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Departments of Biochemistry and Nutrition, and of Biology, Virginia Agricultural Experiment Station,
Virginia Polytechnic Institute, Blacksburg, Virginia
Received for publication August 25, 1958

Reports of viable filterable particles in bacte- filtered on Morton ultrafine bacteriological fritted
rial cultures have often been attributed to glass filters to determine the presence of filterable
artifacts from the media, contaminants, and, in particles of C. gilvus. Filtration was carried out
the case of filtration experiments, to laboratory by suction from aspirator pumps applied through
errors (Frobisher, 1928). Since Kleineberger's a 5-in cotton plug. The assembled filters and
(1935) observations on L forms, they have re- plugs were sterilized as a unit at 121 C for 15
ceived considerable attention, but their signifi- min.
cance and function remain obscure. It has been After the first appearance of growth, the fil-
suggested that they are part of a complex life trates were tested for the presence of E. coli by
cycle serving reproductive or genetic recombina- inoculation into lactose broth. Negative tests
tion functions (Klieneberger-Nobel, 1951b). It indicated that E. coli had been unable to pene-
has been hypothesized that the L forms are trate the filters. Positive controls in which E. coli
aberrant cells or involution forms (Heilman, was added to the filtrate demonstrated that E.
1941). coli could have been recovered had it penetrated
The failure of many filtration experiments to the filter. The filters remained sealed and closed
yield positive data on repetition has presented to the atmosphere until the first appearance of
serious problems in determining the physiology of growth in the filtrate.
filterable forms, and has also raised doubt con- As an arbitrary measure of the size of the
cerning the existence of a filterable stage in particles, a series of filtration experiments was
bacteria. In addition, many reported filterable done with Selas porcelain filters of graded pore
forms required subculture through several serial sizes. These were prepared and used in a similar
transfers before cells reappeared. These manipu- manner and with similar cell suspensions as the
lations increased the possibility of filtrate growth fritted glass type. The E. coli test was carried out
from contamination rather than from particles in these experiments also. The only difference in
(Klieneberger-Nobel, 1951a). procedure was that the cultures were not centri-
The present report concerns viable, filterable fuged previous to filtration. Plate counts were
particles which appear to be produced regularly made from filtrate samples immediately after
and which regenerate the normal, parent type filtration to determine the number of filterable
cell without subculture of the filtrates. particles per ml of 48-hr culture of C. gilvus.
Observations of the living organisms were
METHODS made by use of dark field microscopy. Photo-
A culture of Cellvibrio gilvus (Hulcher and graphs were taken of certain stages of develop-
King, 1958) was observed to produce involution ment of the organism.
forms, particles, and swollen cells even in 16- to RESULTS
24-hr cultures. Cell suspensions of C. gilvus were
grown in 50 ml of cellobiose broth (Hulcher and Of 10 fritted glass filters, 2 were found to be
King, 1958), harvested by centrifugation, and impenetrable to C. gilvus, 3 passed C. gilvus in
mixed with 50 ml of fresh medium. Escherichia every experiment, and the remaining 5 gave vari-
coli cells were harvested from 50 ml of 24-hr able results. Upon inoculation of filtrate samples
cultures and mixed with the suspension to be into lactose broth, negative tests indicated that
certain that the filters were impervious to normal all of the filters were impervious to E. coli. The
cellular forms. The mixed suspensions were time in which growth occurred varied, but was

Dark field observations of living organisms

suggested the existence of a complex life cycle.
This cycle consisted of the enlargement of barely
visible particles into normal cells, the swelling of
normal cells into shapeless, flexible, large bodies,
and the appearance of particles and cells within
the large bodies. These particles and cells became
highly motile and, after 6 to 8 hr, finally were
observed to emerge through the flexible wall of
the large body. The complete process was esti-
mated to take about 48 hr. Binary fission of the
normal cells in the culture was also observed.
Although more attention was given to the large
bodies, binary fission may have occurred with
equal frequency. The high degree of motility
Figure 1. Photomicrograph of a large body in a displayed by the particles and cells interfered
culture of Cellvibrio gilvus under dark field. The with the photomicrographic studies. Complete
body measures approximately 9 , across its largest photomicrographic series of the process could not
dimension (estimated within the limits of dark be made. Figure 1 shows the irregular large body.
field observation). The 2 larger light spots repre- The granules within the large body were motile.
sent normal cells. Motility is evident in the normal The light bodies outside the large body are the
cell in the lower right corner from the light track size of normal cells. What appeared to be granules
made during exposure. The particle above this cell ranged in size down to the limit of visibility, but
appears similar in size to the average particle because of their size and motility they generally
observed. failed to reflect sufficient light to be recorded with
the available equipment. The fragile large bodies
generally within 48 hr. MIorphological examina- were disrupted by staining techniques employed
tion and cultural tests of the growth in the for light field observations.
filtrates revealed pure cultures of C. gilvus. The An attempt was made to concentrate the
cellular morphology in the filtrate cultures was filterable particles for chemical analysis. Centri-
more uniform than that of the parent culture but fugation of 50 ml of the filtrate from the 0.85-,u
after subculture involution forms and swollen maximal pore radius filter for one hour at
cells reappeared. The number of viable particles 23,500 X G failed to sediment quantities of
found in the filtrate by plate count did not vary filterable particles suitable for chemical analysis.
when the organism was cultured at pH 7, 8, 9, or Attempts at differential centrifugation of the
10, when the culture was incubated at tempera- original culture also did not produce macro-
tures of 25, 30, or 37 C, or when the age of the scopic quantities of particle sediment.
culture was 24, 48, or 72 hr at the time of fil-
Plate count data from the filtrates of the These experiments indicate that C. gilvus pro-
graded porcelain filters indicated that an average duces filterable particles. Since, in the critical
of 250 particles per ml of 48-hr C. gilvus culture experiments, the filters remained sealed through-
passed through the 0.85-,u maximal pore radius out the operation, eontamination was improb-
filter, an average of 100 passed through the 0.60-, able. The cotton plug between the filter and
maximal pore radius filter, an average of 20 aspirator remained dry, therefore it is unlikely
particles passed through the 0.45-, maximal pore that contamination could have entered through
radius filter, and 5 through the 0.35-,u maximal the side arm of the flask. The presence of E. coli
pore radius filter. The 0.30-,u maximal pore in the cell suspension and its absence in the fil-
radius filter evidently stopped all viable particles trate indicates that the filters were capable of
since no colonies appeared on the plates and no holding back cellular forms of the bacterium. The
growth appeared in the filtrates. All of the porce- variable results given by the sintered glass filters
lain filters were impervious to E. coli. are probably due to their varying pore size. Those

filters which gave both positive and negative suggested the presence of a life cycle involving
results evidently had a pore size on the borderline the enlargement of minute particles into normal
between the size that admits the smallest particles cells, the swelling of normal cells into irregular
and that which admits none. In the case of one large bodies, and the appearance of motile
or two particles admitted to the filtrate, it would particles and cells which finally emerge from the
be a matter of chance whether they remained large bodies.
viable long enough to regenerate the bacterial
form. Since, during filtration with fritted glass REFERENCES
and porcelain filters the pores would become FROBISHER, M., JR. 1928 On the action of bac-
clogged, the numbers reported here are probably teriophage in producing filterable forms and
somewhat less than the actual numbers of filter- mutations of bacteria. J. Infectious Dis-
able particles in the cultures. No evidence con- eases, 42, 461-472.
HEILMAN, F. R. 1941 A study of Asterococcus
cerning recombination of particles was obtained muris (Streptobacillus moniliformis). I. Mor-
but such a mechanism would further modify phological aspects and nomenclature. J.
interpretation of the number of particles pro- Infectious Diseases, 69, 32-44.
duced. This bacterium seems to give more con- HULCHER, F. H. AND KING, KENDALL, W. 1958
sistent results and is more convenient to work Disaccharide preference of an aerobic cellulo-
with than the majority of filterable bacteria. lytic bacterium, Cellvibrio gilvus n. sp. ,J.
Bacteriol., 76, 565-570.
Particle production is apparently not affected KLIENEBERGER, E. 1935 The natural occurrence
by several changes in environment which still of pleuropneumonia-like organisms in appar-
allow growth of the organism. ent symbiosis with Streptobacillus monilifor-
mis and other bacteria. J. Pathol. Bacteriol.,
SUMMARY 40, 93-105.
Experiments with both fritted glass and porce- KLIENEBERGER-NOBEL, E. 1951a Filterable
lain bacteriological filters indicated that Cell- forms of bacteria. Bacteriol. Revs., 15, 77-
vibrio gilvus regularly produces filterable particles KLIENEBERGER-NOBEL, E. 1951b The L-cycle:
which regenerate normal bacterial cells without A process of regeneration in bacteria. J. Gen.
subculture. Dark field microscopic observations Microbiol., 5, 525-530.