LIFE CYCLES IN THE METHANOGENIC ARCHAEBACTERIUM
RALPH WENDELL ROBINSON
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
This dissertation is dedicated to my parents, for
without their help and encouragement someone else's name
would be on the cover.
I would like to thank my fellow graduate students
Debbie Akin and Donna Williams for their help on this
project. Also, special thanks to my committee and to
Drs. Greg Erdos and Howard Berg.
An unsigned pastel drawing of a thin section of
Methanosarcina mazei by the late Doris Murray 1982
(conpa' Lt'iLh ?ij. '1I).
TABLE OF CONTENTS
1. INTRODUCTION .....................................1
2. LITERATURE REVIEW.................................5
Early Descriptions and Taxonomy...................5
Isolated Strains and Ultrastructure............... 6
3. MATERIALS AND METHODS ..........................13
Cultures and Growth Conditions...................13
Light Microscopy.. ..............................15
Sample Preparation for Electron Microscopy.......15
Method 1. For General Morphology using TEM......15
Method 2. For Gold Labelling
and X-ray Microanaysis..................16
Method 3. For Silver Staining and UV
Method 4. For Scanning Electron Microscopy......17
Method 5. For Freeze-Fracture..................18
Method 6. For Negative Staining................18
Changes in Cytoplasmic Fluorescence............ 19
Cytochemical Analysis of the Cell Surface........ 19
Silver Stain................................. 19
Enzyme Extraction ............................22
Cytochemical Analysis of the Granules
and Polyphosphate Bodies.............23
Enzyme Extraction ............................23
4. RESULTS.......................................... 25
Morphology of the Complex Life Cycle............25
Cocci ....................................... 25
Matrix Development and Division Patterns.....26
Morphology of the Limited Life Cycle............58
Methanosarcina mazei Strain S6...............58
Methanosarcina barker Strain 227 ...........73
Disaggregation in Methanosarcina
strain LYC................. 78
Freeze-Fracture analysis .....................81
Cytochemical Analysis of the Cell Surface....87
Silver Stain............................... 87
Fluorescein-Labelled Lectins............... 90
Gold-Labelled Lectins...................... 93
Enzyme Extractioi. ..........................93
Cytochemical Analysis of the Granules
and Polyphosphate Bodies ..........101
Silver Stain.............................. 104
Enzyme Extraction......................... 104
4. DISCUSSION..................,,............... 110
LITERATURE CITED ................................ 136
BIOGRAPHICAL SKETCH............................. 147
Abstract of Dissertation Presented to the Graduate
School of the University of Florida in Partial
Fulfillment of the Requirements for the Degree of Doctor
LIFE CYCLES IN THE METHANOGENIC ARCHAEBACTERIUM
RALPH WENDELL ROBINSON
Chairman: Henry C. Aldrich
Major Department: Microbiology and Cell Science
Methanosarcina mazei strains S6 and LYC along with
Methanosarcina barker strain 227 were utilized to study
the structure and differentiation of the aggregating
methanogens. Cultures harvested under varying
conditions are described at the ultrastructural level
and qualitatively examined using cytochemical
techniques. Cells are irregular and 1-2 pm in size.
Cells of Ms. mazei strain S6 are enclosed by a protein
layer 12 nm thick in contact with the plasma membrane.
In sarcinal colonies, cells are held in close
association by a fibrous matrix up to 60 nm thick.
Colony maturation was examined in Ms. mazei strain
S6 over a period of one year. Changes occur in the
shape and staining of individual cells. Also, various
inclusion bodies were observed that either persist
throughout colony maturation or are only found at
certain growth stages. The inclusion bodies, cell
surface, and matrix were analyzed with silver methen-
amine, fluorescein- and gold-labelled lectins, X-ray
microanalysis, freeze-fracture, and various enzymatic
Based on electron microscopic examinations, two
life cycles are described for Methanosarcina. A complex
life cycle is described with two variations for Ms.
mazei strains S6 and LYC. In addition, a limited cycle
is described that contains the only morphotypes observed
in Ms. barker strain 227. This limited cycle can also
be observed in the two strains of Ms. mazei studied.
The complex life cycle of strain S6 begins with
single cells that deposit matrix polysaccharide at the
cell surface; this prevents complete daughter cell
separation. These in turn divide forming sarcinae and,
as more matrix material is deposited, become distorted
into compact sarcinal colonies. The colonies dissociate
by degradation of the matrix. When released cells of
strain S6 are placed in fresh media, they can repeat the
Ms. barker is restricted to a limited cycle which
also occurs in the Ms. mazei strains while the matrix
remains intact. These colonies mature from 3-5 microns
into larger masses (>3mm) that multiply by fragment-
ation. When Ms. mazei strain S6 remains in the limited
cycle and does not disaggregate in stationary phase,
several types of possible resting forms are found.
Methanogenic bacteria are strict anaerobes and
generally obtain their energy from the oxidation of H2 to
CH4 by the reduction of CO2. A few genera can also utilize
methanol, format, methylamines, and acetate, forming
methane as a reduced product (Mah et al. 1977, Mah and
Smith, 1981). In general, methanogens can be isolated
from environments that provide H2 and CO2 and exclude 02.
These include the rumen of cattle, the intestines of
other animals and man, decaying tree trunks, stagnant
water, ocean bottoms, thermal hot springs, and sewage
treatment plants. Some sewage plants obtain much of
their operating energy from collected methane.
The morphology of the methanogens is as diverse as
their habitats. Cells are found as regular or irregular
cocci, as rods, spirals, or as sarcinae in aggregates of
large colony clusters. Such diversity in morphology
contrasted with a relatively uniform and unique
metabolic pathway led to the belief that methanogenesis
is a very ancient process, pre-dating the morphological
changes that occurred during evolution (Woese 1982).
Recently, an entire group of bacteria, including
the methanogens, has been recognized as being
phylogenetically separate from other prokaryotes (Balch
1982, Fox et al. 1980, Woese & Fox 1977). This group,
called the archaebacteria, is believed to comprise a
third kingdom, with the eubacteriaa" (other prokaryotes)
and the eukaryotes making up the other two kingdoms.
Archaebacteria are not simply ancestors of the
eubacteria but are a separate and independent group
related to eukaryotes as much as they are to the
eubacteria (Woese et al. 1982). Six characteristics
shared by archaebacteria examined thus far are 1) the
lack of peptidoglycan in the cell wall (Kandler & Koenig
1977, 1978, Kandler 1982, Koenig et al. 1982); 2) the
presence of unusual ether-linked poly-isoprenoid
glycerol lipids in the membrane instead of ester linked
alkane lipids (Tornabene et al. 1978, Langworthy et al.
1982); 3) unique tRNAs and rRNAs (Woese 1982); 4) their
existence in unusual, harsh environments (Woese 1977);
5) a eukaryotic-like DNA-dependent RNA polymerase
structure (Stetter & Zillig, 1980); and 6) unique
cofactors involved in metabolism (Cheeseman et al.
1972). The methanogenic bacteria were the first members
recognized as being uniquely separate from other members
of the Prokaryoteae and they still comprise the largest
and most diverse group in the Archaebacteriaceae (Wolfe
1971, Fox et al. 1977, Mah et al. 1977, Zeikus 1977).
Presently, other members of the archaebacteria include
the aerobic extreme halophiles (e.g. Halobacterium), the
thermoacidophiles (e.g. Sulfolobus and Thermoplasma),
and the thermophilic acidophiles (e.g. Thermoproteus).
Members of the group usually have a low percentage of
rRNA homology with other bacteria and a high percentage
of homology among themselves.
The sarcina-forming methanogens are currently
placed in the genus Methanosarcina. They display a
cellular arrangement that is unique, and several members
exhibit complex life cyles which are rare among isolated
archaebacteria. A life cycle has been partially
described at the light microscope level for Ms. mazei
where sedentary colonies break up into single cells
which can grow into new colonies in fresh media (Mah,
1980). The ability of a colony-forming bacterium to
dissociate into single cells in response to unfavorable
environmental changes provides a unique means for
dispersal. When cells regain a favorable habitat, they
form colonies which attach via sedimentation and
entanglement and are less likely to wash away. Indeed,
colony-forming methanogens are major inhabitants of
fixed bed reactors where the fluid components are
periodically flushed from the system (Zinder and Mah
1979, Robinson et al. 1984).
This group is of prime importance in the final
steps of biomass conversion to methane in mesophilic
environments, with up to 70% of the methane formed
derived from the acetate utilizing methanogens (Mah and
Smith 1981). These two qualities, of displaying a
complex life cycle and being of prime importance in
bioconversion, warrant an in-depth examination of this
group. This dissertation characterizes the structure
and development of these aggregating methanogens at the
ultrastructural level. The general objectives are 1) to
describe the morphology using light and UV microscopy,
scanning electron microscopy, and transmission electron
microscopy; 2) to determine and describe the life cycles
in Methanosarcina mazei; and 3) to obtain qualitative
data on cellular composition using cytochemical
Early Descriptions and Taxonomy
The genus Methanosarcina contains the most
metabolically diverse methanogens isolated thus far. It
was described in 1903 by Maze (cited in Maze 1915 and in
Barker 1936) in mixed cultures as a "pseudo-sarcine"
because of its ability to grow either as large sarcinal
groups or as single cells. Soehngen (1906) later
described a methanogenic sarcina that was more or less
cubical and was not observed as single cells. Smit
(1933) placed both of these bacteria into the genus
Zymosarcina along with other anaerobic sarcinae, with
the type species Z. methanica. In 1936 Kluyver and van
Niel created two genera: Methanosarcina for the methane
producing sarcina described by Soehngen with Ms.
methanica as the type species, and the genus
Methanococcus for the "pseudosarcina" described by Maze
with Mc. mazei as the type species (Barker 1936).
Since Ms. methanica and Mc. mazei had not been
isolated or maintained in pure culture, they were
omitted from the approved list of bacterial names (Ad
hoc committee 1976). Balch et al. (1979) designated
Methanosarcina barker and Methanococcus vannielii as
neotypes. Ms. barker matches the descriptions of Ms.
methanica, but Mc. vannielii is an irregular, highly
motile, marine coccus that does not aggregate and has
little resemblance to the early descriptions for Mc.
mazei made by Maze, Soehngen, or Barker (Jones et al.,
1977). Then, in 1980, Mah reported the first successful
isolation of a pure culture that exactly matched the
"pseudosarcina" descriptions. Thus, the name
Methanococcus mazei was temporarily revived. Recently
though, there has been a re-evaluation of the taxonomic
position of Mc. mazei and its relationship to other
methanococci and methanosarcinae. Consequently,
Methanococcus mazei has been moved into the genus
Methanosarcina as Ms. mazei, with Ms. barkeri designated
the type species (Mah & Kuhn 1984a and b).
Isolated Strains and Ultrastructure
Terminology. Since all methanogenic genera start
with the letter "M," the abbreviations recommended by
Daniels et al. (1984) will be used. These are as
follows: Mbr., Methanobrevibacter; Mb., Methano-
bacterium; Msp., Methanospirillum; Mc., Methanococcus;
and Ms., Methanosarcina.
There is some confusion over the descriptive terms
used for the various morphotypes of Methanosarcina,
especially Ms. mazei. Mah (1980) designates types I,
II, and III (Fig. 1). Zhilina (1976) had earlier
designated different types I, II, and III (Fig. 2). To
further complicate matters, Zhilina and Zavarzin (1979c)
later described their type III as exhibiting a variety
of morphotypes. Except when specifically describing
others' work, this paper will use the 3 forms shown in
figure 3 and refer to them as cocci, sarcinal colonies,
or resting forms. These three morphotypes can be stable
and long lasting in culture. The cocci correspond to
type III described by Mah and by Zhilina. The sarcinal
colony corresponds to type I described by Mah and type II
described by Zhilina.
Mah's isolate of Ms. mazei, designated S6, has been
described only at the light microscope level (Mah 1980).
It exhibits three morphotypes at different growth stages
(Fig. 1). When cultures are young, cells are found in
small sarcinal aggregates (type I) that measure up to 20
pm in size and may form larger aggregations with each
other. As the cultures mature and enter stationary
phase, sarcinal aggregates go through a transitory stage
Type I Type II
to form coccal cysts (type II). These are up to 100 pm
in diameter and ma- form larger aggregates up to several
millimeters in size. Mah reported that when the coccal
cysts are physically disrupted, a cyst "wall" breaks,
releasing individual cocci (type III) that are 1-3 pm in
size. Each of these can grow into sarcinal forms when
placed in fresh medium.
Several other strains of Ms. mazei have been
reported: "MC3" (Touzel and Albagnac 1983) exhibits a life
cycle that is apparently identical to S6; "Juelich"
(Scherer and Bochem 1983a) for which a life cycle is not
described; and "LYC" (Liu et al. 1985). Strain LYC
exhibits only two morphotypes with a unique mechanism of
disaggregation. A sarcinal colony forms when the cells
are grown at pH 6.0 but when the pH is raised to 7.0 the
colony disaggregates into single cells. Apparently, an
enzyme is activated that hydrolyzes the matrix holding
the colony together. Unfortunately, the single cells
cannot presently be induced to form aggregates so a
complete life cycle cannot be delineated. Ms. mazei
strains have not previously been described at the
The ultrastructure of Methanosarcina spp. from
enrichment cultures were examined by Zhilina (1971,
1976), Zhilina and Zavarzin (1979a) and Bochem et al.
(1982). Pure cultures of Methanosarcina spp. not
designated Ms. mazei were examined by Zeikus and Bowen
(1974), Zhilina and Zavarzin (1979b,c), Pangborn (In Mah
1981), Archer and King (1983, 1984), Scherer and Bochem
(1983a,b), Ollivier et al. (1984), and Sowers et al.
Zhilina (1971) first looked at sarcinal aggregates
found in digester enrichments. They are composed of
irregular cells measuring 1-3 pm with unequal division
zones. Colonies are enclosed by a variably thick
lamellar wall. Numerous gas vacuoles were described
that are 70-80 nm wide and 200-400 nm long with a 2.5 nm
membrane. Also present are electron dense granules
measuring up to 100 nm and polyphosphate-like inclusion
bodies of unreported size. Due to the ability to form
gas vacuoles, Zhilina and Zavarzin (1979b) proposed a
new species, Ms. vacuolata, but this epithet has not
been officially recognized.
Zhilina (1976) also reported on three morphotypes
of Methanosarcina in an enrichment culture (Fig. 2).
Morphotype I corresponds to a large sarcinal aggregate
that possesses a central cavity with a single pore.
Morphotype II is a smaller aggregation of irregular
cells tightly enclosed by matrix material. This type is
a sarcinal colony and corresponds to Mah's type I (Fig.
1). Zhilina's type III is a single cell and corresponds
to Mah's type III. Zhilina's morphotype III was later
reported to occur as single cells, in groups forming
loose aggregates, or in tightly packed cysts (Zhilina
and Zavarzin, 1979c). However, these authors do not
indicate any developmental changes that occur between
the various forms. This biotype may be a strain of Ms.
mazei but this has not been confirmed.
Another isolate, designated Methanosarcina strain
MP, which may be a strain of Ms. mazei was recently
described by Ollivier et al. (1984). They describe
sarcinal colonies that can break up into coccoid
elements in old cultures. However, they only examined
the ultrastructure of the sarcinal colonies.
Sowers et al. (1984a) recently reported on the
isolation and ultrastructure of a third species of
Methanosarcina, Ms. acetivorans, that grows in marine
environments as irregular single cells that are motile.
The cytoplasm contains electron dense granules and is
enclosed by a plasma membrane and a closely associated
protein layer. When cultures enter stationary phase the
single cells begin to aggregate into loose associations
that are eventually enclosed by a thin proteinacous cyst
wall. A matrix is not present and when fresh medium is
added the aggregates break up into single cells once
The electron dense granules and polyphosphate-like
inclusion bodies have recently been the focus of several
papers and presentations. Scherer and Bochem (1983 a
and b) used X-ray microanalysis (see Moreton 1981) on
ultrathin sections of 12 strains of Methanosarcina to
determine the partial elemental composition.
Polyphosphate-like globular inclusion bodies 150-250 nm
in diameter contain Ca, P, and Fe. The electron dense
granules have the same elemental composition but in
lower amounts. Murray et al. (1984) examined crude cell
extracts and reported that a carbohydrate is present
when cells are grown under nitrogen limiting conditions
and that the granules may be carbohydrate storage sites.
However, there is little evidence presented and the
granules have not been purified and analyzed.
MATERIALS AND METHODS
Cultures and Growth Conditions
Methanosarcina mazei strains S6 and LYC and the type
species Ms. barker strain 227 were examined. Colony
development was examined only in Ms. mazei strain S6. Two
different cultures of the same strain S6 were utilized.
One exhibited a complex life cycle while the other grew
only in sarcinal colonies in a limited cycle and is a
variant of 56. The variant strain was obtained when
cultures were repeatedly transferred in the laboratory.
Ms. mazei strain S6 and Ms. barker were grown at pH 6.8
in Bl medium (Balch et al. 1979) modified according to Mah
(1980) with H2/CO2, acetate, trimethylamine, or methanol used
as energy sources. Ms. mazei strain LYC was grown at pH
6.0 in a medium described by Liu et al. (1985) with H2/CO2 or
methanol used as energy sources.
Cultures were usually grown in serum tubes (Bellco)
containing 9 ml of medium and sealed with butyl rubber
stoppers. Inoculations consisted of 1 ml of a 10-14 day
culture passed through the stoppers using 20 or 25 gauge
needles. All cultures were incubated at 350 C without
Sarcina development from single cells and matrix
deposition was examined in a series of tubes inoculated
with cultures that had been filtered through a 3 pm
filter to remove any remaining sarcinal forms. Tubes
were incubated from 1-3 days, then prepared for
transmission electron microscopy (TEM) using method 1
Sarcinal colony maturation in the variant strain of
S6 grown on acetate was monitored by inoculating a
series of tubes through a 5 pm filter to select for the
smallest sarcinal forms. No growth occurred when
filters smaller than 5 pm were used (single cells are
not released by this variant). Cells were collected at
5, 10, 12, 15, 21, 30, 90, 105, and 360 days and
prepared for EM using method 1 below.
Colony disaggregation was monitored using strain S6
that spontaneously breaks apart into single cells.
When a 200 ml culture grown in a 600 ml serum bottle
showed the first signs of turbidity, the colonies were
examined using light microscopy and the culture was
prepared for EM using method 1 below.
Colony disaggregation in Ms. mazei strain LYC was
monitored by growing the cultures at pH 6.0 for 5-7 days
and then raising the pH to 7.0 by adding Na2HCO3 (10% in
reduced medium). Cultures were collected at 0, 12, 24,
36, and 48 hrs and prepared for EM using method 1 below.
Two light microscopes were used to examine culture
changes and to record micrographs. A Zeiss WL research
microscope equipped with phase and Nomarski optics was
used to make light micrographs on Kodak Plus-X 35 mm
film. A Zeiss photostrobe was used when photographing
single cells. Alternatively, a Nikon Labophot
microscope equipped with UV epifluorescence was used for
general culture monitoring and the recording of UV
micrographs on Kodak Tri-X film.
Sample Preparation for Electron Microscopy
Method 1. For General Morphology
Samples were fixed in a modified (2.0% form-
aldehyde and 2.5% glutaraldehyde in 0.1M Na-cacodylate
buffer) Karnovsky's fixative (Karnovsky 1965) for 30 min
at pH 7.2 on ice. After washing in buffer for 1 hr,
samples were fixed in 0.1M cacodylate buffered 1% OsO4 at
pH 7.2 for 1 hr on ice. Cells were washed once and then
encapsulated in 1.5% agar (Difco). Samples were then
dehydrated through a graded ethanol/acetone series,
embedded through a resin/acetone series into 100%
Spurr's resin (Spurr 1969), and polymerized in a 600 C
oven for 24 hrs. Thin sections were cut on a LKB
Ultrotome III ultramicrotome using a diamond knife and
collected on copper grids. Sections were poststained
with 1% uranyl acetate for 20 min followed by lead
citrate (Reynolds 1963) for 5 min. Micrographs were
taken on a Hitachi HU-11E or a JEOL JEM 100-CX
transmission electron microscope operated at 75 and 60
KV respectively. For stereo micrographs, semi-thick
sections (gold) were observed at a tilt angle of -5 and
+50 using a goniometer stage (Rieder et al. 1985). The
accelerating voltage was increased to 100 KV.
Method 2. For Gold Labelling and X-ray Microanalysis
Cells were fixed for 15 min in 2% formaldehyde and
0.2% glutaraldehyde in 0.1M cacodylate buffer at pH 7.2
on ice. After washing in buffer, cells were
encapsulated in 1.5% agar then dehydrated in an ethanol
series. Samples were further processed using one of two
methods. In one, they were embedded through a series of
ethanol/Lowicryl K4M plastic (Polaron Equipment Ltd.) to
100% plastic and polymerized by UV radiation overnight.
During this treatment, cells were kept on ice up to the
75% ethanol step and then placed at -400C for subsequent
steps until polymerization was complete. Alternatively,
samples were placed directly in 100% LR White plastic
(Polaron) from the 95% ethanol step and then, after 4
changes of plastic, were polymerized at 60C overnight.
Sections were cut and examined as in method 1 except
they were collected on nickel grids and were not
Method 3. For Silver Staining and UV Fluorescence
This procedure was essentially the same as method 1
except samples were not exposed to osmium, and
epon/araldite plastic (Mollenhauer 1964) was used for
embedding. Sections were collected on nickel grids and
were not poststained.
Method 4. For Scanning Electron Microscopy
Cells were harvested on a 0.2 pm syringe type
filter and fixed with 2% glutaradehyde in 0.1M
cacodylate buffer at pH 7.2 for 20 min. The cells were
washed, treated with 1% buffered OsO4 for 15 min, then
washed again. The filter and cells were dehydrated in
an ethanol series and dried by the critical point method
using CO2. The cells were then sputter coated with gold
and viewed in a Hitachi S-450 scanning electron
Method 5. For Freeze-Fracture
Cells were collected on a 2.3 mm diameter 400 mesh
gold grid, sandwiched between two 3 mm gold discs and
immediately frozen in a propane jet freezing apparatus
(Mueller et al. 1980). The frozen discs were stored
in liquid nitrogen until being separated in a Balzers
BA-360M freeze-fracture machine. The fracture faces
were angle shadowed with platinum-carbon and replicated
with carbon using electron guns. Replicas were cleaned
in 50% aqueous chromic acid for 2 hrs, rinsed, then
followed with sodium hypochlorite (50% household bleach)
overnight before being collected on carbon and Formvar
coated copper grids for viewing using TEM.
Method 6. For Negative Staining
Drops of cultures were placed on Formvar coated
copper grids for 1-5 min depending on culture density.
The medium was blotted away and the adsorbed cells
stained with aqueous 2% phosphotungstic acid or aqueous
1% uranyl acetate for 5 min.
Changes in Cytoplasmic Fluorescence
Twelve-day old colonies of strain S6 grown on
acetate and prepared for TEM using method 3 above were
sectioned using a glass knife to obtain a single
silver-gold thin section. This was poststained and
observed using TEM. When an adequate thin section
through a sarcinal colony was found, a 1 pm thick
section was cut as the very next section. This was
placed on a glass slide and observed with UV microscopy.
A comparison was made between the morphology of cells
observed in the thin section using TEM and the
fluorescent properties of the same cells in thick
Cytochemical Analysis of the Cell Surface
All cytochemical analyses were performed on Ms.
mazei strain S6. Ms. mazei strain LYC and Ms. barker
were not examined.
Sections prepared by method 3 were etched at room
temperature by floating on 1% periodic acid from 1-15
min (Erlandsen et al. 1979). After rinsing, the
sections were stained for 30-45 min at 600C with silver
methenamine (de Martino and Zamboni 1967, modified by
Picket-Heaps 1967). The grids were rinsed and then
floated on Ektaflo fixer (Kodak, diluted 1:100) to
remove non-specific silver deposits. Controls were run
with sections receiving 1) no treatment; 2) periodic
acid only; 3) silver methenamine only; and 4) periodic
acid and silver stain only (no fixer).
Whole colonies of strain S6 grown on acetate were
treated with 0.1M cacodylate buffered 1% OsO4 at pH 7.2
for 1 min to quench autofluorescence and then rinsed
with 0.1M PBS buffer at pH 7.2 supplemented with 0.1 mM
CaCl2, MnC12, and MgC12. The autofluorescence of single
cells lasts only a few seconds, so for single cells this
step was omitted. The samples were then treated for one
hour with one of the following fluorescein-labelled
lectins (Vector Labs, Burlingame CA): concanavalin A
(Con A); wheat germ agglutinin (WGA); soybean agglutinin
(SBA); Dolichos bifloris agglutinin (DBA); Ulex
europaeus agglutinin (UEA-I); peanut agglutinin (PNA);
or Ricinus communis agglutinin I (RCA120). Controls were
run with 0.1 M sugar (D-galactosamine, alpha-methyl-
mannose, alpha-methyl-glucose, or L-fucose) present in
the incubation mixture. Samples were washed in the
supplemented buffer and fixed in buffered 3%
glutaraldehyde for 20 min. After a final wash, samples
were observed with UV microscopy.
Colloidal gold approximately 15.0 nm in size was
prepared by reducing chlorauric acid with sodium citrate
according to the method of Frens (1973). A 4% gold
chloride solution was prepared and 0.5 ml added to 200
ml of double distilled water. Four milliliters 1 of 1%
sodium citrate was added and the solution was refluxed
for 15 min After cooling to room temperature, the pH
was adjusted to 7.0 with 0.1M K2CO 3 The minimum amount of
lectin required for stabilization of the gold was
determined by the technique of Horisberger and Bauer
(1976) as modified by Roth and Binder (1978). A 0.1 ml
aliquot of serially diluted lectin was mixed with 0.5 ml
of colloidal gold. After 15 min, 0.5 ml of 10% NaCl was
added and flocculation due to insufficient stabilization
of the gold was determined by a color change from pink
to blue. A 20% excess of lectin needed for stabil-
ization was added to 30 ml of colloidal gold. A 10%
solution of PEG-20 was added for a final concentration
of 0.5 mg/ml and the solution was centrifuged at 28,000
X g for 60 min. The clear supernatant containing any
unbound lectin was discarded. The gold pellet was
washed once by centrifugation in 20 mM NaHPO4-KH2PO4 at
pH 7.2 containing 0.5 mg/ml PEG-20 and 0.02% sodium
azide and then resuspended in the same buffer to 1/10
the original volume.
Sections of strain S6 prepared using method 2 were
floated for 20 min on drops of the lectin-stabilized
gold which had been serially diluted with PBS. Sections
were washed three times with PBS and then once with
distilled water before viewing. Controls were run with
sections receiving 1) colloidal gold only; and 2)
D-galactosamine (0.1 M) in the reaction mixture.
Sarcinal colonies of strain S6 were exposed to 0.5%
protease (type XIV, Sigma) in 0.1 M cacodylate buffer at
pH 7.0 supplemented with 20% sucrose for 5, 10, and 30
minutes at 370C. These were then fixed and embedded
using method 1 above. Controls were run with colonies
treated only with buffer. Alternatively, cultures
containing single cells were fixed and embedded as in
method 1 above except they were treated with 1.0%
protease for 30 min after fixation. Also, thin sections
of cells prepared according to methods 2 and 3 above
were treated by protease digestion as described below.
Cytochemical Analysis of the Granules and Polyphosphate
Thin sections of cells embedded by methods 2 or 3
were examined by energy- dispersive X-ray microanalysis
(Kevex) in a JEOL JEM 100-CX TEM. Sections were held in
a carbon specimen holder tilted 45-50. The TEM mode
was used at 60 KV and the condenser 1 spot size reduced
to 2 or 3 to restrict the diameter of the area being
Sections prepared by methods 2 or 3 using 12-day
cells of strain S6 were collected on nickel grids and
floated on 1% periodate for 15 min and washed. The
sections were then exposed to one of the following
solutions at 370 C: aqueous 0.5% protease at pH 7.0
(Anderson and Andre 1968); phosphate buffered 1% amylase
at pH 7.0 for alpha amylase and at pH 5.8 for beta
amylase activity (Palevitz and Newcomb 1970); phosphate
buffered 0.5% pullulanase at pH 5.8 (Palevitz and
Newcomb 1970); bicarbonate buffered alkaline phosphatase
(type 1-S, Sigma) at pH 10.4; or phosphate buffered 1%
cellulase (type 2, Sigma) at pH 4.5. Sections were
frequently monitored by TEM during treatment which
lasted up to 24 hrs. Sections were also exposed to
sequential treatments with pullulanase for 6 hrs
followed by alpha and beta amylase each for 24 hrs.
Controls were included with sections floated on their
respective buffers. Positive controls of Dictyostelium
myxamoebae were included to test for glycogen extraction
by amylase. Embedded filter paper was included to test
for cellulase extraction. Sections of Sporosarcina
ureae which has a protein layer outside the gram-
positive cell wall and protein layers on endospres were
included to test for protease activity.
Morphology of the Complex Life Cycle
When viewed with Nomarski optics, free cells of Ms.
mazei strain S6 are approximately 1-2 pm in size (Fig.
4). The cytoplasm often contains 1 or 2 refractile
granules up to 0.5 pm in size (not shown). Under UV
microscopy, cells display moderate fluorescence which
quenches within a few seconds. Scanning electron
microscopy (SEM) shows cocci 1-2 pm in size and roughly
spherical (Fig. 5). Negative staining (Fig. 6) reveals
an uneven surface, and polyphosphate inclusion bodies
can be seen which are up to 0.8 pm in size. Flagella
or pili were not present.
Thin sections show irregular and regular cells up
to 2.0 pm in size (Fig. 7 and 8) that differ in their
degree of cytoplasmic staining. The nuclear region is
not readily apparent. Electron dense granules are
present in the cytoplasm that range from 40-100 nm in
size. These are spheroid with very rough margins and
SSingle cells of Methanosarcina mazei
Fig. 4. Cells viewed with Nomarski optics.
Fig. 5. Cells viewed with scanning electron
Fig. 6. Transmission electron micrograph of
negatively stained whole cells. One to two
polyphosphate inclusion bodies can be seen
in some cells.
Fig. 7. Thin section of cells showing a variety of
4 4 3
:. S .'
Figures 8-11. Transmission electron micrographs of
single cells of Ms. mazei strain S6.
Fig. 8. Cells containing cytoplasmic granules and
polyphposphate inclusion bodies.
Fig. 9. Smooth surface with protein layer in close
contact with the plasma membrane
(arrows). Material with a membranous
appearance may be shedding from the cell
Fig. 10. A cell that has lost most of the protein
Fig. 11. Higher magnification of the cell in figure
10 shows the remaining protein layer as
clumps on the cell surface.
L= protein layer
pm= plasma membrane
to O.iu -
are characteristically surrounded by an electron
transparent halo. In single cells, the granules are not
evenly distributed but tend to group into clusters at
the periphery of the cytoplasm. Larger inclusion bodies
similar to polyphosphate are common. The cytoplasm is
bound by a unit membrane that is not very prominent and
an additional closely associated protein (see below)
layer 12 nm thick (Fig. 9). Frequently, material is
observed that may have sloughed off the surface in
strips. These sometimes have a membranous appearance
(arrowheads Fig. 9). Occasionally cells are seen that
have lost most of this layer (Figs. 10 and 11). The
material on these cells is present as clumps
approximately 14 nm in size just outside the plasma
Matrix Development and Division Patterns
A matrix material coats the outside of the cell
surface and is probably responsible for cell-cell
adhesion during colony formation. Matrix development
begins as a fibrous layer close to the protein layer on
the cell surface. During cell division and subsequent
development into sarcinae, the matrix becomes thicker,
denser, and more regular, somewhat resembling a gram-
positive wall. As sarcinal colonies mature however, the
matrix becomes very irregular as described below.
Figures 12-15. Various aspects of single cell division
of Ms. mazei strain S6.
Different stages of Cell division viewed
under Nomarski optics.
SEM of dividing cells.
Thin section of a cell initiating septum
Thin section showing completed septum.
Matrix material is incorporated in the
With Nomarski optics, cells in two-day cultures
appear like diplococci in various stages of division
(Fig. 12). The first division is by symmetrical binary
fission with the two daughter cells remaining in
contact. In SEM (Fig. 13), the dividing cells appear
similar to those observed in light microscopy. Single
cells initiate division with the plasma membrane,
protein layer, and matrix invaginating as an annulus
across the equator of the cell (Fig. 14). The matrix is
60 nm thick and has a fibrous structure.
After the septum has bisected the cell, the cross
matrix thickens from the periphery inward (Fig. 15)
until it is as thick as the outer matrix. The cells do
not separate, presumably because of the tight
association of the cross matrix. The second divisions
that give rise to four daughter cells are initiated in
the same plane with each other but perpendicular to the
first cross septum (not shown). Sarcinae that develop
from single cells have a regular shape when viewed with
light microscopy and fluoresce brightly under UV
microscopy (Fig. 16). In thin sections of three day
cultures, the sarcinae show a fairly regular shape but
the division septa are askew (Fig. 17). Unlike the
first division where the completed septum is almost in
the same plane, the second divisions are completed in an
asymmetrical manner and two perpendicular septa are
Figures 16-19. Various aspects of Ms. mazei strain S6
A sarcina-containing culture viewed under
Thin section of sarcina showing the
involvement of the matrix in the cross
A culture of cells developing beyond the
Fig. 19. Higher magnification of sarcina developing
into colonies. The division planes are
Overall, the sarcinae are approximately 3.5 pm in
size and the matrix is more condensed. Cytoplasmic
granules are more numerous and tend to group towards the
center of the cells. Further growth results in
irregular masses at four days (Figs. 18 and 19). Each
sarcina is derived from a single cell and is capable of
eventually forming a large sarcinal colony. There is no
evidence that sarcinae coalesce to form colonies. A
large inoculum of single cells results in the growth of
numerous small colonies while a small inoculum (i.e. a
few cells) will give rise to just a few large colonies.
Sarcinal colonies of strain S6 can reach up to 5 mm
(Fig. 20). Light microscopy using Nomarski optics shows
smaller colonies that are approximately 100 pm in size
with an uneven surface (Fig. 21). Under phase and UV
microscopy at lower magnifications, larger sarcinal
colonies up to several hundred pm in size (Fig. 22)
display a strong fluorescence which does not quench
after several minutes (Fig. 23). The larger colonies
are globular and are composed of smaller sized groupings
which may fragment when disturbed (see limited cycle
SEM of broth cultures reveals a configuration
somewhat like cauliflower (Fig. 24). Cells are either
Figures 20-23. Sarcinal colonies of Ms. mazei strain
Fig. 20. Broth-grown culture poured into a staining
dish. The colonies can reach up to 5 mm
Fig. 21. Individual sarcinal colony viewed with
Fig. 22. Aggregates of colonies viewed with light
Fig. 23. The same group of cells seen in figure 22
viewed with UV radiation. The cytoplasm
fluoresces with a bright blue-green color.
Figures 24 and 25. Scanning electron micrographs of
sarcinal colonies of Ms. mazei strain S6.
Fig. 24. Large colony with an appearance somewhat
Fig. 25. Enlargement of boxed area in figure 24
showing the close association of the cells
covered with fibrous matrix material.
7r t A,
closely packed in the colony interior or are arranged in
lobes at the surface. Individual cells can be seen at
higher magnifications but these are difficult to discern
due to the thick matrix material (Fig 25).
In thin sections, colonies are composed of a
heterogeneously staining population of cells that are
more irregular than the earlier sarcinal forms (Fig.
26). The matrix is approximately 40 nm thick and has a
fibrous appearance. The granules are either dispersed
throughout the cytoplasm or are concentrated at the cell
center. The granules possibly come together and fuse to
form the polyphosphate bodies since very compact groups
of granules are sometimes observed which lack the
characteristic electron transparent margins (Fig. 27).
Clear areas are found where polyphoshate bodies have
been removed during processing (Fig. 28). These are up
to 0.8 pm in size and usually possess some residue
around the margins. Occasionally, polyphosphate bodies
are observed where some of the material is present in a
loose reticular network (Fig. 29) or in bodies that are
more condensed (Fig. 30).
A large number of curved tubules 14 nm thick that
appear to contain membrane are trapped between the
protein layer and the matrix (Figs. 27-37). These are
sometimes in close association with the cell surface
(arrows Figs. 31 and 32). When there is a high
concentration of the tubules, they can intertwine with
Figures 26-30. Thin sections of sarcinal colonies of
Ms. mazei strain S6. Numerous inclusion bodies are
shown along with tubules associated with the cell
Low magnification showing the irregular
arrangement of the cells. They contain
numerous inclusion bodies and stain
Thin section showing the cytoplasmic
granules that tend to group toward the
center of the cells in thin sections and
may fuse to form the polyphosphate bodies.
Fig. 28. Section through a polyphosphate body where
most of the material has been removed
Fig. 29. A cell with two polyphosphate bodies that
contain dark staining material in a
characteristic reticular pattern.
Fig. 30. A polyphosphate body containing a dense
arrangement of the dark staining material.
Figures 31-34. Thin sections of Ms. mazei strain S6
showing the cell surface and the close relationship of
the tubules. Bar = 1 pm
Micrograph of tubules found between two
cells. The tubules are found in groups on
the cell surface and appear to be attached
in certain areas (arrows).
Micrograph of the tubules and matrix.
Frequently, the tubules are mixed with
fibers of the matrix material. The
tubules appear attached to the cell
Figs. 33 and 34. Stereo pairs of micrographs that
can be observed using a stereo viewer to obtain a three
dimensional image. A tubular structure is clearly
fibers of the matrix (Fig. 32). Stereo pairs of
micrographs show the irregular arrangement and the
mixing with fibers of the matrix (Figs. 33 and 34). In
cross section (Fig. 35) they are circular and
approximately 15 nm in diameter. They are usually
distributed in clusters at areas on the cell surface;
however, they can be found in grazing sections as
threads stacked roughly parallel to each other and
approximately 20 nm apart (Figs. 36 and 37).
After cells mature into sarcinal colonies during
the complex life cycle, they possess most of the
ultrastructural characteristics of colonies that are
restricted to the limited cycle described below.
After an unspecified amount of time in stationary
phase but usually not before two weeks, the colony
matrix begins shedding from the cells. In dark field
light microscopy at low magnification, single cells are
seen shedding from sarcinal colonies (Fig. 38). Under
phase contrast at higher magnifications, numerous cocci
are present with sarcinal colonies (Fig. 39). It is
common to find intermediate forms where both sarcinae
and cocci are present. Under UV microscopy (Fig. 40),
the colonies appear as coccal colonies held together by
what remains of the matrix. These coccal colonies
Figures 35-37. Thin section micrographs of Ms. mazei
strain S6 showing tubules associated with the cell
Fig. 35. High magnification micrograph of a group
of tubules cut in cross section showing a
hollow tubule structure.
Fig. 36. A cell showing four groups of tubules at
the cell surface. The one at the upper
right is cut in a grazing section of the
Fiq. 37. High magnification of the grazing section
of the cell surface seen in figure 36.
The tubules are in a parallel arrangement.
Figures 38-40. Microscopic examination of gross colony
morphology of Ms. mazei strain S6 during disaggreg-
Dark field light micrograph of two
disaggregating colonies releasing single
Disaggregating culture observed with phase
contrast microscopy shows intact colonies
together with free single cells.
Fig. 40. Disaggregating colonies observed with UV
microscopy. Single cells that fluoresce
brightly are held together by the
remaining matrix material.
usually break up spontaneously or can persist until
In SEM, cells in a cluster of colonies are not
arranged in a tight fashion (Fig. 41) and less matrix
material is present between cells. Individual cells are
easily observed at high magnifications (Fig. 42) and
measure 1.3 to 2.2 pm. Lobes and crevices are not as
apparent as in whole colonies (compare with Figs. 24 and
When observed in thin sections, the matrix has a
loose fibrous nature when the colony degrades into a
coccal colony (Fig. 43). The fibrous matrix has a
laminated appearance at higher magnifications and
involves uncompleted division septa (Fig. 44). The
membrane tubules between the matrix and the protein
layer are scattered throughout this region. The matrix
layers may number 20-30 per cell and shed or peel from
the surface (Fig. 45). At very high magnifications of a
cell nearly devoid of matrix, fibers 4-8 nm thick are
shedding from the surface (Fig. 46). Colony
disaggregation is usually completed 48 hrs after
initiation. However, coccal colonies are sometimes not
completely degraded, with the cells held together by the
remaining fragments of matrix (Figs. 47 and 48). Slight
physical pressure can break these colonies apart.
Single cells within these colonies still possess a
remnant of the matrix (Fig. 49). This layer is dense,
Figures 41 and 42. Scanning electron micrographs of
disaggregating colonies of Ms. mazei strain S6.
A disaggregating colony with little matrix
material holding the cells together.
Fig. 42. Higher magnification of colony with
sarcinae on the surface.
Figures 43-46. Thin sections of Ms. mazei strain S6
showing the matrix material during colony disaggreg-
Fig. 43. Disaggregating colony held together by a
loose fibrous matrix.
Fig. 44. Individual cell arrested during division.
The matrix in the septum has the same
fibrous appearance as the matrix on the
Fig. 45. Micrograph showing the matrix peeling or
shedding from the cell surface in layers.
Fig. 46. High magnification of the matrix on a cell
that has almost lost all of the matrix.
The matrix material is present as thin
fibers and tubules are still associated
with the cell surface.
pm= plasma membrane
'!q$4. pr.i I'
Objn .-h O.lpm
4i I "
S V *
*- P '
Figures 47-49. Thin sections of Ms. mazei colonies in
the later stages of colony disaggregation.
Low magnification of a loose colony held
together by remnants of the matrix.
Higher magnification of a partially
disaggregated colony. A thin layer of
matrix material remains around each cell
in the colony while the associated free
cells lack this material.
A single cell that is surrounded by
remaining matrix. Tubules are present
between the matrix and the cell surface.
L= protein layer
pm= plasma membrane
uneven, approximately 50 nm thick, and is not laminated.
The layer is located several hundred nanometers from the
cell surface. Such cells are less frequent as the
Morphology of the Limited Life Cycle
Methanosarcina mazei Strain S6
A limited cycle occurs in the wild type Ms. mazei
when the colonies are still actively growing before
matrix degradation and colony dissagregation begins.
When the wild type Ms. mazei was repeatedly transferred
in the laboratory, it gradually lost the ability to
undergo the complex life cycle. It failed to
disaggregate into single cells and the matrix remained
intact. This variant strain can only disperse by
fragmentation of the sarcinal colony. Ms. barkeri and
the variant of M. mazei strain S6 are limited to this
cycle and do not break up into individual cells. When
these cultures are passed through 5 pm filters into
fresh media, colonies have the same general morphology
as mature colonies found in the complex life cycle. If
these colonies are disturbed sufficiently by methane gas
evolution or by shaking, they will fragment into smaller
masses. The size depends on the severity of the
shaking, and if very hard (vortexing for 1 min), then
individual units approximately 3.0 pm in size are
released (see boxed area in Fig. 24). These will again
grow into larger masses when placed in fresh media. The
developmental changes of maturing sarcinal colonies of
Ms. mazei S6 were examined in the variant strain over a
period of 360 days. Cultures of Ms. barker were
examined after 14 and 90 days of growth.
In thin sections, the cytoplasm of the cells of
5-day colonies grown on acetate stains rather evenly
(Fig. 50). At this stage the colonies are found singly
in the culture medium and are uniform with a diameter of
3-5 pm. The individual cells themselves are 0.6-1.0 pm
in diameter and are surrounded by a 30-60 nm matrix.
The electron dense granules are scattered throughout the
cytoplasm and polyphosphate bodies are rarely seen.
Membrane tubules between the protein layer and the
matrix are not commonly observed in cultures restricted
to the limited cycle.
Within 10 days, the colonies enlarge to 8-10 pm and
the cells to 1.5 pm (Fig. 51). Cells of this age show a
variability of cytoplasmic staining correlated with
three morphological types. These cell types are
arbitrarily defined and intermediate forms can be found.
The first type (A) has a dense rather dark staining
cytoplasm, while that of the second type (B) appears
less dense and stains lightly. The third cell type (C)
appears highly degenerated and is usually found near the
Figures 50 and 51. Young colonies of the variant
strain of Ms. mazei strain S6 growing in the limited
Five-day old colony with fairly uniform
Ten-day old colony showing a variability
in the cytoplasmic staining of cells.
Cell type "A" stains rather dark while
cell type "B" stains lighter. Cell type
"C" is highly degenerated with numerous
inclusion vescicles. A granule-containing
membrane bound core is present
50 ) <* -i-
^te ---^-- ---- --^^^--^^ --- ^
periphery of the colony. This type has numerous
inclusions and membranous structures and does not show
the cytoplasmic staining homogeneity seen in
Two kinds of membrane cores are found in the
cytoplasm of cells restricted to the limited cycle. One
kind is found in cell types A and B (Figs. 52-55 and
arrowheads Fig. 51). They are 90 nm in diameter and
appear to have a double membrane surface 25 nm thick.
The membranes are straight and have a rigid appearance.
A trilamellar unit structure is only rarely demonstrated
(Fig. 54). There can be up to three cores of this kind
per cell and when more than one is present they are
arranged parallel to each other (Fig. 53). Inside the
cylindrical cores are electron dense granules arranged
in single file. These granules are of the same
appearance as those found in the cytoplasm but are much
more uniform in size at 40 nm. These cores are observed
in less than 1% of the cells in thin sections but almost
traverse the cytoplasm and are closely associated with
the plasma membrane. A connection with the plasma
membrane has not been observed and the ends appear to
open into the cytoplasm (Fig. 55).
The second kind of core is associated with the
plasma membrane in cell type C (Figs. 56 and 57). These
are 100 nm in diameter and are shorter than the cores
seen in younger cells (above). They are more numerous
Figures 52-57. Thin sections of membrane cores found
in Ms. mazei strain S6. Figs. 52-55 are of the
granule-containing cores found in cell types "A" and
"B". Figs. 56 and 57 are of plasma membrane-associated
cores found in cell type "C".
A high magnification of the core present in
figure 51 (arrowheads). The core nearly
bisects the cell and contains a single
file of cytoplasmic granules.
Fig. 53. A cell with two of the granule-containing
cores arranged parallel to each other
Fig. 54. A granule-containing core at high
magnification showing the double membrane
Fig. 55. A granule-containing core with one end
opening into the cytoplasm.
A grazing section of a plasma
membrane-associated core. A regular
pattern is observed in the core surface.
A cell containing several randomly
arranged cores, one of which shows a
connection with the plasma membrane
im= inner core membrane
om= outer core membrane
Iku : 16
however, and are not in an ordered arrangement. These
cores do not possess electron dense granules and appear
empty. The inner membrane is continuous with the plasma
membrane (arrows Fig. 57) and in grazing sections, a
regular helical pattern is observed (Figs. 56 and 57).
This regular pattern is probably due to the protein
layer in close contact with the inner membrane. The
ends of the outer membrane have not been resolved.
Twelve-day sarcinal colonies of strain S6 fixed
only with glutaraldehyde and embedded in epon/araldite
still fluoresce when viewed under UV irradiation. A
comparison of cells observed in TEM (Fig. 58) and the
same cells under UV epifluorescence (Fig. 59) reveals a
correlation between the intensity of fluorescence and
the type of cell observed within the colonies (see
above). The dark staining type A cells observed in thin
sections fluoresce brightly while the lighter staining
type B cells and degenerated type C cells fluoresce very
weakly or not at all.
At 12 days the three cell types within a colony
are present in approximately equal numbers but by day 15
the second lighter staining cell type predominates (Fig.
60) and in cultures older than 21 days the degenerative
cell type is most common (Fig. 61). By 90 days only a
few of the younger cell types can be found (Fig. 62),
and at 105 and 360 days (Fig. 63), which appear the
same, only a few single cells look viable. The matrix
Figures 58 and 59. Sections showing the correlation
between cell type and autofluorescence.
Fig. 58. Thin section of colonies showing the
various cell types.
Fig. 59. Thick section of the same cells in figure
58 but viewed with fluorescence
microscopy. The dark staining cells in
thin sections (type "A") are the same
cells that fluoresce brightly in thick
Figures 60-63. Thin sections of older cultures of the
variant strain of Ms. mazei S6 grown in the limited
Fifteen-day old colony showing the three
cell types (see text).
Twenty-one-day colony with cell type "C"
Ninety-day colony where most of the cells
Fig. 63. One-hunded-twenty-day colony that appears
the same as one-year-old colonies (not
shown). Only a few viable looking cells
between degenerated cells is poorly defined and the
granules and polyphophate bodies persist. Possible
resting forms (see below) can be found in these older
cultures that have thick specialized walls.
Possible Resting Forms
Cultures of the variant Ms. mazei strain S6 older
than three months are largely composed of dead cells.
However, several types of structures which may be
resting forms are found (Figs. 64-66). Islands of
viable looking cells can be found toward the center of
the colonies (Fig. 64). In thin sections these are
usually composed of 10-15 cells each surrounded by a
dense layer of matrix material 100-200 nm thick.
Frequently, individual cells can be found with a
specialized uniform layer 40 nm thick that is apparently
derived from the matrix but appears more dense (Fig.
65). Numerous membrane tuhiles may be found outside the
protein layer and division septa were never observed in
these cells. The single-cell forms are usually
spherical but they are sometimes rod shaped. They may
or may not contain cytoplasmic granules or polyphosphate
bodies. A specialized layer sometimes develop around
cells in younger cultures when the other cells still
look viable (Fig. 66).
Figures 64-66. Thin sections of resting forms found in
old colonies of the variant strain of Ms. mazei growing
in the limited cycle.
Fig. 64. One type of resting form is composed of
many cells enclosed by a thick specialized
Fig. 65. Single-cell resting forms surrounded by a
thick specialized layer.
Fig. 66. A group of viable looking cells with a
specialized layer (arrowheads) around one
R= resting forms
Methanosarcina barkeri Strain 227
Young colonies of Ms. barkeri strain 227 (Fig. 67)
appear similar to the 5-day cultures of Ms. mazei
(compare with Fig. 50) and exhibit morphotypes similar
to those in the limited cycle. The nuclear regions are
prominent and granules are present in the cytoplasm.
The matrix is up to 150 nm thick, approximately twice
the thickness of the matrix in Ms. mazei, and is closely
packed between the cells. An additional layer
approximately 10 nm thick could be observed between the
plasma membrane and the matrix but this is usually
difficult to discern due to the close association of the
matrix (Fig. 68). Colonies at 90 days (Fig. 69) are
highly degenerated, much more so than older cultures of
Ms. mazei (compare with Figs. 62 and 63). A few viable
looking cells are present but these do not have
specialized layers and do not appear to be resting
forms. The degenerated cells contain laminated or
concentric stacks of membranes that form a variety of
structures (Fig. 70). The rest of the cytoplasm is
filled with membrane particles and electron dense
granules (Fig. 70). Occasionally, membrane bound areas
are found with dark staining spherical bodies (Fig. 71).
These are up to 80 nm in diameter and are made of
Figures 67 and 68. Thin sections of 14-day colonies of
Methanosarcina barker strain 227.
Cells within a colony are enclosed by a
closely associated matrix. The cells
stain rather homogenously.
Fig. 68. High magnification of the cell surface
showing a layer in contact with the plasma
pm= plasma membrane
Nu= nuclear region
L= probable protein layer
69-71. Thin sections of 90-day colonies of Ms.
Degenerative cells in these old cultures
do not show any regular structure.
Membrane vescicles are present along with
the electron dense granules.
A higher magnification showing the
vescicles (arrowheads) and granules.
A membrane bound vescicle containing
several very dark staining structures.
These are apparently made of coils of
pm= plasma membrane
m= vescicle membrane
0 pm -
closely packed concentric rings 5.0 nm thick (insert).
The nature of these structures is unknown.
Disaggregation in Methanosarcina Strain LYC
Ms. mazei strain LYC when grown at pH 6.0, has a
morphology and life cycle similar to Ms. barker. When
colonies are viewed in SEM, cells are 1.0-2.5 pm in size
and are held in lobes that make up the sarcinal colony
(Fig. 72). The matrix is not as thick on the surface as
in strain S6 and individual cells can easily be seen.
If cultures are maintained at pH 6, the larger colonies
will fragment into smaller colonies when agitated. When
the pH of a culture is changed to 7.0 however, single
cells begin shedding from the colonies.
When the disaggregating colonies are observed in
SEM, cells on the colony surface are not tightly
associated (Fig. 73). These are similar to cells in
disaggregating colonies of strain S6 (compare with Figs.
41 and 42). Thin sections, however, show that cells are
being released from the matrix at the colony surface and
only the outermost cell layers are affected (Fig. 74).
The cells in the colony interior have a homogeneous
staining cytoplasm which contains only a few electron
dense granules and polyphosphate-like bodies. The
matrix is dark staining and is approximately 50 nm
Figures 72-75. Disaggregation in Methanosarcina mazei
SEM of a sarcinal colony growing at pH
SEM of a colony after changing the pH of
the medium to 7.0. Free single cells are
apparent on the colony surface.
Thin section of disaggregating colony.
The matrix is only being degraded at the
High magnification of the colony surface
showing a single cell breaking through the
remaining matrix. The single cells are
osmotically sensitive and the cell surface
is not well preserved. However, dark
areas are present (arrowheads) that may
correspond to a protein layer.
72 ~ ~ i
pI ,II .~ ,
$ ~ -
thick. It is fibrous and generally not in close
association with the plasma membrane. The matrix has a
loose appearance in the colony periphery and is
approximately 150 nm thick. At the very outer edge, the
matrix thins (Fig. 150) until the underlying cell breaks
through and migrates away from the colony. The cells
become contorted in this process and retain an irregular
appearance. A protein layer was not observed outside
the plasma membrane, which is poorly preserved. The
cytoplasm stains lighter than that of cells within the
colony and there are regions along the cell surface that
stain darker than the cytoplasm but lighter than the
matrix (arrows Fig. 75). It is possible that the cells
are osmotically sensitive and a layer, if present, was
damaged during processing.
The sarcinal colonies of strain LYC will sometimes
completely dissociate over a period of 48 hours.
Usually though, degradation stops before the entire
colony is broken up and colonies, although reduced in
size, are still seen at the bottom of culture tubes.
Unfixed, propane jet frozen, sarcinal colonies of
Ms. mazei strain S6 and Ms. barker strain 227 show a
tendency to fracture through the cytoplasm rather than
the plasma membrane and membrane faces are only rarely
Figures 76-81. Freeze-fracture analysis of the variant
strain of Ms. mazei strain S6 and of Ms. barker strain
Figs. 76 and 77. Cytoplasmic fractures through Ms.
mazei grown in the limited cycle showing two types of
A high magnification of the smaller class
of cytoplasmic particles.
Low magnification with larger particles
(arrows) which probably correspond to the
Figs. 78 and 79. Plasma membrane fractures through Ms.
mazei growing in the limited cycle revealing a high
number of intramembrane particles.
High magnification of intracytoplasmic
Low magnification of plasma membrane
fractures of several cells.
Cytoplasmic fracture through Ms. barker
showing irregular particles and a smooth
fracture surface through the matrix.
Plasma membrane fracture through Ms.
barker showing a high number of membrane
particles arranged in regular patterns.
pf= protoplasmic fracture
observed. Replicas of the variant strain S6 growing in
the limited cycle show two classes of intracellular
particles (Figs. 76 and 77). One ranges in size from 10
to 20 nm and the other measures from 80 to 200 nm. The
latter number 1-2 per cell and are probably the
polyphosphate bodies. A dense arrangement of protein
particles 9.0 nm in size was found in the plasma
membrane (Figs. 78 and 79). Cultures of S6 examined at
5, 10, 14, and 21 days showed no difference in the
particle distribution in the fracture planes. Free
single cells of strain S6 were not observed to fracture
through the plasma membrane.
Ms. barker strain 227 possessed a heterogeneous
population of cytoplasmic particles ranging from 10 to
30 nm. with approximately the same distribution as
observed in Ms. mazei. The larger class of particles
corresponding to the polyphosphate bodies was not
observed (Fig. 80). The matrix is approximately 120 nm
thick and fractures with a smooth surface. The plasma
membrane contains a large number of particles 9.0 nm in
size that are commonly arranged in regular arrays in
many areas (Fig. 81).
Colonies of strain S6 growing in the complex life
cycle showed a similar tendency to fracture through the
cytoplasm rather than the plasma membrane. However
these showed a more complex structure than those of
colonies growing in the limited cycle. Fracture planes
Figures 82-85. Freeze-fracture analysis of Ms. mazei
strain S6 grown in the complex life cycle.
Figures 83-85. Specialized areas found associated with
the plasma membrane.
Fracture through the cytoplasm and a large
A fracture through a stack of laminated
A fracture through several laminated areas
that curve around and back on the stack.
A fracture through a lamminated region
where individual layers can be clearly
observed. The layer folds back on itself
and may be one continuous layer.
cy= cytoplasmic fracture
would frequently pass through polyphosphate bodies (Fig.
82). These are very large up to 0.8 pm in size with a
rough even fracture surface. Specialized laminated
structures are associated with the plasma membrane
(Figs. 83-85). These usually consist of stacks of
material in curved or convoluted forms with individual
layers approximately 12 nm thick. Sometimes the
structures appeared as tightly packaged folded layers
11.4 nm thick arranged in parallel coils (Fig. 85).
These areas may correlate to groups of tubules observed
at the cell surface in thin sections.
Cytochemical Analysis of the Cell Surface
When thin sections of 12-day sarcinal colonies of
strain S6 are etched with 1% periodic acid for one min
and then stained with silver, the colonies show moderate
labelling of the matrix material (Fiq. 86) indicating a
general polysaccharide composition. The layer just
outside the plasma membrane does not label. When
sections of disaggregating colonies were etched for 15
min before staining, the matrix shows a much higher
level of staining (Figs. 87 and 88). The matrix
material is evident and fibers throughout the
extracellular space are prominent. Again, the layer
Figures 86-88. Siver methenamine staining of Ms. mazei
Sections of cells etched for 1 min show a
light labelling of the matrix
(arrowheads). The cell surface has a dark
appearance due to the etching but does not
label with silver (arrows). The
cytoplasmic granules show heavy labelling.
Thin section showing an intact colony,
disaggregating colony, and free cells.
The sections were etched for 15 min and
the staining is much heavier than in
figure 86. Only the matrix material
stains. The free cells do not stain.
Higher magnification of a cell with some
remaining matrix material. The cell
surface and associated tubules do not
stain while the matrix stains heavily
I pm -
close to the plasma membrane and the associated membrane
tubules do not stain. Free cells do not show any
specific labelling of the cell surface, indicating a
lack of matrix material. Controls not treated with
fixer after silver staining had the same level of
non-specific background labelling and this step was
subsequently eliminated. The other controls did not
A strong fluorescence was observed at the colony
surface when strain S6 was treated with fluorescein-
labelled PNA, SBA, DBA, or RCA120 lectins (Fig. 89).
These lectins are specific for galactosyl groups.
Fluorescence was not observed when galactosamine was
present in the reaction mixture. Free cells generally
did not label with only an occasional cell showing
fluorescence (not shown). This is expected due to the
lack of matrix material. No fluorescence was observed
in samples treated with ConA, UEA I, or WGA lectins.
These are specific for mannosyl and glucosyl, fucosyl,
and glucosaminyl groups respectively.
Figures 89-91. Lectin labelling of the matrix of
intact colonies of Ms. mazei strain S6.
Fig. 89. UV micrograph of fluorescein-labelled
soybean lectin binding to whole cells.
Peanut, Dolichos, and Ricinus lectins
showed a similar fluorescence (not shown).
Fig. 90. Unstained thin section treated with
colloidal gold-soybean lectin. The
labelling is heaviest over the matrix
Fig. 91. Control section with the blocking hapten
sugar galactosamine added to the reaction
mixture. The labeling is non-specific.
Based on the results obtained using fluorescein-
labelled lectins, an attempt was made to stabilize
colloidal gold with PNA, SBA, and RCA. PNA and SBA
stabilizations were successful but RCA-gold would
flocculate during centrifugation and could not be used.
PNA and SBA-gold treated cells are labelled lightly over
the entire matrix (Fig. 90). These lectins are specific
for N-acetyl galactosamine. Only non-specific labelling
was observed on thin sections of single cells. The
controls that included galactosamine in the labelling
mixture (Fig. 91) show only non-specific labelling.
Sections receiving colloidal gold only did not label
The layer just outside the plasma membrane can be
removed when colonies are treated with protease before
fixation. Colonies treated for 5 or 10 min show
protoplasts enclosed by the intact matrix which is 20-70
nm thick (Fig. 92). At higher magnifications (Fig. 93),
the cytoplasm is bound solely by the plasma membrane,
and the region between the cell surface and the matrix
is free of particulate matter. Extensive cell damage