Observations on Bacillus Typhosus in its Filterable State

OBSERVATIONS ON BACILLUS TYPHOSUS IN ITS FILTERABLE STATE

A Preliminary Communication

By Arthur Isaac Kendall, Ph.D., Chicago, Illinois — and Royal Raymond Rife, Ph.D., San Diego, California

California and Western Medicine, Vol. XXXV, No. 6, December 1931, pp. 409–411. From: The Rife Research Laboratory, San Diego; the Laboratory of Research Bacteriology, Northwestern University Medical School, Chicago; and the Pathological Laboratory of the Pasadena Hospital, Pasadena. Presented at a meeting of the Bacteriological Section of the Los Angeles Clinical and Pathological Society, November 20, 1931.

It seems improbable that viable bacteria in the filterable state have ever been unequivocally seen. Nevertheless, the theoretical and practical importance of filterable forms of bacteria in theoretical and applied biology cannot be denied.

Recently, through the simultaneous availability of the Rife microscope, an instrument combining very high magnification with coördinated resolving power, and a simple procedure for inducing the filterable state in bacteria at will, the possibility of actually demonstrating organisms in this hitherto illusive condition very obviously presented itself.

Two features of the Rife microscope, full details of which will be presented elsewhere, must be specifically mentioned here. First, the entire optical system, including not only the lenses but also the illuminating unit, is made of quartz. In addition, a double wedge quartz prism is mounted between the illuminating unit and the quartz Abbé condenser. The latter can be rotated, with vernier control, through 360 degrees, thereby affording readily controllable polarized light at any required angle. The import of this polarization unit will be discussed later. Inasmuch as this microscope magnifies from 5,000 to 17,000 diameters, it is obviously very necessary to have it mounted upon an immovable foundation.

The organism selected for these experiments was the well known Rawlings strain of B. typhosus. The immediate history of the culture used is as follows: [a dated sequence of cultures from October 29 through November 12, 1931, each thrice-filtered through Berkefeld "N" and "W" filters, is given in the original.]

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.

In this viable, filtered state the culture was taken to Pasadena, California, and, through the instrumentality of Dr. Milbank Johnson, the coöperation of Dr. Alvin G. Foord, and the courtesy of the Pasadena Hospital, the necessary space and equipment for mounting the microscope and continuing the cultures were made available.

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 5,000 diameters.

The double wedge quartz prism referred to above was set by means of the vernier to minus 4.8 degrees. Examined in this polarized light, this thrice filtered culture of B. typhosus cultivated in K (protein) Medium showed small, oval granules, many of them 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 granules in the uninoculated control K Media.

From the two facts thus far arrived at, namely, that the small, oval, turquoise-blue bodies were actively motile and also that they were cultivable from K Medium to K Medium, it is surmised that these small, oval, motile, turquoise-blue bodies are indeed the filterable forms of the B. typhosus.

From the fact that these small, oval, turquoise-blue bodies could be seen both in the parent rod and free swimming in the medium, it is assumed that these small, oval, actively motile, turquoise-blue bodies are indeed the filterable form of B. typhosus.

Laboratory of Medical Research, Northwestern University Medical School, 303 Chicago Avenue, Chicago, Illinois. Rife Research Laboratory, 712 Electric Building, San Diego.