VOLUME XXXV - DECEMBER, 1931 - NO. 6
OBSERVATIONS ON BACILLUS TYPHOSUS IN ITS FILTERABLE STATE
A PRELIMINARY COMMUNICATION
By ARTHUR ISAAC KENDALL, PH.D. Chicago, Illinois
ROYAL RAYMOND RIFE, PH.D.
San Diego, California
The Rife Research Laboratory, San Diego, California; The Laboratory or Research Bacteriology, Northwestern University Medical School, Chicago, Illinois; and The Pathological Laboratory of the Pasadena Hospital, Pasadena, California.
Presented at a meeting of the Bacteriological Section or the Los Angeles Clinical and Pathological Society, November 20, 1931.
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November 2, 4 p. m. Inoculated six cubic centimeters of K (protein) Medium 2 from the agar slant culture.
November 3, 10 a. m. Filtered this culture in K Medium of November 2, through a Berkefeld "N" filter. (The culture was diluted with four volumes of sterile physiological saline solution; the vacuum used was less than four inches of water; the total time of filtration was less than ten minutes.)
November 3. One drop of filtrate, representing one-fifth drop of the original culture, was introduced into six cubic centimeters of K Medium. Incubated at 37 degrees centigrade. The filtrate was also tested for purity as follows: (1) cultural reactions; (2) sugar fermentation reactions; (3) agglutination with specific typhoid serum. All were typical.
November 5. The forty-eight-hour culture of November 3 in K Medium was filtered, as above, through a Berkefeld "N" filter. One drop of the filtrate was added to six cubic centimeters of K Medium and incubated at 37 degrees centigrade.
November 9. The culture was again transferred to K Medium.
November 12. Still another culture was made, in every instance using three loops of culture for the inoculum.
It is worthy of note that this thrice filtered culture of B. typhosus grew quite readily in K Medium as above outlined: after the second filtration it failed to grow in peptone broth. In other words, the organism having become filterable and accustomed to protein media (proteophilic) lost its ability to grow in ordinary peptone containing nutrient broth.
The cultures of November 9 and November 12 were examined under the microscope and there were no discernible bacilli, although the cultures were markedly turbid. Darkfield illumination revealed very small, actively motile granules, and direct observation of these with the oil emersion lens confirmed the presence of these motile granules, without, however, affording any indication of their structure; Therefore, these granules for obvious reasons could not be unequivocally diagnosed as the filterable form of the bacillus.
When a culture of B. typhosus in the filterable state, grown as above indicated in K Medium, was examined with this micropolarimeter, it was observed that the plane of polarization of the light passing through the culture was deviated plus 4.8 degrees, with the simultaneous appearance of a definite blue spectrum. With this observation in mind, the culture was next studied with the Rife microscope at 5000 diameters.
The double wedge quartz prism referred to above was set by means of the vernier to minus 4.8 degrees (The reason for setting the quartz wedge in the reverse direction will be discussed in another place). Examined in this polarized light, this thrice filtered culture of B. typhosus cultivated in K (protein) Medium showed small, oval granules, many of them quite actively motile. These motile granules when in true focus appeared as bright turquoise-blue bodies, which contrast strikingly, both in color and in their active motion, with the noncolored, nonmotile débris of the medium.
These observations were repeated eight times, using in each instance growth of the filterable organisms in K Medium. The cultures examined were both twenty-four and forty-eight hours old. The qualitative results were always the same, namely, the occurrence of small, oval, actively motile, turquoise-blue bodies in the cultures and the absence of these small, oval, actively motile, turquoise-blue bodies in the uninoculated control K Media.
There is another even more direct procedure for establishing the identity of these small, oval, motile, turquoise-blue bodies. It has been shown in previous communications 3 that agar cultures, or better, broth cultures of B. typhosus inoculated into K Medium, become filterable within eighteen hours' growth at 37 degrees centigrade. It should follow, inasmuch as not all of the bacilli appear to become filterable under these conditions, that at least some of the bacilli should have similar turquoise-blue granules within their substance if they are indeed passing to the filterable state. Also the free swimming filterable forms, the small, oval, motile, turquoise-blue bodies described above, should be simultaneously present.
Dark field examination of such a culture eighteen hours old revealed unchanged, actively motile bacilli, bacilli with granules within their substance, and free swimming, actively motile granules. This culture examined in the Rife microscope with the quartz prism set at minus 4.8 degrees and with 5000 diameters magnification, showed very clearly the three types of organisms just described, namely:
First, unchanged bacilli: These were relatively long, actively motile, and almost devoid of color.
Laboratory of Medical Research, Northwestern University Medical School, 303 Chicago Avenue, Chicago, Illinois.
Rife Research Laboratory, 712 Electric Building, San Diego.
2. Northwestern University Medical School Bulletin, Vol. 32, No. 8, (October 19), 1931, for full details.
3. Op. cit.
Abb. 1: Foto des Mikroskops und seines Erfinders. Königlicher Raymond Rife. Ph. D. In der Abbildung ist die Beleuchtungsquelle ganz links, das Licht geht durch den Kondensor der Unterstation und dann durch das optische System. Der vertikale Tubus ist der Beobachtungstubus. Die drei Linsen sind auf die Kamera gerichtet. Die Kamera ist eine spezielle Stop-Motion-Kamera für Standardfilme. Hinter der Kamera befindet sich der Motor, der die Kamera antreibt. Der Tisch, auf dem das Instrument steht, ist so angeordnet, dass das Mikroskop um jede Achse von der Horizontalen in die Vertikale gekippt werden kann.
Abb. 2: Dr. Arthur Isaac Kendall, Direktor der medizinischen Forschung. Northwestern University Medical Co-Autor mit Royal Raymond Rife, Ph. D., der Arbeit "Beobachtungen zum Bacillus Typhosus in seinem filtrierbaren Zustand".