INFLUENZA.

On the first occurrence of influenza in the winter of 1889-90 the numerous bacteriologic investigations undertaken failed to yield conclusive results. In the secretions of influenza-patients the ordinary exciting agents only of inflammation, especially streptococci and lanceolate diplococci, were encountered. Of the latter it was stated that in appearance and growth they presented certain points of distinction as compared with ordinary pneumococci. A specific bacterium was, however, not found. In subsequent epidemics (1891 and 1892) Pfeiffer, in the Hygienic Institute at Berlin, recognized a special bacillus as the exciting agent of influenza, and developed it in pure culture. The statements of Pfeiffer have since been completely confirmed.

Morphology of the Influenza-bacillus.-The influenzabacillus is an extremely small organism (0.2 μ thick and 0.5 μ long), in thickness not quite equaling the slender bacilli of mouse-septicemia, and being only twice or thrice as long as wide. Its extremities are rounded. Rarely in the sputum and more commonly in recent pure culture, the bacilli form short pseudo-filaments. Long bands in cultures three or four days old are to be considered as beginning involution-manifestations. The influenza-bacilli possess no capsule, and are without movement of their own. Frequently, two especially short bacilli lie close to each other (division-forms). This may readily give rise to confusion with diplococci.

Influenza-bacilli appear not to possess spores.

Spore-like

formations have never been found in the secretions or in cultures, and, besides, the bacillus is but little resistant to the influence of temperature, drying, etc.

Staining of Influenza-bacilli.-The bacilli take the stain with considerable difficulty. Löffler's solution of methyleneblue may be employed, and, still better, a dilute, pale-red solution of carbolfuchsin in water. The preparation must be exposed to the action of the stain for from five to ten minutes. If the exposure is of shorter duration, or if other stains are employed, the central portion of the bacillus is often more feebly stained than the extremities. The bacilli are not stained by Gram's method.

Cultivation of the Influenza-bacillus.-The influenzabacillus is strictly aerobic, and it develops only in the presence of hemoglobin or of leukocytes. The latter fact explains why cultivation of the influenza-bacilli remained for so long a time

unsuccessful. Pfeiffer also was often able to cultivate the bacilli from the sputum or pus from the lungs directly upon agar, but this did not take place invariably, and at times it was quite impossible to continue the growth of such cultures in any way. The explanation for this fact is that the bacilli developed in the first culture when with the infecting material a trace of blood was simultaneously transferred to the culture-medium. Growth failed to take place, however, when the blood was wanting, and thus also in all daughter-cultures.

The development of influenza-bacilli takes place regularly, and the culture obtained may be continued indefinitely, if the infecting material is inoculated upon a culture-medium containing blood, and best upon blood-agar tubes (p. 82). For the

development of pure cultures Pfeiffer recommends the following method: The infecting material-bronchial sputum or fluid from a bronchopneumonic infiltrated portion of the lung in a case of influenza-pneumonia-is rubbed up with one or two cubic centimeters of bouillon to a homogeneous emulsion. By means of a platinum loop some of this is inoculated upon blood-agar, and also for control-purposes upon ordinary glycerin-agar, tubes, care being taken to secure uniform distribution of the infecting material upon the entire surface. The dilution of the sputum in the bouillon is intended, in the first place, to separate the influenza-bacilli to such a degree that isolated colonies will form upon the blood-agar tubes. In the second place the hemoglobin that may be present in the infecting material will be so greatly diluted that the influenza-bacilli will be incapable of

developing in the control-tubes not previously treated with blood. The inoculated test-tubes are placed in the thermostat. After the lapse of twenty-four hours the influenza-colonies will become visible in the blood-agar tubes as densely packed, watery drops, whereas the control-tubes will be either sterile or present isolated colonies of streptococci, diplococci, or other bacteria, which were present in the infecting material in addition to the influenza-bacilli.

The water-like drops of influenza-colonies are usually so small that they are only visible with the aid of a lens. They exhibit little tendency to become confluent. Should they be especially numerous and close together, they coalesce to form larger drops with curved margins, but these permit a recognition of the aggregation of individual colonies. If the colonies are isolated and widely separated from one another, they may grow to the size of a pinhead, but in this case also they retain a vitreous transparency. The water of condensation of influenza-cultures generally remains clear. When mixed with blood derived from the oblique surface of the culture, delicate white flocculi form in it. In bouillon mixed with blood and spread in a thin layer the influenza-bacillus thrives quite abundantly.

The plate-procedure is greatly to be recommended for the isolation of influenza-bacilli and for diagnostic purposes, if some blood is added to the liquefied agar before inoculation, or if agar is permitted to solidify in Petri dishes and blood is added, and the diluted sputum, etc., is smeared upon the surface in several streaks. The colonies present the same appearance as those in agar-tubes.

The temperature-optimum for the cultivation of influenzabacilli is that of the body. The upper limit for growth is 42° C. (107.6° F.), the lower between 26° C. (78.8° F.) and 27° C. (80.6° F.). The bacilli do not grow at room-temperature.

Oxygen is always necessary for the development of the influenza-bacilli; they do not grow in an atmosphere of hydrogen or of carbon monoxid, even in the presence of blood.

Pfeiffer undertook to determine what constituent of the blood the influenza-bacilli require for their development. On making transfers from blood-serum or blood-fibrin to agar-tubes no growth took place. Red blood-corpuscles were invariably necessary, and particularly the hemoglobin contained in them, as was later shown, was the active substance. Hemoglobinagar (p. 82) is equally adapted with blood-agar for the cultivation of influenza-bacilli. Pfeiffer endeavored to associate this indispensability of hemoglobin for the growth of influenzabacilli at first with its relations to oxygen, with its faculty of acting as an oxygen-carrier. He succeeded, however, in obtaining growth upon an agar-layer in the presence of carbon

monoxid hemoglobin. Exposure of blood-agar tubes to a temperature of 70° C. (158° F.), and even boiling of the hemoglobin, failed to prevent entirely the development of the influenza-bacilli. Pfeiffer was then led to believe that the ironcontent of the hemoglobin was the important factor, but he was unable to cultivate the bacilli in culture-media containing iron other than that of the blood.

It may be mentioned further that all kinds of blood exhibit the same specific activity with relation to influenza-bacilli. Pfeiffer obtained growth upon the blood of rabbits, guineapigs, pigeons, and fish, and in more luxuriant degree and more speedily on pigeon's blood-which is rich in hemoglobin—than on human blood.

Resistance of Influenza-bacilli.-Influenza-bacilli are. destroyed in a few minutes when exposed to a temperature of 60° C. (140° F.). They cease to develop at a temperature of 43° C. (109.4° F.), but they are only coagulated, not destroyed, for if tubes that have been exposed for forty-eight hours to a temperature of 43° C. (109.4° F.), and have remained sterile, are then exposed to a temperature of 37° C. (98.6° F.), colonies will yet develop abundantly. In unsterilized drinking-water the bacilli die quickly-in from twenty-four to thirty-six hours. Upon blood-agar and in bouillon they retain their vitality for from fourteen to eighteen days, and in moist sputum they appear to preserve their infectivity for at least fourteen days. The influenza-bacilli are quite sensitive to drying. When dried in blood or sputum at a temperature of 37° C. (98.6° F.), they succumb in an hour or two, and when dried at roomtemperature, within not more than from thirty-six to forty hours.

Occurrence of Influenza-bacilli.-The influenza-bacilli occur regularly in the secretions of influenza-patients. In the secretion of the nasal cavities the specific bacilli have been found in enormous numbers; although generally associated with other microorganisms, yet, however, in preponderating number. The secretion in a case of ordinary coryza, on the other hand, is remarkably free of bacteria, being almost sterile. The sputum in cases of bronchitis and pneumonia complicating influenza is viscid, mucopurulent, globular, and not seldom also purulent and confluent, in color often yellowish green, not rarely pure white and only seldom rusty brown, and it contains the

influenza-bacilli in almost absolutely pure culture, and always in surprising number. The bacilli usually lie in the mucous ground-substance arranged in nests and groups; they are found also within the pus-corpuscles, at the beginning of the disease in small number, and during convalescence in preponderating number. In the latter condition they surround the nucleus and are not included within it. Sputa containing influenza-bacilli are often ejected for days and months. Especially in cases of tuberculosis have such chronic complications of influenza with bronchopneumonic localization not been rare. The bacilli have been found in the bronchopneumonic foci in cases of influenza-pneumonia, rarely in the pus in cases of influenzaCanon observed in the blood of influenzapatients delicate bacilli resembling those of influenza. According to Pfeiffer's investigations, these organisms, if influenza-bacilli at all, are exceptional, for, as a rule, the organisms are not present in the blood.

empyema.

With regard to the localization of the bacilli in the gastric and nervous forms of influenza, unequivocal investigations are wanting. Likewise, the numerous complications and sequelae of influenza have thus far been little studied from the bacteriologic standpoint, so that it has not yet been determined whether they represent results of the activity of the influenza-bacillus or its toxin, or are secondary infections. In a case of influenza-aortitis Pfeiffer found, in addition to the diplococci of Fränkel, numerous influenzabacilli, and in the exudate from a case of influenza-meningitis diplococci exclusively.

The bacillus of influenza has never been found outside the human body, in the earth, or in water. It could scarcely persist for a long time under these conditions on account of its feeble powers of resistance.

Portals of Infection for, and Distribution of, the Influenza-bacillus.-The influenza-bacillus is probably taken up by the air-passages exclusively. Its distribution by means of dried and powdered sputum can play an etiologic rôle only in restricted degree, as the bacillus withstands drying so badly. The ordinary mode of conveyance is certainly by means of the moist nasal and bronchial secretions of influenza-patients. The widespread and often pandemic distribution of influenza may be explained by the fact that in the first place the susceptibility of human beings to