Professional Documents
Culture Documents
GILVUS
J. D. TUCKETT AND W. E. C. MOORE
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
227
228 TUCKETT AND MOORE [VOL. 77
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-
103.
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.