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Proceedings
OF THE
STAFF MEETINGS OF THE MAYO CLINIC
Published
Weekly for the Information of the Members of the Staff and the
Fellows of The Mayo Foundation for Medical Education and
Research
Vol. 7
Rochester, Minnesota, Wednesday, July 13, 1932
No.28
Observations
on Filter-Passing Forms of Eberthella Typhi (Bacillus Typhosus)
and of the Streptococcus From Poliomyelitis
E. C. Rosenow, M. D., Division of Experimental Bacteriology:
Interest in the series of reports by Dr. Kendall ( note 3,4) on
the filtrability of bacteria by the use of his protein-rich,
peptone-poor, K medium reached its climax, it might be said,
following publication of the report on the successful
visualization, with the high-power Rife microscope, of the
filter-passing form of Eberthella typhi in cultures of K medium,
and in the corresponding filtrates.(note 5) Discussion over
these important, and in some respects revolutionary, findings
has become widespread. Since I, as well as others. (note
3,6,7,8) have been attempting, and with some success, to make
the streptococcus, especially that from poliomyelitis, filtrable
and to develop the virus of poliomyelitis from the streptococcus
so consistently isolated in this disease, I accepted a recent
kind invitation of Drs. Kendall and Rife to share with them
their observations in a restudy of the filter-passing forms of
Eberthella typhi as seen with an improved model of the Rife
microscope. They asked me also to bring with me my cultures of
the streptococcus from poliomyelitis.
It
is the purpose of the report to record the more important
observations made during three days, July 5 6, and 7, 1932,
spent with them in Dr. Ken all’s laboratory at
Northwestern University Medical School, Chicago. Owing to the
novel and important character of the work, each of us verified
at every step the results obtained. Microscopic examination of
suitable specimens was made as a routine by Dr. Rife with his
high-power microscope, by Dr. Kendall with the oil immersion
dark field, and by myself with the ordinary Zeiss microscope
equipped with a 2 mm. apochromatic oil immersion lens and x10
ocular giving a magnification of about 900 diameters. Most
obesrvations with the Rife microscope were made at 8,000
diameters. In order to check the magnification, gram and
safranin stained films of cultures of Eberthella typhi, of the
streptococcus from poliomyelitis, and stained films of blood,
and of the sediment of the spinal fluid from a case of acute
poliomyelitis, were examined. Bacilli, streptococci,
erythrocytes, polymorphonuclear leukocytes, and lymphocytes were
clearly seen, and in each instance were, as near as could be
estimated, about nine times the diameter as when examined with
the 2 mm. oil immersion at about 900 diameters.
The
following principles and methods were stated by Dr. Rife
as being essential in order to visualize clearly the objects at
this and higher magnification by direct observation. Spherical
aberration is reduced to the minimum and magnification greatly
increased by using objectives in place of oculars. Proper
visualization, especially of unstained objects, is obtained by
the use of an intense beam of monochromatic polarized light
created by rotating wedge-shaped quartz prisms placed between
the source of light and the substage quartz condensor.
Dispersion of the transmitted rays of light, as they pass upward
to the eye, is prevented by passing them through a series of
quartz erecting (90 deg) prisms. Projection of the rays of light
through air is not greater than 30 mm. at any point.
The
oval, motile, turquoise blue bodies described previously by
Kendall and Rife were demonstrated unmistakably under the Rife
microscope in numerous hanging drop, between slide and cover
glass, preparations of old and young cultures and corresponding
filtrates of two strains of Eberthella typhi. One of these
strains represented the one studied by them previously, the
other was isolated a short time before from the blood in a case
of typhoid fever. The bodies were numerous in cultures of the
bacilli in the K medium but were also found in smaller numbers
in peptone broth cultures. The motile forms, as a rule, were of
a deeper blue-green color than nonmotile forms. In several
instances they were seen attached to, or were in the swollen
ends of, bacilli having a similar turquoise blue color. In one
instance, the turquoise blue body was seen to be extruded or
detached and as this occurred the bacillus lust its
bluish-green color. The bacilli not containing granules seemed
to be of different density and were of a grayish-brown color
instead. No bacilli were found in clouded cultures in K medium
inoculated previously with filtrates of cultures; only large
numbers of the bluish-green bodies were seen. This was true of
the strain examined last November and which has been kept in
filtrable, nonbacillary form through a series of transfers ever
since, as well as their recently isolated strain. Stained and
hanging drop preparations of clouded filtrate cultures examined
under my microscope were uniformly negative, despite the fact
that the blue bodies, as seen under the Rife microscope,
appeared not less in diameter than the cross-diameter of the
bacilli. Dancing bodies were seen under dark field illumination.
These were considered as the filtrable, turquoise blue bodies.
The turquoise blue bodies were not found in filtrates of the
uninoculated K medium. Bodies resembling those of Eherthella
typhi, but of grayish-brown color were seen in K medium cultures
of Eschericbia coli and corresponding filtrates.
It
was next agreed to filter cultures of the streptococcus from
poliomyelitis which I brought with me, for the purpose of
controlling the results obtained with Eberthella typhi, as well
as to see what the Rife microscope might reveal. This strain was
isolated June 18, 1932, from the nasopharynx of a boy aged ten
years in the acute stage of a typical attack of epidemic
poliomyelitis. The strain had been passed consecutively through
three sets of rabbits, and produced death from flaccid paralysis
of all of the ten rabbits given intracerebral injections. It has
been recovered from filtrates of emulsions of brain and cord of
paralyzed rabbits three consecutive times and once from a
filtrate of a chick infusion broth culture. It had been
subcultured by rapidly repeated transfers of glucose-brain broth
twelve times between the second and third animal passages, and
subsequently. The cata- phoretic velocity remained poliomyelitic
throughout. The streptococcus had become smaller through
repeated filtratious, animal passages and gowth in special
mediums. It grew readily on aerobic blood-agar plates when first
isolated in glucose-brain broth but lost this power after
repeated filtrations and growth in special mediums. Two cultures
were filtered through new Berkefeld N filters, one in K medium,
the other in chick infusion broth. (The chick infusion broth is
prepared in the same way as meat infusion broth but instead of
using ground meat, ground chicks within the shells of twenty day
incubated, fertilized eggs are used.) Both sets of cultures had
been incubated forty-eight hours and then kept at room
temperature in transit for eighteen hours longer. Smears from
the former, stained by gram and safranin, revealed a moderate
number of diplococci and short chains; the cocci were spherical,
varied considerably in size and were characteristic of growth in
this medium. Smears from the latter revealed numerous diplococci
and short chains, the cocci being elongated in the long axis of
diplococci and chains, but which also varied considerably in
size. Exceedingly small, gram negative forms were readily
detected. These were more numerous and smaller than the smallest
forms in the K medium. Hanging drop preparations of these
cultures under the Rife microscope revealed cocci, diplococci
and streptococci of varying size, shape and density. Filtration
was done exactly as in cultures of Eberthella typli. The culture
was diluted four times with salt solution. Only moderate suction
was used and filtration was completed in about (en minutes,
Cultures
of the filtrates were made immediately into glucose-brain broth,
chick infusion broth and into chick infusion, egg yolk, and K
mediums. Hanging drop preparations of these filtrates made
immediately and the following day, being kept at room
temperature meanwhile, revealed cocci and diplococci of
approximately the size and shape of those seen in the cultures
and were characteristic of the medium in which they were grçwu.
Concentration by centrifugation was attempted without success.
In
no instance were turquoise blue bodies observed in these
filtrates. The cocci and diplococci were of a brownish-gray
color, of more uniform intensity than those seen in the
corresponding cultures, and were almost always surrounded by a
clear halo about twice the width of that seen at the margins of
debris and Eberthella typhi. Stained films of the filtrates and
filtrate sediments examined under (he ordinary microscope never
revealed organisms. Hanging drop, dark field preparations
revealed nothing distinctive of diplococci.
On
the basis of these startling results, I had my assistant at
Rochester prepare and send three filtrates, one from
glycerolated brain and cord of a monkey that died of
poliomyelitis following inoculation of adapted filtered virus,
one from the glycerolated brain of a rabbit that died following
inoculation of virus of herpes, and one from the brain of a
normal rabbit. Approximately 5 per cent emulsions in sodium
chloride solution of the brain and cord substances were
prepared, as is our custom in order to avoid contamination from
the air, by shaking with glass beads in a shaking machine,
instead of grinding in a mortar with sterile quartz sand. These
were centrifuged and filtered through Berkefeld N filters.
Strong suction (19 cm. mercury) was Used and shipment was made
at room temperature in scrupulously clean, sterile vials sealed
with a rubber disk cap.
Hanging
drop preparations, under the Rife microscope, of the filtrate of
poliomyelitic virus made twenty-four hours previously, revealed
readily as high as three per field of brownish-gray cocci and
diplococci identical in size, density, and color, to those found
in filtrates of the cultures of the "poliomyelitis"
streptococcus. The hanging drop preparations of the filtrates of
the virus of herpes also revealed a considerable number of cocci
and diplococci but of a bright to pale pink, somewhat smaller
than those found in the filtrates of the streptococcus and of
the virus of poliomyelitis. The objects resembling cocci and
diplococci were easily distinguished from debris; they, as the
diplococci and streptococci in the cultures, were non-motile, of
fairly even density, size and form and were surrounded by a
light halo suggesting a narrow capsule. Prolonged search for
similar forms in hanging drop preparations of the filtrate of
the brain of the normal rabbit proved unsuccessful. Nothing
resembling the turquoise blue bodies of Eberthella typhi was
seen in these filtrates, and in no instance were cocci and
diplococci found in filtrates of uncontaminated cultures of
Eberthella typhi. Attempts with the Rife microscope to find the
coccus and diplococcus forms in increased numbers of hanging
drop preparations from the bottom and top of the centrifuged
"virus" filtrates were only mildly successful. Stained
and hanging drop preparations examined with the ordinary
microscope failed to reveal organisms and the identity of small,
glistening bodies in hanging drop preparations under dark field
illumination was uncertain.
During
the course of examination of old cultures of filtrates of
Eberthella typhi in K medium, two tubes that had been repeatedly
opened previously for demonstrating turquoise blue bodies and
that had become very turbid, were found to contain large gram
positive to gram negative diplococci of uniform size, sometimes
in short chains. These were both filtered through Berkefeld N
filters and one also through a Berkefeld W filter. Examination
under the Rife microscope of hanging drop preparations of all
three filtrates revealed unmistakable nonmotile, bluish-green
cocci and diplococci resembling those in the cultures. Those in
the W filtrate seemed definitely smaller than the ones in the N
filtrates. They were larger than those seen in filtrates of the
cultures of the poliomyelitis streptococcus, virus of
poliomyelitis and of herpes. The color resembled that of the
turquoise blue bodies of Eberthella typhi making it difficult,
in some instances to distinguish between nonmotile forms of
these and cocci. Bacilli and actively motile bodies were not
found.
Subcultures
in glucose-brain broth and chick infusion medium, from the K
medium and chick infusion broth cultures that were filtered,
yielded a pure growth of the streptococcus whereas cultures in
the K medium and ordinary plain broth remained sterile. All
culture of the filtrates of the poliomyelitis streptococcus
cultures proved sterile. The two tubes of chick infusion layered
with oil, inoculated one each with the culture filtrates, became
clouded in six days hut no bacteria could be found in stained
smears. Chick infusion cultures of this streptococcic strain
have since been filtered twice. The strain retained
characteristic cataphoretic velocity as well as characteristic
infecting power.
Cultures
of the filtrates of the virus of poliomyelitis in glucose-brain
broth, layered with oil, became slightly cloudy in five days,
and smears revealed a moderate number of gram positive diplococci
sometimes in short chains. Chick infusion and K medium cultures
remained sterile and free from clouding. Cultures of the
filtrate of the virus of herpes in chick infusion medium layered
with oil became cloudy in six (lays and smears revealed large
numbers of small gram positive diplococci, sometimes in short
chains characteristic of strains which Evans and we sometimes
isolate from herpetic virus. Cultures of the filtrates in K
medium and glucose-brain broth remained sterile.
The
rabbit given intracerebral injections with the herpes virus
remained free from symptoms for four days. It died during the
night of the fifth day after injection. Cultures from brain and
cord have remained sterile thus far. Frozen sections of brain
and cord revealed typical lesions of encephalitis in which
diplococci resembling those isolated from the filtrate injected,
were found. The Macacus rhesus monkey given intracerebral
injections with the adapted virus from poliomyelitis remained
well for eight days and became ill on the ninth day with classic
symptoms of experimental poliomyelitis, that is fine tremor of
face and head, excitation, staccato voice, and complete flaccid
paralysis of hind extremities. The spinal fluid was slightly
turbid and contained 1,147 cells per cubic millimeter.
Polymorphonuclear leukocytes and lymphocytes were present in
about equal numbers. The animal was etherized. A small cyst
containing chocolate colored fluid was found at the point of
injection in the right frontal lobe. Moderate congestion of the
vessels of the cerebral cortex was found, but no other lesions.
Smears from the centrifugated spinal fluid revealed not less
than fifty undoubted (liplococci, varying greatly in size from
exceedingly small, elongated forms, such as commonly seen in
cultures in chick infusion medium, to large, deeply stained gram
positive diplococci in which each member was oval or round. Many
of these were found within polymorphonuclear leukocytes and
large lymphocytes. In several, large numbers of diplococci were
found. A few gram positive diplococci, sometimes in chains of
two or three, were found on prolonged search, in direct smears
of pipettings of the cerebrospinal fluid, in material from the
anterior horns of the cervical and lumbar regions of the spinal
cord, and in contact smears from the cerebral cortex. A search
of similarly prepared smears made from pipettings from the
liver, spleen, and kidneys did not show organisms. Cultures in
chick infusion medium of pipettings of the cerebrospinal fluid
admixed with brain substance, and cultures of the emulsions of
portions of the spinal cord, revealed a pure growth of
streptococci resembling those seen in direct smears. Frozen
sections of the lumbar and cervical cord disclosed typical
lesions of poliomyelitis.
On
the basis of these findings, there can be no question of the
existence of the filtrable turquoise blue bodies of Eberthella
typhi described by Kendall. They are not visible by the ordinary
methods of illumination and magnification, not because they are
too small, but rather, it appears, because of their peculiar
nonstaining hyalin structure. Their visualization under the Rife
microscope is due to the ingenious methods employed rather than
to excessively high magnification. Examination under the Rife
microscope of Specimens containing objects Visible with the
ordinary microscope, leaves no doubt of the accurate
visualization of objects or particulate matter by direct
observation at the extremely high magnification (calculated to
be 8,000 diameters) obtained with this instrument.
The
findings under the Rife microscope of cocci and diplococci in
filtrates of cultures of the streptococcus from poliomyelitis,
and in filtrates of the viruses of poliomyelitis and herpes
encephalitis, not detectable by the ordinary methods of
examination, and which resembled in form and size those found in
the respective cultures, and the absence of minute forms,
suggests that the filtrable, inciting agent of these diseases is
not necessarily extremely small, as is universally believed.
Indeed, the filtrable, inciting agent may be the nonstaining,
highly plastic, hyalin state of the visible, stainable,
cultivable organism, the streptococcus.
It
is of course, possible, that these unstainable, invisible forms
revealed by ordinary methods of examination, arc not (lie
inciting agents or ‘‘viruses’’ of these diseases and
that they represent merely the filtrable, or other state of the
streptococcus. A consideration of the great difficulty one has
in isolating the streptococcus and demonstrating diplococci in
lesions in these diseases and the ease with which the bodies are
found in the filtrate, indicate clearly that the
"invisible" forms of the streptococcus, if such they
be, are present in large numbers in the host, as in positive
cultures of the streptococcus. Their form, size and color are
too characteristic and true to type to permit considering them
as artefacts or as being expressive of etiologically unrelated,
contaminating streptococci. Noninfectivity of the filter-passing
forms, except in the cases of virus diseases, their presence in
large numbers in filtrates, both of cultures and of infected
tissues, and the great difficulty in obtaining the visible form
in cultures of filtrates,(note 2,3,7,8) indicates that
‘‘invisible,’’ filter-passing forms represent a certain
stage in the development of microorganisms.
BIOLIOGRAPHY
- Evans, Alice
C.: Studies on the etiology of epidemic encephalitis. II.
Virulent bacteria cultivated from so-called herpetic and
encephalitis viruses. Pub. Health Rep. 42: 171-176 ( Jan.
21) 1927.
- Hadley, P.,
Delves, E. and Klimek, J.: The filterable forms of bacteria.
I. A filterable stage in the life history Of the Shiga
dysentery bacillus. Jour. Infect. Dis 48: 1-159 (Jan.) 1931.
- Kendall, A.
I.: Observations on the filtrability of bactera, including
filtrable organisms obtained form cases of influenza;
studies in bacterial metabolism CI. The James A. Patten
Lecture in Bacteriology, Northwestern Univ. Bulll., 32, No.
5, 1931; Science, 74: 129-139 (Aug 7) 1931.
- Kendall, A.
I.: Mediums for the isolation and cultivation of bacteria in
the filtrable state. Studies in bacterial metabolism CII.
Northwestern Univ. Bull., 32, No. 8, 1931.
- Kendall, A.
I., and Rife, R. R.: Observations on Bacillus typhosus in
its filtrable state: preliminary report.California and West.
Med., 35: 409-411 (Dec.) 1931
- Richardson,
Ruth D. and Mellon, R. R.: Experiments on the filtrability
of the Rosenow streptococcus of anterior poliomyelitis.
Proc. Soc. Exper. Biol. And Med. 29: 451-454 (Jan.) 1932
- Rosenow, E.
C., and Towne, E. b.: Bacteriologic observations in
experimental poliomyelitis of monkeys. Jour. Med. Res., 31:
175-186 (March) 1917
- Rosenow, E.
C., and Wheeler, G. W.: The etiology of epidemic
poliomyelitis. Jour. Infect. Dis., 22: 281-312, 1918.
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