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VOLUME
XXXV
DECEMBER, 1931
NO.
6
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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
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 coordinated
resolving power, and a simple procedure for inducing the
filterable state in bacteria at will,1 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 Abbe
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:
October 29, 1931. An agar
slant was made of a thrice-plated culture of B. typhosus,
Rawlings strain. (Editor's Note: This agar slant was made in the
Laboratory of Research Bacteriology, Northwestern University
Medical School, Chicago, Illinois.)
*From:
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.
November 2, 4 p. m. Inoculated six cubic centimeters of K
(protein) Medium2 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 6.
The twenty-four-hour culture of November 5, which grew well, was
again filtered. This time the finest Berkefeld filter,
"W," was used. As before, a drop of this filtrate was
added to six cubic centimeters of K medium and incubated at 37
degrees centigrade. Growth was abundant November 7.
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.
Fig.
l.--Photograph of the microscope and its inventor. Royal Raymond
Rile. Ph. D. In the illustration the source of illumination is
to the extreme left, the light passing through the substation
condenser and then through the optical system. The vertical tube
is the observation tube. The
three lenses are directed toward the camera. The camera is a
special stop motion camera for standard films. Beyond the camera
is the motor to drive it. The stage on which the instrument is
placed is so arranged that the microscope can be tilted through
any axis from horizontal to vertical.
In
this viable, filtered state the culture was taken to Pasadena,
California, and, through the instrumentality of Dr. Milbank
Johnson, the cooperation 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. The subsequent developments, which are the immediate
subject of this discussion, are as follows:
November
16. The cultures of November 12, made in Chicago, were
transferred to fresh K Medium and incubated at 37 degrees
centigrade overnight.
November
17. The Rife microscope was installed and the first cultures,
those inoculated November 16, were examined. The preliminary
observations of these cultures were made with a polarizing
microscope with a spectroscopic attachment. It should be borne
in mind that the entire optical system of this micropolarimeter
was of quartz. A one-eighteenth-inch apochromatic oil immersion
lens was used, with a 20x quartz ocular.
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
debris 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.
Fig.
2.--Arthur Isaac Kendall, Ph. D., Director of Medical Research.
Northwestern University Medical co-author with Royal Raymond
Rife, Ph. D., of the paper on "Observations on Bacillus
Typhosus in Its Filterable State."
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.
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
communications3 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.
Darkfield
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.
Second, long, actively motile bacilli, each with a rather
prominent granule at one end.
The granule in such an organism was turquoise blue,
reminiscent in size, shape, and color of the small, oval,
actively motile, turquoise-blue granules found in the protein
medium (K Medium) where, it will be recalled, no formed (rod
shaped) bacteria could be demonstrated. These bacilli having the
turquoise-blue granules were colored only at the granule end,
the remainder of the rod being nearly colorless, in this respect
corresponding to the unchanged (nonfilterable) bacilli just
mentioned.
Third,
free swimming, small, oval, actively motile, turquoise-blue
granules, precisely similar, apparently, in size, shape, and
color to those seen in the granulated bacilli just described.
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.
REFERENCES
1.
James ,4. Patten Lecture, Northwestern University Bulletin, Vol.
32, No. 5 (September 28), 1931.
2. Northwestern University Medical School Bulletin,
Vol. 32, No.
8, (October
19), 1931,
for full details.
3. Op. cit.
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