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BATTLE CREEK, MICH., U. S. A., FEBRUARY, 1893.
BY PAUL PAQUIN, M. D.
(Illustrations in Frontispiece.)
So much is being said and printed now about cholera, that physicians, in view, of the possibility of its appearance in this country next spring, are all very anxious to inform themselves as much as possible on the most recent discoveries concerning it. It may not be amiss, therefore, to publish at this time, some notes on the most important points concerning its cause, development, and
At the outset, it is well to bear in mind that Asiatic cholera is, as the medical profession well knows, endemic in India, and reaches Europe usually by way of Egypt. It never arises spontaneously in Europe or America.
As far back as 1848, the parasitic nature of cholera was suspected. In that year Virchow, and in the next year Pouchet and Swayne, found vibrios in abundance in the dejections of choleric patients, without attributing to them any. specific action. From that time until the discovery of the comma bacillus by Koch, numerous investigations were made without definite results. In 1884 Koch became positive that cholera was due to this parasite, which he termed the comma bacillus because of its peculiar curved appearance, something the shape of a comma.
The definition of Asiatic cholera may be given as follows: An essentially infectious disease, apparently contagious under certain circumstances, and epidemic. It may or may not be attended
by premonitory diarrhoea; its invasion is sudden, accompanied by copious evacuations, vomiting, diarrhoea, cramps, and algidity, which frequently end in death, or in a reaction in which various symptomatic manifestations occur, followed by recovery or death. It must not be confounded with cholera nostras or cholera morbus, which sometimes present many of the foregoing characteristics, but which are produced without the comma bacillus, by different micro-organisms. These other organisms appear and do mischief in certain seasons, contrary to the Asiatic cholera, which may appear with more or less violence at any time of the year. It is modified but not destroyed by low temperature.
The Germ of Cholera.- As we have mentioned, the comma bacillus of Koch is the parasite which produces cholera. It is found in the inodorous aqueous liquid dejections in which float whitish riziform grains. The comma bacillus is almost always present in these particles.
The bacteria can be demonstrated by spreading on a cover-glass a fragment of riziform particle, allowing it to dry, and then coloring it a few seconds with methyl-violet or methyl-blue. The preparation is washed and examined with an immersion objective and an Abbe condensor. This method, however, shows numerous other bacteria, among which it may be difficult to discover the comma bacilli. To find these bacilli in large quantities, it is necessary to study a case very rapidly in the earliest stage, when they may be as plentiful as in a new, pure culture.
The best means of seeing these parasites in liquid .fæcal matters containing many, is to spread a bit of a small mucous particle on a glass slide, allow it to half dry, and then cover it with a few drops of weak solution of methyl-violet
(6 B) in distilled water. A cover-glass is applied, and pressed down with bibulous paper. The preparation is then examined with a high power objective, dry or in immersion. The bacilli are found still alive, though stained; they are very active, and retain their movements for some time.
This mode of preparation, say Cornil and Babes, is better for a delicate examination than a complete desiccation with coloration and mounting in Canada balsam. It is well established that during and by manipulation and dehydration, the bacteria of cholera contract, as do most other bacteria under the same influences. The result is that they decrease in size, while their movements are of course completely destroyed.
The average comma bacillus is about Iμ,5 to 2μ,5 in length by oμ, 5 to oμ,6 in thickness. The germs are well named, "like a comma; " their sides are smooth and their extremities either blunt or a little pointed and thickened. They are not so long, but broader than the bacilli of tuberculosis. Occasionally two rods are observed end to end, in such a shape that the two united form the letter S. They are perhaps the most characteristic of the comma bacilli. (See Figs. 2 and 3, frontispiece.)
After the second or third day of cholera, when the period of reaction begins, the comma bacilli are scarce, and stained by bile; it is then difficult to see them, and it is sometimes necessary to make cultures if we wish to ascertain their presence. After a long period it would be difficult, if not impossible, even by cultures, to ascertain if they were present, because at the beginning of the period of recovery they disappear, and soon they are entirely absent. In rapid cases, however, in which death occurs, the germs may be cultivated from the fæces immediately after death. The secondary lesions which are found on the mucous membrane of the intestines, etc., such as ulceration and gangrene, are attended by a development of numerous bacteria of decomposition, which eventually destroy the bacilli of cholera. This point should not be forgotten in making investigations.
Numerous methods of growing cultures have been recommended for this germ, as for other microbes; but for practical use the two following seem to have given the most satisfaction:
The first consists in inoculating one tube of liquefied gelatine with a platinum. wire. This tube is shaken, and from it a second inoculation is made in a second tube with a curved platinum wire, using three drops for inoculating material; the second tube is shaken and mixed as the first. Five drops are then taken from this tube and mixed with the liquid gelatine of a third one. The contents of each tube are then poured on three plates superposed, the first of which should be the inferior one.
The second method consists of taking a few riziform particles of the fæces, mixing them well with liquid gelatine, and making from this some gelatine solutions by Koch's method, i. e., successive solutions with 1, 3, 5, and 10 drops. These different culture solutions are poured on plates. The development is allowed to progress for two or three days, during which colonies of microbes may appear on the first plate as slight liquefied spots. They may be plain enough on some or all after two or three days. The colonies may be scarce or numerous; of course they are more numerous in the more concentrated of the culture solutions.
Another series of similar dilutions is then made, by using a little taken from the liquefied spots. The substance may
also be cultivated in beef broth at the temperature of the body. If it contains any cholera bacilli, they will develop on the surface in the form of a white scum.
In examining with a magnifying glass or microscope at 55 diameters, 24 hours after sowing a plate of gelatine, the colony of bacilli may be recognized by the following characteristics, which we quote word for word from Cornil and Babes's writings :
"In the center of the colony there exists a spot as if formed by a mass of dust surrounded by a granulous circle, and a second circle clear and nongranulous. (See Fig. 1.) Between the center and the first circle the gelatine is liquefied. These cultures have a yellowish appearance: they are more transparent than most of the cultures of the germs of the fæces. The other bacteria of the fæces usually form larger colonies, round, dark, and brown, which do not liquefy gelatine. We have found twice, among the bacilli of cholera, some other curved microbes, which are described in our clas
sification. One of the microbes found, liquefies gelatine, more slowly, however, than the comma bacillus, and it is distinguishable from it because it is not curved or motile.
"If the colonies of the cholera bacillus are very close to one another, they soon become united, and give rise to irregular figures. The gelatine is liquefied, and in this liquid zone swim little opaque flakes.
"In order to isolate this bacterium, it is necessary to take material from the center, magnifying the colony to 50 diameters for that purpose. A slight particle taken from this spot with a sterilized platinum needle, is transferred to a gelatine or agar-agar tube. Two days later a grayish mass may be seen in the gelatine; it is transparent, its surface is somewhat concave, granulous, and conical, with the point directed toward the bottom of the tube; in other cases it penetrates into the gelatine in the form of whitish prolongations. Liquefaction takes place slowly, but a considerable quantity of it is present after a few days. The liquid portion is somewhat milky and granulous at its lower part, and in the full development of the culture its liquid portion is all whitish and opaque. A distinguishing point between the liquefied action of the comma bacillus and that of other bacteria on liquid gelatine, is, that in the former the gelatine beneath the puncture of the inoculation remains solid for a long time, while almost every other liquefying bacteria liquefy the gelatine in a few days in the form of a sack. Generally speaking, the bacillus of cholera liquefies gelatine much more slowly than any other bacteria perhaps, for it takes at least one week to liquefy a tube completely."
If a gelatine culture medium of 10 per cent is used, a characteristic point worthy of notice presents itself: It is the reaction of the gelatine in the form of an air bubble. In less concentrated gelatine, or in a culture exposed to a higher temperature, the gelatine liquefies it sooner in the form of a sack.
In liquefied gelatine the culture examined is found to contain very motile bacilli. They may be studied by placing a small drop of the liquid with a platinum wire on a cover-glass, placed on the excavation of a glass slip containing a drop of a very weak solution of methyl-violet (1 to 2000). The edges of the coverglass should be smeared with oil or vase
line, to prevent evaporation. Microscopic examination should be made at once. The germs take on coloration this way without dying. Their movements are very rapid, and they may be seen at an ordinary temperature. The movements consist chiefly in oscillations more or less analogous to those of spermatozoids. 66 The curved bacilli contract by bringing the two curved extremities together, and diminish their curvature in turning." The culture begins to show germs inclined to curvature of the adult form, after 10 hours. The curvature comes gradually as the culture grows older. The forms of the germs in the cultures as well as in the dilute liquids may vary according to age and circumstances, from almost straight rods in the beginning to spiral shaped bacteria in old cases. (See Fig. 2.) The spiral forms are similar to several bacteria, but are chained together by the extremities, giving the appearance of several s's end to end.
The Vitality of the Comma Bacilli.The comma bacilli are aërobic, and may live on and in other substances than the material of the alimentary canal and the ordinary culture media. It has been established that they grow easily on damp clothing, and in milk without altering its color, aspect, etc. They also grow at the temperature of the body on cooked potato, on which they appear in the form of a grayish-colored coat. These germs have also been cultivated with success on meat, in broth, on carrots, cabbages, moist bread, eggs, cheese, and in sweet water. The best temperature for their growth is 186° to 104° F., but they multiply very well on gelatine at 68° F. They do not grow below 60° F. They do not die, however; in fact, they preserve their first vitality. Cold weather even, does not destroy them, for they remain alive at a temperature below the freezing point, although they are inactive.
The germs thus preserved may exhibit their vitality again at an ordinary temperature, either in cultures, moist clothing, or in specimens of earth. Indeed, under such circumstances they may multiply so rapidly as to predominate over other germs. They are usually destroyed, however, two or three days afterward, by the action of other bacteria growing with them. This is probably what takes place, in this malady, in the fæces, on the sur
face of the intestines where the bacilli of cholera disappear entirely in a few days after the attack. It is established that even when the small intestine is inflamed and dispossessed of its epithelium, and covered more or less with blood from capillary hemorrhages, the germs of putrefaction flourish, and the symptoms that result are analogous to intoxication by septicemia.
Cholera germs were successfully culti vated while exposed the whole winter to the temperature of the exterior air at Berlin, being renewed from time to time. The best days possible were chosen for this operation. This demonstrates that it is possible for these germs to pass the winter alive, provided it occurs in conditions offering sufficient nutrition.
On the other hand, the comma bacilli stand the heat pretty well for several days; 112° to 113° F., for instance, does not effect them until after a very long time, but they were killed at 122° F. in a very few days. Cultures heated slowly to about 147° F. or very rapidly to about 167°, soon become sterile.
In water they may live a certain time, but water as a rule contains no substance capable of nourishing them sufficiently, and as a result they soon perish. Of course this does not apply to stagnant water containing organic matters, nor does it apply to water of rivers which are fed by rich deposits of organic substances from the banks and small streams from the country. Such waters make very nutritive places for the accumulation of germs of many kinds, including probably the comma bacillus. The germs may live in ordinary drinking water several days, but in distilled water they die in twelve hours. Seltz water kills them in a day. Koch claims that most acids are deadly to them, but that they can live in contact with a few of them. He says that the acids of the gastric juice are deadly to them, till they reach the alimentary canal; if Koch's theory is correct, it must be that to reach the bowels they pass the stomach in a large quantity of liquid or in solid material, being thus protected by the mixture and dilution, and not stopping sufficiently long to come in contact with the gastric juice.
Various substances have been tried with a view to destroying the comma bacillus, and it was found that vegetation was arrested or prevented by the follow
ing substances in the proportions indicated: Alum, at 1 to 100; camphor, I to 300; carbonic acid, 1 to 400; essence of mint, I to 2000; sulphate of copper, I to 25,000; quinine, 1 to 5000; corrosive sublimate, 1 to 100,000.
Other experimentalists put the figures as follows: Corrosive sublimate, 1 to 20,ooo; carbonic acid, 1 to 1000; sulphate of copper, I to 3000 to 1 to 5000; salicylic acid, 1 to 800 to 1 to 900; thymol, 1 to 900 to 1 to 10,000; iodine, 1 to 500; bromine, I to 600; alcohol, 1 to 15; quinine, 1 to 800; and acetic acid, 1 to
Desiccation is also deadly to the comma bacillus. Koch found that choleric fæces spread on linen and allowed to dry a few hours to a few days, destroyed the life of the cholera germs. Dejections buried in the ground and then dried at the surface of damp or dry earth, furnished dead comma bacilli. It is believed, therefore, that this parasite does not possess spores capable of resisting such influences and living for years in a latent condition, as do spores of charbon, for instance. From Koch's experiments, desiccation of infected objects would be a very practical and safe mode of disinfection.
The vital action of other microbes is another mode of destruction of the comma bacillus. Several kinds interfere with rapid multiplication, if they do not. actually stop the growth of this microbe.
Experimental Cholera. The general phenomena of cholera are explained by two theories, both of which seem correct. The first theory is that the comma bacillus produces very rapidly some local intestinal lesions, and possibly some toxic substances, conditions which are attended with certain serious local and general disturbances, and sometimes death; the second theory is that septic microbes produce powerful septic agents, and complicate the cholera infection at an early period, resulting in the paralysis of the walls of the blood vessels and other most serious sapremic, or septic, phenomena. All the lesions and symptoms of cholera seem to depend directly or indirectly on these two far-reaching factors. The observations and experiments of the most conscientious and accurate investigators and practitioners seem to support these views. The production of experimental cholera was the only link necessary to complete the evidence against the
suspected specific germs causing the various complications which are a part of cholera. This has been done to some degree of satisfaction. Guinea-pigs and other small animals have been infected, and the disease has been studied in its various stages, both in rapid and slow
Pathology of Cholera.- (Figs. 4 and 5, representing sections of intestine.) Just a few words on this point. I had not intended to touch on it at all, but it is of value in demonstrating the truth of several preceding statements.
The chief microscopic lesions are: A special mucous condition of the peritoneum; severe congestion of the intestines, which have a peculiar roseate color; a creamy serous liquid in the mucous surface of the intestines; pronounced congestion of the vessels of the periphery of the glands of Peyer in ordinary cases; often swelling and ulceration of the follicles. The creamy liquid consists chiefly of disintegrated cells of the epithelium of the intestinal villosites.
The fæcal matters are odorless, or emit a flat smell; they consist of watery matter in which float many grayish or whitish flakes riziform grains which form a deposit in the bottom of the vessel, leaving the upper portion barely clouded. They are neutral or slightly alkaline. In the first and sometimes the second day there is no bile in the fæces. The biliary and urinary secretions seem to be suspended during the period of algidity.
Microscopically, the liquid of the fæces. and the intestinal surface contains numerous mortified, loose epithelial cells, "cylindrical, or tumefied, or granulous, or hyaline."
The lower part of the ileum is usually the most affected, and in that region may be found the comma bacilli in the greatest quantities. It seems that the liquid substances ingested carry the germs to this point. The comma bacilli penetrate through the villosities into the layers of the mucous membrane, the glands, and the connective tissue, as may be seen in proper sections. The microscope has demonstrated numerous comma bacilli in the first stages of the cultures; in the second, many other germs are seen with them; and at a certain moment, these invading microbes predominate, and arrest the growth of the comma bacillus, as stated above. Among these microbes
was found a curved individual resembling the comma bacillus, but easily distinguished from it. Another resembles the staphyloccocus aureus, and is pathogenous to mice and probably to man. Technique. This has been touched upon in the foregoing explanations. It is easy to stain fresh comma bacilli, particularly if 24-hour cultures of a moist chamber are used. The germs are dried slightly on cover-glasses, and stained with a weak solution of methyl-violet. After complete desiccation, the germs resist the stain, and on the other hand they are contracted and appear smaller; it is then difficult to determine their structure. If it is desired to color dried germs, it is necessary to place them in an aqueous solution of anilized fuchsin slightly alkaline, and place the whole in a damp chamber covered with a glass bell. They should be mounted in Canada balsam.
Another idea is that dried substances containing germs of cholera may be transferred from place to place and then produce infection. This cannot be a common cause, since desiccation means death to these parasites. It is man himself that is the chief medium for the propagation of cholera, notwithstanding the fact that the germs he carries may not propagate on his person; he may carry them in his bowels in active vegetation, or they may be preserved for a time on his clothing, in damp habitations, in stagnant water, on the surface of vegetables, and in food, such as milk, bread, etc.
The development of the bacillus of cholera depends on many conditions. It has been seen that it may live at winter