disease have kept things moving at a lively pace. Some of the principal contributions to the history of the disease have been given by Trousseau, Bourgeoise, Rilliet and Barthez, Bamberger, Buhl, Virchow. In 1847 the latter first made the anatomical distinction between catarrhal, croupous, and necrobiotic varieties of laryngeal affection. Among the following workers may be mentioned West, Sanderson, Billroth, Oertel, Eberth, H. C. Wood and Formad, and many others. Here begins a period to which well fits the quotation of von Ziemssen at the opening of the late congress at Munich: "Rerum cognoscere causas," or, to go a step further, one quoted by the late Dr. J. A. Broadus, of Louisville, in his last address to the graduating class of the Kentucky School of Medicine: "Prius cognoscere, deinde sanare." Most all investigators recognized the presence of micro-organisms in the pseudo-membrane, and sometimes in the tissues and organs; all of them, however, speak only of micrococci, and while some have thought these to be in etiological relation to the disease, others have looked upon them as simply accidental in development, an effect rather than a cause. Thus Wood and Formad (in 1880) declare it altogether improbable that bacteria have any direct function in diphtheria, that is, that they enter the system as bacteria and develop in the system and cause the symptoms. In a later publication of a number of other experiments. and conclusions, as late as 1882, the same authors say: "There is no proof as yet that the micrococci are the cause of the disease. Their presence in the exposed dead tissue is no evidence, for the membrane. represents but the necrotic mucous lining, etc."

Up to this time the only organisms attracting attention are the different micrococci. In 1883 Klebs found constantly present, in the pseudo-membranes of patients dying with true diphtheria, bacilli of a peculiar and striking appearance. In 1884 Loeffler published the results of a very thorough and extensive series of investigations on this subject, having made use of the modern methods of bacteriological examination, as initiated by the greatest of all living bacteriologists, Robert Koch. His examinations really mark the beginning of the new era in the study of diphtheria. He found the bacillus described by Klebs in most, but not in all, cases of throat inflammations which had been diagnosticated as diphtheria. He separated these bacilli from other bacteria present and obtained them in "pure culture." Inoculation of these bacilli into an abraded mucous membrane of susceptible animals produced pseudo-membranes, and frequently death followed.

Subcutaneous injection of a bouillon culture caused death with characteristic diphtheritic lesions. Loeffler did not find these bacilli in every case examined, and also found them in one instance in the throat of an apparently healthy child. This failure to find them in all cases seems to be now explained by the fact that certain varieties of pseudomembranous inflammations are not due to the Loeffler bacillus, but to other organisms. In several later communications, in 1887 and 1890, Loeffler has come to the positive conclusion that the bacillus is the true and only primary cause of diphtheria. Many investigators have since proven the presence of, in the majority of cases of suspected diphtheria, a bacillus corresponding in every way to that described by Loeffler. In 1888 Roux and Yersin published experiments, which not only fully corroborated Loeffler's views, but which furnished most important additional proof. They caused the characteristic lesions and symptoms of diphtheria, not only by the inoculation of the bacilli themselves upon an abraded surface, but also by filtered cultures, showing that the bacilli during their growth produced poisonous products, which were capable of causing the general lesions, except possibly the pseudo-membrane. Furthermore, they were able to produce in animals the characteristic post-diphtheritic paralysis. Roux and Yersin thus conclude from their observations: "The occurrence of these paralyses, following the introduction of the bacilli of Klebs-Loeffler, completes the resemblance of the experimental disease to the natural malady and establishes with certainty the specific rôle of this bacillus."

Finally, the macroscopical and microscopical changes in the tissues of animals dying of experimental diphtheria have been shown by Welch and Flexner, by Babes, Benda, and others to be essentially the same as those produced in man, thus giving further proof of the specific rôle of this bacillus. Thus the three strictest requirements of proof that a certain bacterium is the specific cause of a specific bacterial disease seem to have been fulfilled, that is, (1) The constant presence of the organism in the diseased animal; (2) the isolation of the organism in pure culture, and (3) the reproduction of the disease by inoculations of these pure cultures and a similar distribution of the bacteria in the experimental and in the natural disease. If that be the case, we are now justified in saying (Prudden) "that the name diphtheria, or at least primary diphtheria, should be applied and exclusively applied to that acute infectious disease usually associated with a pseudo-membranous affection of the mucous membranes, which is primarily caused by the

bacillus, called the bacillus of Loeffler." All other cases of pseudomembranous or exudative inflammations of the mucous membranes, in which the Loeffler bacilli are positively absent, are classed to-day as "pseudo or false diphtherias," or, as Heubner proposes, as “diphtheroids." Not including here those cases caused by different chemical agents, these pseudo-diphtherias are supposed to be caused by a bacillus resembling in some respects the true diphtheria bacillus of Loeffler, but which can be positively distinguished by careful methods, called the pseudo-diphtheria bacillus, and in addition to these the different. pyogenic cocci, pre-eminently the streptococcus pyogenes.

If we consider the definition above given, and knowing the close clinical and pathological resemblance of the different exudative inflammations of the mucous membranes, we can see the importance of becoming perfectly familiar with the characteristics of the different organisms; for it is of the greatest importance, from a prognostic standpoint, to make the distinction between true diphtheria, a most malignant disease, and its comparatively harmless neighbors. Another important point is to be able to make the diagnosis early, because of the extreme contagiousness of diphtheria, so that we may use preventive measures as well as curative. We will therefore study for a few moments the principal morphological, biological, and cultural characteristics of the bacilli, as well as their pathogenetic qualities.

The Loeffler organism is a bacillus about as long as a tubercle bacillus and twice as wide as this. Its morphological behavior, however, is very varying as found in the exudation or artificial culture media. This depends very likely upon their age and the character of the soil. Some are seen to be spindle-shaped, others like little dumbbells, others lancet-shaped, and again short rods appearing to be made up of segments will be seen, while, in addition, a number of irregular, clubbed, flask-shaped, bizarre varieties may be detected in the field of the microscope (involution forms?). The bacillus is non-motile. It is aerobic, and grows at temperatures between 20° and 42° C., the most favorable temperature being 37° to 38° Celsius. The culture media ought always to be slightly alkaline in reaction. Growth takes place on different culture media, such as agar-agar, with or without glycerine, nutritive bouillon, milk, alkalinized potato, gelatine, but that best suited for a rapid and luxuriant growth is the coagulated blood-serum mixture of Loeffler, made up of blood serum (best of beef or mutton), three parts, nutritive beef bouillon, one part, with addition of 1 per cent

peptone, 0.5 per cent NaCl, and I per cent grape sugar. This is poured into sterilized culture tubes, and, after plugging these with sterilized cotton, is coagulated and sterilized in a slanting position, either by the original method of Koch, or by the rapid (American) method of Councilman, viz., rapid coagulation by heat with subsequent sterilization by steam. For the purpose of isolation of the bacillus out of the diseased body we inoculate several tubes with a particle of exudate from the mucous membrane, either by means of a platinum noose, or by means of a cotton swab fixed to a stiff steel wire, as devised by Prudden, and followed systematically in the examinations by the New York Board of Health, by Park and Beebe, under the direction of Biggs. This swab is rubbed well against the throat of the patient and then gently over the inclined surface of the culture medium. This latter method has also been followed in the large number of examinations made in Louisville during the epidemic of diphtheria, in the fall of 1894 and the spring of 1895, by Drs. Louis Frank and H. H. Koehler at the laboratory of the Kentucky School of Medicine, and in isolated cases by myself and others.

These inoculated tubes, put in the brood-oven at a temperature of 37° to 38° C., will develop on the surface of the serum, in from twelve to eighteen hours, a number of dull, dense, whitish or yellowish-white, slightly elevated points of from one half to one millimeter in diameter. These as a rule represent diphtheria bacilli, from which now it is easy, by the well-known methods, to obtain the organisms in pure culture, either for more accurate study or for the purpose of testing their virulence by inoculation into proper animals, especially guinea-pigs. There may be seen, even at this time, other bacterial colonies with which the bacillus diphtheria was associated, but they are rarely so far advanced or so conspicuous as the Loeffler bacilli, and that is in fact one great advantage of the blood serum as a culture medium for the diphtheria bacillus. It grows much more rapidly as a rule on blood serum than do the other bacteria with which it is commonly associated. On account of the great importance of making a certain and early bacteriological diagnosis, I will mention that Ohlmacher, in a recent article (Med. News, May 4, 1895), states that four hours' growth on blood serum in the incubator has been found to develop sufficient growth of bacilli to enable him, without waiting for visible colonies, to make the diagnosis by a microscopic examination of a stained specimen. Baginsky (B. Klin. Woch., pp. 1174-94) recommends very warmly the

Pure

method of D' Espine and de Marignac, that is, removal of a piece of the membranous exudation by a forceps, and washing it in two-per-cent boracic acid solution before inoculating the culture tube. cultures on blood serum, kept at a temperature of 37.5° C. for three or four days, present individual, round, elevated, grayish-white or yellowish-white colonies, about one to three millimeters in diameter. They are usually slightly elevated in the center, where they are denser than at the periphery. The surface is at first slightly moist, but gradually becomes dull and dry in appearance.

Upon glycerin-agar their growth is quite characteristic, but slower and more uncertain. They appear at first, when on the surface, as very flat, almost transparent, glistening, round points, not elevated above the surface; they have a darker, granular central portion and a lighter peripheral one. The latter becomes broader as the colony grows in size, is usually marked with ridges or cracks, and notched at the periphery; when growing deep down in the agar-agar the colonies are coarsely granular and rarely more than three millimeters in diameter. (Demonstration of culture tubes.)

On gelatine plates they form, at a temperature of 22° to 24° Celsius, small round colonies which do not liquefy the gelatine.

On bouillon they grow well, cause a slight cloudy deposit, change its reaction to an acid one within twenty-four hours, which, after a week or ten days, gives way to permanent alkalinity. Milk offers a favorable soil.

The bacilli seem not to develop spores, but they may remain living for a considerable time even outside of the body. Thus they were found to live on different culture media, by Hoffmann one hundred and fifty-five days, by Loeffler and by Park seven months, by Klein (on gelatine) eighteen months. On bits of dried membrane, by Loeffler fourteen weeks, by Park seventeen weeks, by Roux and Yersin twenty weeks, by Abel, on a toy building-block, for six months.

The D. B. stains readily with all the basic aniline dyes; the one most used is Loeffler's alkaline methylene-blue. Strange to say, I find, in looking over the subject, a difference of statement as to the staining faculty by Gram's method. Thus, Sternberg, Welch and Abbott, Roux and Yersin, and Weichselbaum state that it stains by Gram, while C. Guenther, in his excellent book, and Lenhartz are of opposing views. Time has prevented me from making personal inquiry as to the explanation for this difference of statement.