CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
THE OCCURRENCE OF METABACTERIUM POLYSPORA
I\
-
IN GUINEA PIG FECES AND A MICROSCOPIC
STUDY OF ITS ENDOSPORES
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Roger Alvin New
June, 1979
The thesis of Roger Alvin New is approved:
Dr. Charles R. Spott
Dr. Richard L. Potter
Committee Chairman
California State University, Northridge
June, 1979
ii
Curiosity . . . . . the mother of discovery
iii
ACKNOWLEDGMENT
I wish to express my sincere gratitude to my
advisor, Dr. Daisy A. Kuhn, for the patient understanding,
encouragement, and assistance that has contributed so
greatly to my academic development at California State
University, Northridge.
I acknowledge with gratitude
my committee members,
Dr. Charles R. Spotts and Dr. Richard L. Potter, for their
evaluation and critique of my thesis, and Richard Chao and
Delta Mishler for their expert advice and assistance in
electron microscopy.
I am appreciative of the outstanding examples and
encouragement of.my colleagues Sam Chitjian, Michael Nyby,
Christie Jenkins, and David Gregory.
I am eternally greatful to my dear wife Robyn
whose loving support and unceasing encouragement has made
all this possible.
iv
TABLE OF CONTENTS
Page
DEDICATION.
iii
ACKNOWLEDGMENTS
iv
LIST OF FIGURES AND TABLES.
vi
ABSTRACT
vii
INTRODUCTION.
1
MATERIALS AND METHODS
3
RESULTS . .
22
DISCUSSION.
43
REFERENCES.
53
v
LIST OF FIGURES AND TABLES
Page
FIGURES
1.
2.
3.
4.
5.
Phase-contrast photomicrographs of
M. polyspora. . . . . . . .
. . . . . .
30
Scanning electron micrographs of
M. polyspora sporangia. . .
33
. . . . . .
Scanning electron micrographs of
morphological stages of ~· polyspora.
35
Scanning electron micrograph of
mechanical enrichment . . . .
38
Scanning electron micrograph of
subpolar_appendages . . . . .
38
TABLES
Incidence of M. polyspora in guinea
pig feces . -. . . . . . . . . . .
23
Germination of ethanol-treated spores
41
III. Heat resistance of ethanol-treated spores
42
I.
II.
vi
ABSTRACT
OCCURRENCE OF METABACTERIUM POLYSPORA
IN GUINEA PIG FECES AND A MICROSCOPIC
STUDY OF ITS ENDOSPORES
by
Roger Alvin New
Master of Science in Biology
Fecal material from 39 guinea pigs was collected
and microscopically surveyed for the presence of Metabacterium polyspora, a large, Gram-positive bacterium
uniquely forming many endospores.
It was found in the
feces of 9 of 11 adult female guinea pigs, 5 of 14 adult
males, and 3 of 14 infants.
The abundance was high in
pregnant sows near the time of delivery.
The persistence
in newborn infants depended on continued contact with
feces containing Metabacterium.
Mechanical enrichments of M. polyspora were prepared from fecal suspensions by filtration to remove
coarse plant debris followed by centrifugation to eliminate
smaller bacteria.
vii
M. polyspora sporangia measured from 10 - 25
length by 5
pm in
pm in width and enveloped 2 - 8 endospores.
Individual endospores measured 8
pm
by 2 Jlm.
Scanning
electron micrographs of sporangia displayed a relatively
smooth cell surface.
In some cases the outer envelope
was folded and snugly contoured the endospores.
Subpolar
appendages were commonly present at one end of the sporangia; they measured about 60 nm in width and up to 720 nm
in length.
The spores were brightly refractile in phasecontrast illumination and retained Wirtz-Conklin's spore
stain.
They could not be resolved in Sacks and Alderton's
a.queous polymer two-phase system.
Germination depended on
nutrients, was activated by ethanol, and was decreased by
heating at 80°C for 30 minutes.
M. po1yspora failed to
grow on a variety of media under aerobic
and anaerobic
conditions.
The common occurrence of M. polyspora within the
indigenous microflora of guinea pigs and current knowledge
justifies that Metabacterium Chatton and
P~rard
1913 be
described and be placed as a genus of uncertain affiliation
with the endospore-forming rods and cocci in future editions of Bergey's Manual of Determinative Bacteriology.
viii
1
INTRODUCTION
Chatton and Perard (1913a) observed an exceptional
bacterium in guinea pig cecal contents.
It was unique in
its large size ( 10 - 25 pm in length by 5 pm in width) and
its ability to form 1 - 8 spores, each measuring 5 pm by
1 pm, per cell.
They named a new genus and species,
Metabacterium polyspora.
In 1951, Robinow (1951) demonstrated similarities
between spores from Metabacterium polyspora and Bacillus
species in their response to strong oxidants and acids.
Later, Robinow (1957) reaffirmed the original description
of Chatton and Perard (1913a) and in addition described
the organism having motility.
He observed locomotion to
be accompanied by rotation around the long axis, and he
demonstrated peritrichous flagella by Loffler's stain
(Robinow, 1957).
vegetative cells.
not observed.
Sporangia were as actively motile as
The life-cycle of the spore-former was
The apparent absence of internal differen-
tiation, especially of a recognizable cell nucleus suggested the bacterial nature of M. polyspora (Chatton and
Perard, 1913a; Robinow, 1957).
Vuillemin (1913) recommended that Metabacterium be
regarded as a genus conservandum.
Although "scarcely
2
sufficiently known to make accurate diagnosis possible",
Buchanan (1918) placed Metabacterium in the family
Bacillaceae and distinguished it from the other genera in
that family by its multiple spore formation.
Metabacteri-
um was not described in the 6th edition of Bergey's
Manual (1948), however, it was referred to as an anaerobic
spore-forming bacterium of which little was known.
The
genus did not appear in the 7th edition of Bergey's
Manual (1957), nor was the name listed in the Index
Bergyana (Buchanan, Holt, and Lessel, 1966).
In the 8th
edition of Bergey's Manual, Gibson and Gordon (1974) did
not present a description of the genus Metabacterium;
they merely referred to it in further comments on endospore-forming bacteria of uncertain taxonomic position.
The present investigation was undertaken to systematically survey guinea pigs for Metabacterium, and to
examine the biological properties of M. polyspora and its
endospores.
3
MATERIALS AND METHODS
Survey of guinea pigs.
Fecal material from 39 guinea pigs
(25 adults and 14 infants) was collected and surveyed for
the presence of M. polyspora.
Some guinea pigs were lo-
cated in pet stores, some in laboratories, and others were
obtained from commercial breeders.
If Metabacterium was
present in the feces, the animal was usually purchased and
maintained in small wire mesh bottom animal cages for long
term observations.
These cages permitted the feces to
fall through the mesh, minimizing repeated contact of the
animals with their feces.
The feces was collected on
pine shavings, sawdust or newspaper that had been placed
in a tray under the cage.
Animals were generally fed with a clay animal dish
(seven inches in diameter and two inches deep) which enabled the animals to sit in their food while feeding.
In
a few cases, animals fed with a trough-type feeder affixed
to the side of the cage, with gravity feed flow from an
upright reservoir which unlike the dish feeder, protected
the food from fecal contamination.
The standard diet for the animals, whether
main~
tained at home or in the laboratory, consisted of small
4
dry Rabbit Chow alfalfa pellets (Raulston and Purina, St.
Louis, Missouri) and water, supplemented occasionally
with fresh lettuce.
Small dry alfalfa pellets were also
the staple diet given at pet shops by breeders.
Examination of feces.
Fresh moist fecal pellets
from a given guinea pig were suspended in approximately
equal volume of sterile distilled water in a 16 x 150 mm
culture tube.
They were broken up with a clean glass
stirring rod, and the suspension briefly shaken by hand
for a few seconds.
A drop of the fecal suspension was
transferred to a microscope slide (Corning Glass Works
Co., Corning, New York) and sealed under a coverglass
with vaspar (an equal mixture by weight of petroleum jelly
and paraffin).
The wet mounts were examined microscopically with
either a Zeiss GFL microscope at 500x and 1250x magnification, or a Wild M-20 microscope at 200x, 400x and 1000x
magnification in bright-field (positive) phase-contrast
illumination..
The Zeiss GFL microscope was equipped with
a liZ condenser (numerical aperture 0.9), and 40x Neofluar
(n.a. 0.75) and 100x Neofluar (n.a. 1.30) objective
lenses.
'fr.te ocular magnification was 12. 5x.
The Wild
M-20 microscope was equipped with 20x Wild Fluotar (n.a.
0.60), 40x Wild Fluotar (n.a. 0.75), and 100x Wild Fluotar
5
(n.a. 1.30) objective lenses.
The ocular magnification
was lOx.
The spores of M. polyspora were readily identified
at low magnification (200x, 400x, or 500x) because they
·were large in size and brightly refractile in bright-field
(positive) phase-contrast illumination.
Higher magnifica-
tion (1000x or 1250x) facilitated the resolution of the
outer covering which enveloped the spores.
Mech~nical
enrichment of M. polyspora spores.
Freshly voided guinea pig feces, 2.5 g, was suspended in
40 ml of distilled water and passed through a primary
single layer of Handi Wipe
flask.
1
filter into a 125 ml Erlenmeyer
The Handi Wipe filters used to remove coarse plant
debris were made from Handi Wipe commercial cloth towels
(Colgate-Palmolive Company, New York, New York), cut into
4 x 4 inch squares.
They were wetted with tap water prior
·to being placed into glass funnels.
It was important that
these towels not be wrung out as that tended to distort
the weave and "pore-size".
The primary filtrate was
passed through a secondary four-layer thick Handi Wipe
filter, and the secondary filtrate in turn was filtered
1.
Handi Wipe is a registered trademark
6
through an eight-layer Handi Wipe filter.
The resulting
filtrate, approximately 35 ml, was transferred to a 50 ml
screw cap conical tube (Product 25330, Corning Glass Works,
Corning, New York) and centrifuged at 100 rpm for 5
minutes in an International swinging bucket centrifuge
(Model V, size 2, radium 26 em, International Equipment
Company, Needham Heights, Massachusets).
The supernatant
was carefully removed with a pipette and Propipette pipette
bulb (Spectronics Corporation, Westbury, New York).
The
sediment was resuspended in 40 ml of sterile distilled water and again centrifuged at 100 rpm for 5 minutes.
procedure was repeated three additional times.
This
The final
sediment containing spores was resuspended and fixed in 10
ml of phosphate buffered glutaraldehyde at room temperature
for eight hours.
Fixative solution.
Spore enrichments were fixed
in the following solution (Hyatt, 1970):
Glutaraldehyde fixative solution
Glutaraldehyde,
8%
Pennsylvania.
.
.
(Polyscience Inc., Warrington,
.
. .
.
.
.
.
.
.
.
.
1 ml
.
.
• .
4 ml
Sorenson's phosphate buffer, 0.2 M
pH 7 . 2 •
•
.
.
•
•
•
•
.
•
•
Distilled water
Sorenson's phosphate buffer stock solution
0.2 M, pH 7.2
5 ml
7
a)
Dibasic sodium phosphate 0.2 M
Na HP0 2.68 g in 50 ml H 0 . . . . . . . 36.4 ml
2
4
2
b)
Monobasic sodium phosphate
0.2 M NaH P0 ·H 0 1.38 g in 50
2 4 2
. . . 13.6 ml
Phase-contrast photon microscopy.
The fixed spores
were observed routinely in sealed wet mounts and phasecontrast photomicrographs were made on a Zeiss photomicroscope equipped with a VZ oil immersion aplanatic phasecontrast condenser (n.a. 1.40) and a 100x Neofluar phasecontrast objective lens (n.a. 1.30) used in combination
with the 1.25x Optovar and the 3.2x projective lens.
Illumination was provided by a 12 volt 60 watt, tungsten
filament lamp with a regulating transformer.
Photomicro-
graphs were recorded on 35 mm Kodak Plus-X panchromatic
film (Eastman Kodak Company, Rochester, New York).
Diagnostic stains.
Hucker's modification of Gram's
stain and Wirtz-Conklin's spore stain were applied to air
dried, heat fixed smears of fecal suspensions (Paik and
Suggs, 1974).
Scanning electron microscopy (SEM).
A coverglass
was coated with poly-D-lysine to facilitate the adhesion
of cells (Mazia, Schatten, and Sale, 1975).
One drop of
0.1% poly-D-lysine solution was placed on a coverglass and
allowed to wet the entire surface ( 22 mm x 22 mm) by
8
tilting the coverglass.
Once air dried, the coverglass
was washed with distilled water, and again air dried.
A drop of the specimen suspension was placed on the
coverglass and after about one minute, the specimens were
dehydrated by passing the coverglass through a series of
50%; 70%, 80%, 95%, and 100% v+v ethanol allowing 5 minutes
at each concentratioh.
The preparation was dried by
the critical point method (Anderson, 1951), and coated
with gold and palladium with a Hummer II coating device
(Technics Inc., Alexandria, Virginia).
The shadowed speci-
mens were stored in a desiccator over Drierite desiccant
(W. A. Hammond Drierite Co., Xenia, Ohio).
Scanning electron micrographs were made on an International Scientific Instruments Super Mini Scanning Electron Microscope (International Scientific Instruments,
Santa Clara, California).
Photographs were taken with a
built-in Polaroid 545 Land film camera, and recorded on
Polaroid type 55 Land film,
(Polaroid Corporation, Cam-
bridge, Massachusetts), producing a positive print and a
negative.
Transmission electron microscopy (TEM).
Twenty
grams of guinea pig feces were suspended in 150 ml sterile
distilled water and processed by the mechanical enrichment procedure previously described.
The spore-rich
sample which sti.ll contained considerable plant material
9
and crystalline debris (to be discussed in results), was
further cleansed by gravitational sedimentation in a 52%
w+w polyethylene glycol (PEG) solution.
A 10 ml calibrated glass centrifuge tube was filled
with 8.8 ml 52% PEG at 25°C.
About 1.0 ml of specimen
suspension was carefully overlaid, and sedimentation by
gravity was allowed to proceed for 80 minutes.
tinct bands formed:
Two dis-
a light band remained at the inter-
face of the aqueous specimen and the PEG, and a dense band
settled out through the organic PEG solution.
Each band,
about 0.4 ml in volume, was carefully removed, diluted in
12 ml distilled water and centrifuged for 5 minutes at
half maximum speed in an International clinical centrifuge
(International Equipment Co., Needham Heights, MA).
The
preparations were observed microscopically after each
separation.
The isolated bands were each resuspended in
1 ml sterile distilled water and again, carefully overlaid
on 8.0 ml 52% PEG.
After sedimentation of plant and
crystalline debris, the light, spore-rich band was removed and washed in distilled water by centrifugation.
The sample was transferred to a 4 mm x 45 mm polypropylene
conical microcentrifuge tube (Pelco, Tustin, California)
and packed by centrifugation in a clinical centrifuge for
10 minutes at maximum speed.
The specimens were removed
from the microcentrifuge tube and embedded in a drop of
-
-------
·- - - - - - ___'-"Ot:·~---
10
molten 1.5% Bacto-agar (Difco Laboratories, Detroit,
Michigan).
Once the agar had solidified, it was cut into
blocks about 1 mm
3
in size.
The specimens were post-
fixed, dehydrated, and embedded according to standard
procedures outlined by Hyatt (1970).
The samples were post-fixed in phosphate buffered
1% Oso4 for one hour at room temperature.
The post-
fixative solution was prepared by mixing equal parts by
volume of 2% Os0
0.2 MpH 7.2.
4
solution and S5rensen's phosphate buffer
The agar blocks were washed by centrifuga-
tion to remove the post-fixative solution, and were transferred to 50% ethanol for 7 minutes, and dehydration continued for 7 minutes each in 70%, 80%, 95%, and twice in
100% ethanol.
The specimens were washed three times in
propylene oxide for 7 minutes each time and placed in a
50:50 mixture of propylene oxide and EPON embedding mixture
for 1 hour.
The 50:50 plastic mixture was replaced with
100% EPON embedding mixture.
The embedding material. had
the following composition (Burke and Geiselman, 1971):
EPON 812.
. . . 52.9 g (43.6 ml)
Dodecenyl succinic anhydride
(DDSA).
.
Methylnadic anhydride (MNA)
.18.4 g (18.4 ml)
.28.7 g (23.2 ml)
Eleven to fifteen drops of Tridimethyl amino methyl
phenol (DMP-30) was added per 10 ml of the plastic
mixture.
11
The 1 mm
3
.
agar blocks were transferred to Pelco 105
embedding molds (Pelco, Tustin, California).
The molds
were filled with embedding mixture and allowed to stand at
room temperature for 12 hours before being heated at 60°C
for 8 hours for polymerization to take place.
The procedures of trimming and facing the EPON
blocks, and of preparing glass knives and troughs were outlined by Hyatt (1970).
Thin sections were obtained on a Sorvall Ultra
Micro tome (Model MT 2 B, Ivan Sorvall, Inc., Norwalk,
Connecticut), at motor speed 0.45, and picked up on Formvar
·coated 200 mesh copper grids.
Thin sections of specimens
were stained with saturated acetone solution of uranylacetate for 30 minutes at room temperature, and leadcitrate (Reynolds, 1963) for 10 minutes.
The specimens
were examined in a Zeiss electron microscope model EM 9S2
(Carl ·zeiss Inc., Oberkocken, West Germany).
Electronmicro-
graphs were recorded on Kodak 4489 EM film (Eastman Kodak
Co., Rochester, New York) and printed on Kodabromide F-2
paper (Eastman Kodak Co. , Rochester, New York).
Slide culture technique.
The behavior of individual
Metabacteriu.m spores was observed on Brain Heart Infusion
Agar (Difco) (BHI), and as a control on 1.5% Bacto-Agar (in
water) in a slide culture.
The slides were prepared by the methods described
12
by Kuhn and Starr (1972):
Five milliliter portions of
the media were melted in a boiling waterbath, and poured
into sterile plastic petri dishes.
The molten agar was
spread over the plate surface by gentle circular rotation
of the dishes on a flat bench top.
Once cooled and soli-
dified, the thin layer of agar medium was cut into small
2
blocks about(20 mm) in size (just smaller than a microscope coverglass), and a small agar block was scooped from
underneath with a clean coverglass and transferred to a
cleaned sterilized microscope slide.
The inocula were prepared by suspending about one
gram of guinea pig feces in 30 ml of sterile distilled
water, briskly mixing them by hand for a few seconds and
passing the suspension through a single layer Handi Wipe
filter to remove the coarse undigested plant debris.
The
filtrate was then passed through a four-layer Handi Wipe
filter, collected in a 50 ml polypropylene screw cap
conical tube, and settled in a swinging bucket centrifuge
for 5 minutes at approximately 400 rpm.
The crude suspension of M. polyspora was stored for
up to two days in 95% ethanol at 4°C to inhibit bacterial
growth.
Prior to inoculation of media a portion was
washed three times in 50 ml volumes of distilled water by
centrifugation.
For determination of heat resistance, the
inoculum was heated in an 80°C waterbath for 30 minutes.
13
The agar blocks were inoculated with a small drop
of the spore suspension with a sterile pasteur pipette and
sealed under an alcohol cleaned coverglass with vaspar.
Cultures incubated in air were sealed on all four sides
of the coverglass to prevent desiccation and to permit
the establishment of microaerophilic conditions.
Cultures
incubated anaerobically in a Gas-Pak anaeroJ?ic jar
(Becton, Dickinson and Company, Cockeysville, MD), were
sealed on three sides only to establish anaerobic conditions.
Incubation was at 37°C with daily microscopic ob-
servations.
The culture slides were mounted on a V-shaped glass
rod in a glass petri dish containing a round moistened
filter paper to preserve humidity.
Selected individual spores were located microscopically and their position recorded on a calibrated stage
to allow repeated observations of the same specimens.
Anaerobic culture technique.
A procedure of enrich-
ment and isolation of spore-forming anaerobic bacteria
was tried with a few fecal suspensions rich in M. polyspora.
The fecal suspension was heat treated in a boiling
waterbath for 20 minutes to kill vegetative cells and to
induce spores (Robinow, 1960).
Nutrient
Broth (Difco),
and Bacto-Brewer Thioglycollate Broth (Difco) were inoculated with the fecal suspension, and incubated anaerobi-
14
cally at 37°C in a BBL Gas-Pak anaerobic jar for 7 days.
The anaerobic roll tube procedure (Hungate, 1950)
was used with media developed at the Virginia Polytechnic
Institute, Anaerobe Laboratory in Blacksburg, Virginia
(Cato, Cummins, Holdeman, Johnson, Moore, Smibert and
Smith, 1970).
Prereduced peptone-yeast extract (PY) agar
served as a basal medium to which were added 1% w+v glucose (PYG), 0.8% w+v Bacto-Nutrient Broth (PYNB), and 10%
v+v Bacto-horse serum with 1% w+v glucose (HSPYG).
The
PY basal medium had the following composition (Cato et.
al., 1970):
Bacto-peptone (Difco).
. _.
2.0 g
.
Bacto-yeast extract (Difco) . .
1.0 g
Resazurin solution (25%) .
0.4 ml
Salts (Cato et. al., 1970)
4.0 ml
Distilled water.
100.0 ml
Cysteine HC1-H 0 (Calbiochem, La Jolla,
2
California).
.
0.5 ml
Bacto-agar (Difco) .
1.5g
The salts solution was prepared as follows:
CaC1
2
(anhydrous) .
.
MgS0 .
4
K HP0
2
4
. .
.
0
.
.
0
0
0
0
. . .
. . .
. .
0
0.5 g
0.2 g
1.0 g
15
KH 2 Po 4 .
1.0 g
NaHC0 3 .
10.0 g
2.0 g
NaCl
1000 ml
Water
First, the CaC1
2
and MgS0
300 ml of distilled water.
4
salts were dissolved in
Then, 500 ml of water were
added while swirling.the flask slowly, and the remaining
salts added.
dissolved.
Swirling was continued until all salts were
Finally, the remaining 200 ml of distilled
water were added and mixed.
The salts solution was stored
0
at 4_C,
The resazurin stock solution (25%) was also prepared according to Cato et. al. (1970).
Ten grams of
resazurin (Matheson, Coleman and Bell, Norwood, Ohio) was
0
dissolved in 40 ml of distilled water, and stored at 4 C.
The PYG and PYNA media were prepared according to
the following procedure quoted from Cato et. al. (1970).
Oxygen-free
"1.
2.
3.
co 2
was used in place of N .
2
Mix all ingredients, except cysteine, in
Erlenmeyer flask. Flask should have a small
head space to minimize air volume that must
be purged during cooling. Fit a removable
chimney to the boiling flask to prevent media
from boiling over.
Boil (10 to 20 minutes) until medium changes
from pink to yellowish.
Cool in ice water under 0 -free co . Flow of
2
2
should cause gentle bubbling (sufficient
to exclude air) .
co2
16
4.
When cool, remove from ice bath.
5.
Add cysteine. Adjust pH, using 8 N NaOH or
5 N HCl.
(After autoclaving pH should be 7+
0 .1).
6.
Change to 0 -free N gas and dispense media
2
2
into tubes; stopper with butyl rubber stoppers.
Nitrogen prevents pH change during storage.
7.
Place rack of tubes in press. Autoclave for
12 to 15 minutes at 121oc. Autoclave may be
exhausted rapidly."
Upon removal from the autoclave, the rack was transo
.
ferred to a 50 C waterbath,
which prevented solidification
of the agar until each tube could be individually rolled.
The roll method consisted of cooling the warm tube in ice
water, and quickly rolling it along a table top until the
agar set, coating the inside surface of the tube.
Cato's outline was modified for the
pr~paration
of HSPYG:
1.
All ingredients except serum and cysteine were
mixed in an Erlenmeyer flask and boiled to
dissolve.
Boiling was continued until the
indicator changed from pink to yellow (about
20 minutes).
2.
A gas cannula was inserted, introducing a flow
of oxygen-free co
2
gas, while the mixture
cooled.
3.
The flask was stoppered and placed
~n
to prevent loss of the stopper during
a press
1'7
sterilization in the autoclave.
4.
After the medium was cooled to 45-50°C in a
waterbath, serum and cysteine were added while
gassing.
5.
A second cannula was used to gas culture tubes
while aseptically dispensing 6 ml of the medium to each tube.
6.
Stoppered with butyl rubber stoppers, the
tubes were held in a 45°C waterbath until they
were rolled individually.
In place of the cannulas described by Cato et. al.
(1970), glass pasteur pipettes, 5.75 inches long
(Van
Waters and Rogers Scientific Inc., San Francisco, California), were heated and 2 inches of the tipped end bent to a
30
0
- 45
0
acute angle.
A non-absorbing cotton plug filter
was inserted in the mouth of the pipette and the cannulas
were placed in a glass petri dish or individually wrapped
for sterilization.
The air in culture tubes was replaced with a gas
mixture of 97%
co 2
and 3% H (Valley Welding Supply,
2
Canoga Park, California).
The mixture was passed through
a DEOXO cold catalyst gas purifier (product #32565,
Englehard Industries, East Newark, New Jersey) to remove
traces of molecular oxygen.
-·-..%~·-----
-----
18
Freshly voided feces containing M. polyspora was
used to inoculate PYG and PYNA roll tube cultures.
The
fecal suspension was heated in a boiling waterbath for 10
minutes to destroy vegetative bacterial cells; an unheated
portion served as a control.
Cultures were incubated at
37 °c f or as 1 ong as 14 days.
The HSPYG medium was inoculated with a fecal suspeno
'
sion which had been pasteurized at 60-65 C for 30 minutes.
An unheated fecal suspension served as a control.
In
addition, HSPYG medium was inoculated with fecal material
that had been freshly obtained directly from the intestinal tract of an animal which had died of unknown cause,
and had been diluted in an approximately equal volume of
sterile 0.9% NaC1 solution, while being gassed with oxygenfree
co2 .
Incubation followed at 37°C with daily observa-
tions over a period of 7 days.
Spore separation by aqueous polymer two-phase systern.
Separation of Metabacterium
spores from cellular
debris was also attempted with an aqueous phosphate,
organic polymer two-phase extraction (Sacks and Alderton,
1961).
A lysozyme treatment was incorporated to facilitate
release of endospores from Metabacterium sporangia, and to
eliminate vegetative cells of other bacteria (Brown,
Ordal, and Halvorson, 1957) ..
19
The behavior of Metabacterium spores
was observed
in the aqueous polymer ·two-phase system prior to and
following lysozyme treatment.
A fecal suspension was filtered and washed with
distilled water as previously described in the slide culture procedure to produce a concentrated suspension of
Metabacterium spores and sporangia.
divided into two portions:
The suspension was
one portion was directly
subjected to the extraction of Sacks and Alderton (1961),
the other portion was first treated with a 0.05% lysozyme
solution for 2 hours at 37°C.
The lysozyme solution was prepared by adding 99 ml
sterile distilled water, at room temperature, to 50 mg of
lysozyme (Product no. 16876, Sigma Chemical Company, St.
Louis, MO).
When the enzyme was completely dissolved,
1.0 ml of the specimen suspension was added to the culture
flask, and incubated at 37°C for 120 minutes on an incubator shaker (Model G 27, New Brunswick Scientific Company
Inc., New Brunswick, New Jersey).
Following incubation,
protein solubilization was inhanced by adjusting the
suspension to pH 11 with 10 N NaOH (about 6 drops per 100
ml) (Brown et. al., 1957).
The pH was checked with
pHydrion pH indicator paper with a range from pH 10 pH 12 (Micro Essential Lab., Inc., Brooklyn, New York).
Incubation was resumed for five minutes after which time
20
the lytic treatment was concluded, and the pH of the solution was neutralized with a few drops of concentrated hydrochloric acid.
The samples were washed twice in H o by centrifuga2
tion at 1000 rpm for 15 minutes in the swinging bucket
centrifuge.
mounts.
Microscopic examinations were made on wet
The samples were resuspended in an equal volume
of water, and served as the lysozyme treated sample in the
two-phase system.
Reagents of aqueous polymer two-phase systems.
A
50% (w+w) solution of polyethylene glycol 3000-37000
(Product Px 1289-20, Matheson, Coleman and Bell, Norwood,
Ohio), was made by dissolving 5.56 g of PEG in 5.56 ml of
sterile distilled water (Sacks and Alderton, 1961).
Four
to six hours were required to dissolve the polymer at room
temperature.
The resulting volume was about 10 ml.
A 53%
(w+w) PEG solution was made by dissolving 5.56 g in 5.0 ml
of water at room temperature.
about 9.5 ml.
The resulting volume was
The 3M phosphate buffer pH 7.1 was pre-
pared by dissolving 30.7 g K HP0 and 16.9 g KH Po in
2 4
2
4
100 ml of sterile distilled water (Albertson; 1958).
The
pH was verified with a Zeromatic pH meter (Beckman Instruments, Inc., Fullerton, California).
Two milliliters of an unfixed fecal suspension and
6.8 ml of phosphate buffer were shaken by hand until well
"-"~---
---------
-·---
-
--------~'-"·
21.
mixed in a 20 ml screw cap tube.
Either 4.0 ml of 50% PEG
solution or 3.6 ml of 53% PEG solution was added.
Distil-
led water was then added to a volume of 20 ml, followed
by additional shaking for 5 minutes.
The phases were
separated by centrifugation at 800 rpm for 2 minutes in
the swinging bucket centrifuge.
The organic (upper) layer
was carefully removed and transferred to a clean 50 ml
conical tube, taking care not to disturb the interface.
The organic layer was diluted to 40 ml (about an 8-fold
dilution) with sterile distilled water to reduce the
viscosity, and centrifuged in a Sorvall Ultracentrifuge
(Model RC-2, Ivan Sorvall Inc., Norwalk, Connecticut) at
10,000 rpm for 20 minutes.
The materials from the organic phase and from the
interface were observed microscopically in wet mounts.
~------
----
------
--------------
----
~';;,.'·----
-----
-
---------
22
RESULTS
Incidence of M.
~ora.
The incidence of Meta-
bacterium was greatest among adult female guinea pigs.
It was observed in the feces of 9 of the 11 sows (animals
3,4,12,14,15,19,20,34,39).
In the adult males it was ob-
served in the feces of 5 of the 14 boars surveyed (8,17,31 1
32,38).
Metabacterium was observed in the feces of only 3
of the 14 piglets (animals 4,5,27).
determined on all 14 infants.
The gender was not
The results of the surveys
are summarized in Table I.
Animal 3 was an adult female, pregnant on acquisition from a pet shop.
The first examination of fecal ma-
terial, one week before giving birth to two offspring,
displayed sporulated and presumed vegatative forms of M.
poly_spora (Figure 1).
. When maintained on a diet of fresh
lettuce for 5 days after giving birth to the litter, there
appeared to be a decline in the number of M. polyspora with
respect to the normal intestinal flora in the feces.
Re-
turned to a diet of dry alfalfa pellets, an increase was
seen in the number of M.
poly~pora
within 3 days.
A con-
stant diet of dry alfalfa pellets was continued for eight
months, at which time, this animal was in seriously poor
health with paralysis of the posterior limbs and diarrhea.
23
Table I.
Incidence of Metabacterium polyspora
in guinea pig feces.
Animal no.
and sex
I.
Source
Observation
period
Presence of
M. polyspora
Comments
Single Observation
Adults
a)
'7 M
CSUN
once
b)
rare
b)
M
"
II
9
M
II
II
b)
10
11
13
M
II
"
b)
M
II
II
14
16
F
"
II
rare
F
II
"
rare
"
II
"
II
23 nd
24 nd
25 nd
35 M
II
II
II
"
"
II
II
d)
36
"
"
d)
8
F
Infants
21 nd c)
22 nd
M
pet store
II
rare
b)
II
a) Animals located in animal laboratory facility.
unknown.
Origin
b) Housed in wire bottom cage with trough-type feeder.
c) Gender not determined.
d) Housed in open pen with sawdust covered floor with dishtype feeder.
-;,cs,·
~~~~----
---~
-~-
--~
- -------
-~----
-----------
~Av
-~~·-
- ------
24
Table I.
Animal no.
and sex
Source
continued.
Observation
period (weeks)
Presence of
M. polyspora
Comments
II. Long Term Observations
Adults
1
M
pet store
2
3
M
F
"
6
F
12
F
15
17
18
19
20
F
"
M
"
M
unknown
F
"
13
F
"
4
31
32
33
34
37
38
39
unknown
pet store
M
CSUN a)
M
"
M
"
M
pet store
M
breeder
M
"
"
F
e)
21!
25
II
34!
4!
5!
4
2
4
3
2
2
8
48
2
2
+
+
+
+
+
+
+
+
+
f)
f)
g)
g)
f)
f)
d)
d)
d)
+
f)
+
+
f)
a) Animals located in animal laboratory facility.
unknown.
f)
Origin
d) Housed in open pen with sawdust covered floor with dishtype feeder.
e) Autopsy showed no M. polyspora in samples from intestinal contents.
f) Case history described in text.
g) Autopsy confirmed M. polyspora.
25
Table I.
Animal no.
and sex
II.
Source
continued.
Observation
period (weeks)
Presence of
M. polyspora
Comments
Long Term Observations, cont'd.
Infants
born at home
+
+
f)
4
M
5
F
It
1
26
M
It
4
27
F
11
4
II
4
11
4
f)
It
4
f)
c)
28 nd
29 nd
30 nd
1
c) Gender not determined.
f) Case history described in text.
f)
f)
+
+
f)
f)
26
The animal was autopsied within a matter of hours after
death and the intestinal contents were examined.
The
caecum contained thick brown mucus; bacteria could not be
seen in wet mounts:
Animals 4 and 5 were the newborn offspring of animal
3; they were housed with the mother and observed for seven
days.
Fecal droppings became available on the fourth day
after which time they were collected and surveyed daily. M.
polyspora was not evident in the feces of either of these
animals between the fourth and sixth day, but by the seventh
day of life, M. polyspora was seen in the normal flora.
Animal 12 was an adult female.
M. polyspora was pre-
sent in the first fecal samples after the animal was obtained from the pet store.
After 5! weeks the animal was
sacrificed, at which time M. polyspora was no longer found
in either the feces or the caecal and intestinal contents.
Animal 19 was an adult female.
During the initial
month of observation, M. polyspora was not detected in her
feces, after which time she was force-fed a pasteurized enrichment of Metabacterium spores.
The animal was observed
for an additional month at which time she gave birth to a
litter (animals 28,29,30).
M. polyspora appeared in the
feces of this animal at the time of giving birth.
The
mother and her three offspring were maintained in a wire
bottom cage with a trough-type feeder, and they had no
27
appreciable contact with their feces.
M. polyspora
diminished in number in the feces of the mother during the
two weeks after birth, until it was no longer microscopically detectable.
No further evidence of M. polyspora was
found in the randomly collected samples of feces from the
mother and from each of her offspring during
th~
final
month of observations.
Animal 20 was obtained as a pregnant adult female
and observed repeatedly for a period of one month.
Metabac-
terium was observed for the first time on the day after the
offspring (animals 26,27) were born.
It is of interest to
note that multicellular filaments of Oscillospira were also
observed in the feces of this animal.
Infant 26 was re-
moved from the mother's cage as soon as it began feeding on
dry alfalfa.
It did not display evidence of M. polyspora
during the course of observation.
Infant 27, which was
kept with the mother and was removed from the cage only for
collection of fecal samples, did develop a flora which included Metabacterium and Oscillospira.
The mother became
sick, and upon her death, M. polyspora was not detected in
her intestinal contents.
When the offspring 27 was main-
tained in a separate mesh bottom cage with a trough-type
feeder, the presence of M. polyspora
and eventually disappeared.
Oscillospira, however, re-
mained in this animal through the
tions.
in its feces declined
fin~l
month of observa-
~
· - - · - - - - - - '----=-:,__
- - - ---~
- - - -----~---- ·-
0:'c _ _ _ _ ~~--·-
28
Initial examination of fecal suspensions from animal
34, an adult female, revealed the presence of M. polyspora,
which was observed with regularity for about a two-month
period.
The animal became ill and was given a large dose
of sulfonamide (obtained from a pet shop).
terial was voided for about one week.
No fecal ma-
The first fecal
samples excreted thereafter were collected and routinely
examined microscopically.
debris was present.
Considerable undigested plant
Many yeast-like budding cells and
numerous bacteria were evident, but no M.
served in the fecal samples.
polyspor~
was ob-
This animal died shortly
thereafter.
Animals 38 and 39 were respectively an adult male and
a pregnant adult female obtained from a commercial breeder.
Up to the time of acquisition, they were housed together in
a sawdust covered wooden floor hutch and maintained on a
diet of frequent servings of fresh lettuce in addition to a
daily diet of alfalfa pellets and water.
Fecal samples were
obtained from each of these animals and examined routinely.
Both animals demonstrated the presence of M. polyspora
within the feces.
Even though the number was somewhat
greater in the female, there were only 2 or 3 Metabacterium
sporangia per wet mount.
Then, the animals were separated
from each other in wire bottom cages with dish-type feeders
and placed on diets of dry alfalfa pellets and water for
29
three days.
At that time, the number of M. polyspora had
increased in the intestinal microflora Qf the female to
approximately one sporangium per microscope field of view,
using a 40X objective lens,while the number of M. polyspora
had remained fairly constant in the male.
Enrichments of Metabacterium.
A mechanical enrich-
ment of M. polyspora from voided feces was obtained by the
process of filtration through Handi Wipe cloth towels to remove coarse plant debris and washing by slow speed centrifugation to eliminate most of the smaller bacteria.
The re-
sulting enrichment of sporangia, spores, and vegetative
forms is compared to an unfiltered fecal suspension in
Figure 1.
Metabacterium spores were not resolved in Sacks and
Alderton's aqueous phosphate organic PEG
two~phase
system.
Neither was Metabacterium spores resolved in a PEG solution
of higher vicsocity (53%).
They were collected at the in-
terface mixed with tbe cellular and plant debris.
The 0.05% lysozyme solution released the endospores
from the Metabacterium sporangia and digested nearly all
bacterial cells in the fecal suspensions.
spores remained
fractile.
mo~phologically
The Metabacterium
intact and optically re-
There was no significant difference in the be-
havior of the lysozyme-treated spores and the untreated
spores in the aqueous polymer two-phase system.
30
FIGURE 1
Phase-contrast photomicrographs of M.
polyspor.~.
sporangia and presumed vegetative cells in a fecal suspension before and after mechanical enrichment.
Magnification
775X.
(Upper):
M. polyspora sporangia recognizable in fecal suspensions by their size and many spores.
(Lower):
Mechanically enriched suspension of M. polyspora
sporangia and a presumed vegetative cell (center
of lower portion).
32
Morphology and cytology.
The large size and unique
morphology of M. polyspora made it easily recognizable microscopically, even among the densely populated microbial
flora of the intestinal tract (Figure 1).
In phase-contrast
illumination, the large endospores were brightly refractile, and stood out distinctly against the grey background.
The individual spores measured approximately 8
pm
with sporangia measuring from 10 to 25 pm by 5 pm.
by 2 pm
The
sporangia enveloped most commonly 2-5 endospores, and
occasionally as many as 6,7 or 8.
spores seen within a sporangium.
Rarely were single
Presumed vegetative
cell~
of M. polyspora (Figure 1), concentrated from freshly voided
feces, stained intensely Gram-positive.
The spores re-
mained unstained, or had a slighly blue tinge, possibly
due to a residual sporangium.
The
endospore~
demonstrated a positive reaction to
Wirtz-Conklin's spore stain (green) as did other bacterial
spores present in the preparation.
the pink counterstain.
The sporangium displayed
Structures resembling what appeared
to be fore-spores, also retained the green spore stain.
The scanning electron micrographs of M. polyspora
sporangia displayed a relatively smooth cell surface.
In
some sporangia the outer envelope was folded and snugly
contoured the endospores (Figures 2,3).
Some M. polyspora
·displayed at one end of the sporangium, subpolar appendages
33
FIGURE 2
Scanning electron micrographs of Metabacterium
p_~!Y­
spora obtained from guinea pig feces, illustrating the
smooth surface and range in morphology of sporangia.
Magni-
fication 5600X.
(Upper Left) :
Subpolar appendages on sporangium enclosing two or three endospores.
(Upper Right):
Folded outer envelope closely contouring
four distinct endospores.
(Lower Left) :
Sporangium enclosing three endospores.
(Lower Left of
Center):
Folded outer envelope, contouring three
endospores.
(Lower Right of
Center):
Folded outer envelope contouring four
endospores.
(Lower Right):
Subpolar appendages present.
Folded outer envelope contouring four
endospores.
35
FIGURE 3
Scanning electron micrographs illustrating topography
and range in morphology of Metabacterium polyspora obtained
from guinea pig feces.
(Upper Row):
Magnification 7840X.
Presumed vegetative forms of Metabacterium
polyspora, displaying subpolar appendages
and folded outer envelope.
(Lower Left):
Folded outer envelope closely contouring
three distinct endospores.
(Lower Right):
Shape and dimensions suggest this structure
be interpreted as a free endospore.
~------·--
37
measuring about 60 nm in width and up to about 720 nm in
length (Figure 5).
Free spores also appeared to have a
smooth surface when observed by SEM (Figure 3).
Spores
measured 7.4 - 8.0 pm in length by 1.8 - 2.2 pm in width.
A portion of the mechanical spore enrichment used
in phase-contrast photomicroscopy and SEM was embedded in
EPON for TEM.
the samples.
Difficulties were encountered in sectioning
Crystalline debris, seen in the phase-contrast
and scanning electron micrographs (Figure 1, Figure 4),
shattered the glass knives.
The density gradient provided
by the PEG solutions permitted the spores to float while a
large percentage of the crystals settled out.
This process
reduced the amount of debris to avoid knife damage sufficiently to obtain a few preliminary thin sections.
TEM
suggested the prokaryotic nature of M. polyspora.
Germination of spores.
Individual Metabacterium
spores lost refractility in bright field (positive) phasecontrast microscopy, on BHI agar media in slide cultures.
Nutrients were needed for the loss of refractility to occur.
Such an optical change was interpreted as germination of
the spores.
The percentage of germinated spores was the
fraction of the total number of refractile spores that lost
refractility within a given period of time.
Up to, but not
more than 30 minutes may have elapsed from the time the
spores were placed on the medium, to the initial observation
(t=O hrs).
38
FIGURE 4 (UPPER)
Scanning electron micrograph of a mechanical enrichment of Metabacterium obtained from guinea pig feces.
Illustrated are a free endospore (lower left), a presumed
vegetative cell (center), a sporangium (upper right) and
plant material and crystalline debris.
Magnification 1120X.
FIGURE 5 (LOWER)
Enlarged scanning electron micrograph of sporangium
in Figure 2, showing the subpolar appendages.
16800X.
Magnification
40
The requirement of nutrients for germination was
demonstrated by comparing the behavior of ethanol-washed
spores on water agar and on BHI agar, in air and under
anaerobic conditions at 37°C.
The results are presented
in Table II.
Germination of ethanol-washed Metabacterium spores
on BHI was reduced by heat treatment (80°C for 30 minutes)
in cultures incubated in air for 196 hours at 37°C.
The·
results are presented in Table III.
Anaerobic cultivation.
After 72 hours of incubation
at 37°C in a Gas-Pak anaerobe jar, Nutrient Broth (Difco),
Thioglycollate Broth (Difco), and AC Medium Broth (Difco),
that had been inoculated with a pasteurized fecal suspenSlon rich in
~-
polyspora showed microbial growth as judged
by the appearance of turbidity.
However, microscopic exami-·
nation made at 96 hours and at seven days did not reveal
the presence of M. polyspora.
~!·
~yspo_ra.:_
was not cultivated in PYNA roll tubes
inoculated with unboiled fecal suspensions, nor was it
found among the variety of organisms cultivated in HSPYG
roll tubes inoculated with suspensions of voided feces or
feces obtained directly from the intestinal track, known
to contain M. polyspora sporangia.
TABLE II.
Microscopic observation of germination of ethanol-treated
Metabacterium spores in slide cultures on BHI agar· at 37°C
Medium
Total no.
of spores
No. of non-refractile spores (percentage germinated)
24 hours
48 hours
0 hours
12 hours
BHI
52
6 (11.5%)
42 (80.8%)
46 (88.5%)
46 (88.5%)
Water agar
56
1 ( 1.8%)
1 ( 1.8%)
3 ( 5.4%)
3 ( 5.4%)
BHI
36
0(
O%)
9 (25.0%)
17 (47.2%)
27 (75.0%)
Anaerobic
water agar
42
4 ( 9.5%)
4 ( 9.5%)
4 ( 9.5%)
4 ( 9.5%)
I~
Anaerobic
fJ:>.
!-"
TABLE III.
Microscopic observation of heat resistance of
ethanol-treated Metabacterium spores in slide
cultures on BHI at 37°C.
Spore
treatment
Total no.
of spores
No. of non-refractile spores (percentage germinated)
0 hours
24 hours
120 - 196 hours
Heat
(80°C 30 min.)
72
9 (12.5%)
16 (22.2%)
28 (38.9%)
No heat
76
9 (11.8%)
66 (86.8%)
74 (97 .4%)
Neither heat
nor ethanol
39
0 (
0%)
9 (23.1%)
14 (35.9%)
~
~"1
43
DISCUSSION
Metabacterium polyspora was consistently observable
in the feces of certain guinea pigs but not in that of
others even during long term observations.
as a resident of the indigenous flora,
seen with regularity.
Once established
~etabacterium
was
M. polyspora was transient in in-
fants when contact with their mothers' feces was discontinued, and in adults, when it was introduced by oral inoculation.
Perhaps M. polyspora must be introduced during in--
fancy and given time to develop as a member of the indigenous flora in order to remain as a resident within the host
animal.
The sudden and transient appearance of Metabacterium
in the feces of sows at the time of giving birth to a
litter is not understood.
Perhaps proliferation of the mi-
crobe is accelerated during late pregnancy, or perhaps a
more vigorous physical expulsion of the feces is responsible
for the appearance of Metabacterium near the time of birth.
The finding of a Metabacterium sporangium
in an oral
swab of a guinea pig led to the study of the mode of transfer of M. polyspora from mothers to their offspring.
The
incidence of Metabacterium within the intestinal flora of
infants seems to be dependent upon continued contact and
44
probable ingestion of feces containing Metabacterium.
There appeared to be a correlation between the diet
of the host animal and the abundance of M. polyspora in its
feces, as can be seen from the noted increase in the relative number of Metabacterium when animals were switched
from a diet of predominately fresh green lettuce to a diet
of dry alfalfa pellets.
Perhaps the animals ingest a
greater quantity of their own feces when feeding on dry alfalfa pellets from dishes in which they sit, thus receiving
a higher reinoculation of the microbe, than when they eat
fresh lettuce.
More likely, an alfalfa diet establishes
more favorable ecological conditions for the proliferation
of Metabacterium in the intestine of the guinea pigs.
'fhe
dry alfalfa diet did not initiate the appearance of M.
polyspora 1 if it was not already present.
Because M. polyspora occurs in relatively low numbers,
a process of filtration and low speed centrifugation was
developed to mechanically concentrate Metabacterium from
feces in order to facilitate the microscopic and physiological studies.
Handi Wipe cloth towels were found to be
well suited for the removal of coarse undigested plant material from feces.
As long as the Handi Wipes were not
wrung out, they had a close, uniform weave which yielded
consistent results.
It was found that multiple layers, up
to 8, increased the effectiveness of the filter.
More than
45
8 layers greatly decreased the volume of the filtrate without any visible advantage with regard to removal of plant
debris.
Fecal suspensions did have a tendency to clog the
Handi Wipe filters, thus the initial filtration was done
through a single layer of towels.
3.0, 5.0 and 8.0
Millipore filters with
pm pore sizes were quickly clogged by the
fecal suspensions, and cheesecloth had a coarse non-uniform
weave; they were thus not effective in the removal of plant
debris from the micro-organisms in the feces.
The large
Metabacterium sporangia were found to settle out of suspension by gravity sooner than nther bacteria.
was employed to speed this process.
Centrifugation
The speed and dura.tion
of centrifugation yielding an acceptable separation of
Metabacterium from smaller bacteria was determined by
experimentally using various combinations of speed and time·.
The spore extraction in an aqueous polymer two-phase
system relies in part upon
th~
surface affinity of the spore
coat for the organic PEG phase (Sacks and Alderton, 1961).
The envelope that usually surrounds the set of Metatacterium
spores had been removed by the lysozyme treatment as employed by Sacks and Alderton (1961) and should not have
interfered.
The free spores of Metabacterium apparently
did not possess the same surface properties as did the
spores of Bacillus and Clostridium species separated from
feces by Sacks and Alderton (1961).
Their behavior
46
resembled the spores of Putrefractive Anaerobe 3679 (Brown,
Ordal, and Halvorson, 1957) which according to Sacks and
Alderton (1961), remained at the interface in their twophase system.
The two-hour long treatment with 0.05%
lysozyme did not appear to have an adverse effect upon the
spores.
However, 9-hour long treatments caused the loss of
refractility in most spores, probably due to the spore coat
having been partially digested and allowing water to enter
the spore.
Questions might be raised regarding the preservation
of the specimen integrity during the preparation of the
mechanical enrichment.
About three hours elapsed from the
initial suspension of fecal material to the fixation of the
specimen concentrate for scanning electron microscopy and
transmission electron microscopy.
Judged by phase-contrast
microscopy, the sporangia did not undergo any visible
changes during the mechanical enrichment process.
Dry fecal
pellets have been stored at 4° - 8°C for over 5 years without visible deterioration of Metabacterium.
Attempts to
concentrate spores from fecal material fixed in glutaraldehyde seconds after defecation by the guinea pig were not
as successful.
Fixation of feces aggregated bacterial
cells and other fecal contents, and changed the differential filterability of M. polyspora so that it could not be
separated from other bacteria.
4'7
The morphological terms used in photon and scanning
electron micrography assume that Metabacterium is a bacterial spore-former as had also been done by Chatton and
Perard (1913a) and Robinow (1957.).
The photon-optical
properties of Metabacterium spores, refractility under
bright field (positive) phase-contrast illumination, and
their ability to retain the diagnostic spore stain are features suggestive of bacterial endospores.
nature of M.
polyspo!~
The prokaryotic
is suggested also by the preliminary
transmission electron micrographs.
The varying appearance of the outer envelope seen in
phase-contrast photomicrographs, and the increasing degree
of folding and contouring seen in scanning electron
micro~
graphs (Figure 2) may represent a sequence in the lifecycle of Metabacterium.
Conclusions cannot be reached at this time with regard to the identity or significance of the subpolar appendages seen occasionally at 6ne pole of the cell in scanning ·
electron micrographs.
by Robinow (1957).
Peritrichous flagella were described
However, the structures observed in the
current study are wider than would be expected for bacterial
flagella.
Metabacterium was reportedly motile only in fresh
caecal contents prior to exposure to air (Robinow, 1957).
It is not possible to assess the degree of anaerobiosis that
48
existed in Robinow's wet mounts.
In voided feces Metabac-
terium was not motile (Chatton and P~rard, 19J.3a; Robinow,
1957).
In the current study, motility of Metabacterium was
not observed in numerous phase-contrast observations of
freshly removed intestinal contents and freshly voided
feces suspended in distilled water.
had not
b~en
Perhaps the specimens
sufficiently protected from air.
cells generally appeared
intact~
Although the
it is possible that the
flagella had been sheared off, if indeed they had been present, during elimination from the intestinal tract or during the preparation of the specimens for photon and electron
microscopy.
There may also exist non-motile populations,
differing genetically from those observed by Robinow (1957).
Several factors may account for the failure to cultivate M. polyspora in vitro.
anaerobic:
The organism is presumably
Its apparent habitat is the intestine, and it
reportedly lost motility on exposure to air (Robinow, 1957).
The culture systems utilized in the course of this investigation were not strictly anaerobic, with exception of the
roll tube method.
have been met.
Thus, the proper redox potential may not
The roll tube technique achieved strictly
anaerobic conditions, however, the M. polyspora within the
inocculum was sparse, and, if viable under the culture
conditions, could undoubtedly have been overgrown by other
members of the normal flora.
49
The heat resistance of prokaryotic endospores is
generally attributed to a state of extreme dehydration, and
formation of a dipicolinic acid core (Warth, 1978).
The
use of heat shock to eliminate susceptible vegetative bacteria and to induce the heat resistant spores to germinate
did not lead to success in cultivating Metabacterium.
Slide
culture experiments indicated that the spores of Metabacterium were not as heat resistant as are the spores of
Bacillus or Clostridium species.
decreased the percentage of
refractility.
Heat treatment actually
Metabacteriu~
spores that lost
The optical loss of refractility in bright
field (positive) phase-contrast illumination is defined as
spore germination and indicates the irreversible and degradative conversion from a dormant to a metabolically active
state (Hanseni Spiegelman, and Halvorson, 1970).
The slide
culture experiments indicated that germination (loss of
refractility) of Metabacterium spores is independent of
oxygen tension, and requires nutrients.
Development is
arrested before vegetative outgrowth occurs, because the
redox potential or fastidious nutrient requirements of this
organism are not met.
Germination was apparently induced
by washing the spores in ethanol prior to inoculating
nutrient slide cultures.
Ethanol-washed spores used as con-
trols on water-agar slide cultures did not lose refractility.
50
Variable percentages of spores were non-refractile
at the time of the initial observations (0 hours).
The
loss of refractility may have occurred in the cultures
prior to the initial observation.
In some cases there was
a 15 - 30 minute lag in time between the placement of
spores on the medium and the initial observation.
This
time lag could have accounted for a loss of refractility in
cultures of rapidly germinating ethanol-treated spores on
BHI, however, nearly the same percentage of non-refractile
spores were seen in the initial observation of water-agar
cultures and in BHI cultures inoculated with heat treated
spores, which demonstrated a slower rate of germination.
It is likely that the spores were non-refractile prior to
inoculation.
The unusually large dimensions of Metabacterium do
not exclude it from the bacteria.
Other voluminous pro-
karyotes include for instance Achromat_ium and Cristispira.
Metabacterium
p~lyspora
is unique in the formation
of usually more than two endospores in a single cell.
few bi$porulating bacteria are known.
two genera,
Fusosporu~
A
Delaporte described
(Delaporte, 1964a) and Sporospiril-
lum (Delaporte, 1964b.).
Filaments of Oscillospira
(Chatton and Perard, 1913b) are believed to form two endospores on occasion.
However; none are known to form
several endospores like Metabacterium.
Even though members
51
of actinomycetes, e.g., Micropolyspora (Lechevalier, Solotorovsky, and McDurmont, 1961) is described as forming multiple "spores", these structures are clearly not bacterial
endospores.
'fhe formation of multiple endospores may play a reproductive function in Metabacterium.
In the opinion of
Chatton and Perard (1913a), this organism may have given up
transverse fission of vegetative cells.
As yet, the life-
cycle of Metabacterium has not been elucidated.
The genus Metabacterium was not described in Bergey's
Manual of Determinative Bacteriology (eighth ed., 1974),
while Oscillospira.:_ (Chatton and Perard, 1913b) was included
as a genus of uncertain affiliation, based upon microscopic
features.
In view of the current knowledge, the following
description of Metabacterium polyspora is offered.
Metabacterium is a large somewhat fusiform, Grampositive rod, measuring 5 pm by 10 - 25 pm in size.
be motile by peritrichous flagella.
cess has not been observed.
It may
The reproductive pro-
Two to eight endospores, mea-
suring 2 pm by 8 pm, are formed per non-septate cell.
The
spores are refractile and retain the spore stain (resistant to cold stains).
It has not been grown in axenic cul-
ture, however, spores do germinate (lose refractility) on
nutritive media.
Its habitat in the intestine and loss of
motility in air suggest it is anaerobic.
It is found in
52
the caecum and feces of the guinea pig.
The common occurrence of M. polyspora within the
indigenous microflora of guinea pigs and current knowledge
justifies that Metabacterium (Chatton and Perard, 1913) be
described and be placed as a genus of uncertain affiliation
with the endospore-forming rods and cocci in future editions
of Bergey's Manual of Determinative Bacteriology.
53
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