute too much, and that commabacilli require a certain concentration of nutritive substance in which to grow. liquid, and then it was seen that the anthrax-bacilli were killed by an extremely small quantity of iodine; but in practice the conditions are quite the reverse. We never have to stop the develoment of infectious matters in pure water, but in the alkaline contents of the intestines, or in the In these cultivation-experiments, it was juices of the tissues, and the iodine does further seen that the nutritive substances-not remain free in these, but combines at at least, the gelatine and meat-broth-must once with the alkalies. The investigation not be acid. As soon as the gelatine shows of the influence of iodine on the commaonly a trace of acid reaction, the growth bacilli was made by adding iodine-water to of the comma-bacilli is very stunted. If meat-broth, which was just suitable as a the reaction be in a marked degree acid, nutritive liquid. Iodine dissolves in water the development of the bacilli completely in the proportion of about 1 in 4,000. One ceases. It is at the same time noteworthy cubic centimetre of this iodine-water was that it is not all acids that seem to be unfav-mixed with ten cubic centimetres of meatorable to the comma-bacillus ; for the surface of a boiled potato, where it is cut, is known to have an acid reaction, in consequence, if I am not mistaken, of its containing malic acid. Nevertheless, commabacilli grow very luxuriantly on potatoes. Hence, one cannot say, straight off, that all acids hinder the growth; but, in any case, there are a number of acids which have this effect. In meat-broth it is probably lactic acid, or an acid phosphate. As the influence of substances that prevent the development of the growth of the comma-bacilli is one of no small interest, a number of other substances have been examined with regard to this point. I must observe here that the prevention of development does not imply disinfection. In these experiments, it is only intended to determine that amount of substance which is sufficient to hinder the growth of the bacteria. But with this the bacteria are by no means killed outright, as should be done in disinfection. We had experienced something similar in the experiments on the influence of carbonic acid on commabacilli, in which also the growth was re But broth, but this did not hinder the growth Alcohol stops the development of comma- Common salt was used in the proportion of two per cent. without the growth of the comma-bacilli being hindered. Sulphate of iron only hinders the growth when two per cent, is added to the nutritive fluid. In regard to this substance, which is very much used for purposes of disinfection in times of cholera, I venture to remind you that a proportion of two per cent. is necessary before it acts as a preventive to development. The comma-bacilli are not yet killed by the sulphate of iron in |