The obtaining of a pure culture and the further cultivation of the influenza bacilli upon artificial culture media at first offered great difficulties. The ordinary culture media all proved to be insufficient. Although Pfeiffer occasionally and exceptionally succeeded in cultivating one generation of the bacilli upon ordinary agar at incubator temperature, and although Kitasato states that he succeeded in obtaining several generations upon glycerin agar, nevertheless these culture media must be considered as inadequate and only occasionally sufficient.
Subsequently Pfeiffer, after " innumerable futile attempts," found a suitable culture medium for easily and certainly cultivating the influenza bacilli, in agar streaked with sterile blood. The procedure is as follows:
The surface of a slanting agar tube or the surface of agar in a Petri dish is streaked with blood obtained under aseptic precautions, most conveniently blood from the finger tip. All varieties of blood are efficacious, but the cultivation upon agar streaked with blood obtained from the pigeon is characterized by a particularly rapid and luxurious growth (Pfeiffer).
The prepared blood agar tubes are provided before use with rubber caps, and are placed for twenty four hours in the incubator. It is a fundamental rule, as far as the sputum is concerned, to use only fresh bronchial secretion obtained in sterile dry receptacles.'
R. Pfeiffer's method for the obtaining of pure cultures is as follows: " Bronchial sputum or fluid from lung areas infiltrated with pneumonic exudate is rubbed up with one to two cubic centimeters of bouillon (sterile normal salt solution may also be employed) until an evenly distributed, only slightly turbid emulsion is produced. Loopfuls of this emulsion are transferred to blood agar and controls also on common agar or glycerin agar. The emulsion should be evenly distributed upon the surface. The test tubes are now placed in the incubator (37° C). After twenty to twenty four hours the influenza colonies are seen on the blood agar, looking like fine drops of dew which, microscopically, are seen to be made up of fine rods. The control test tubes will have remained either sterile or contain only isolated colonies of other forms of bacteria, usually streptococci or Frankel's diplococci."
W. Kruse describes a " brush method." Several tubes containing 2 per cent, agar, after pouring off the condensation water, are melted, and while hot poured into Petri dishes. These are allowed to cool uncovered under glass shade, to prevent the subsequent formation of condensation water. Then, with an ordinary paint brush which has been sterilized in steam, pigeon blood is brushed over the surface of the agar plate. In the same way, also with a brush, the material to be examined for influenza bacilli is distributed over the surface, either direct or after dilution in sterile bouillon. From the finished plate, with a new brush, some of the material may be taken off and placed upon a second blood agar plate, etc. In this way any dilution required may be obtained. [Symbiosis, especially with Staphylococcus aureus, favors the cultivation of Bacillus influenzas (Grassberger and others). A simple, good nutritive medium can be made by boiling up blood, without the serum, with normal NaOH, adding this to liquefied agar, and shaking thoroughly. The medium remains efficient and reliable for a long time (Ghon and Prevss).- Ed.]
The influenza colonies growing upon blood agar are so characteristic that they are easily recognized among several hundred other colonies of bacteria. They usually occur as clear, limpid droplets lying close together, with only a slight tendency to coalesce. Usually the colonies are so small that a hand lens is necessary to see them clearly. With only a few and widely separated germs, from which the colonies can readily expand, they often attain considerable size
-about the diameter of a small pin's head; but even these, the largest colonies, show a characteristic glassy transparency. We may further mention the following cultural characteristics of influenza bacilli: 1. They are strict aerobes. 2. They grow only at blood temperature (37° C.)-the maximum temperature limit 42°, and the minimum, 28° C.; at room temperature, from 23° to 24°, there is not a sign of development even after four clays. 3. Their period of viability in bouillon is considerable under ordinary circumstances; they do not die off until from fourteen to eighteen clays. For the same period of time, and sometimes even for twenty clays, they retain their vitality on blood agar. On this medium it is immaterial whether the developed cultures remain in the incubator or are kept at room temperature. Nevertheless it is well to make subcultures every four or five days. 4. The blood agar cultures in the incubator are fully developed in from twenty to twenty four hours. 5. A persistent form, spores, have not yet been demonstrated. 6. In drinking water the bacilli very rapidly lose their vitality in from twenty four to thirty six hours. 7. In sputum that has been kept moist the saprophytes which are present soon overgrow the influenza bacilli, though, according to Pfeiffer, they retain their infectiousness in moist sputum at least for fourteen days. 8. The influenza bacilli are conspicuously sensitive to drying. Blood agar cultures which have been streaked upon glass plates and have been dried at a temperature of 37° C. become sterile in from one to two hours; dried at room temperature, in from eight to twenty hours. Sputum from grip patients dried at the ordinary room temperature was sterile in from thirty six to forty hours.
True influenza bacilli, according to Pfeiffer, are found exclusively in endemic and epidemic influenza. In the secretions of an ordinary coryza influenza bacilli are never found. E. Neisser found them once in the sputum of a phthisical patient who, although not affected by influenza himself, was in a bed in the same ward with influenza patients.