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ELECTRON MICROSCOPY OF PLASMOLYSIS IN ESCHERICHIA COLI
EUGENE H. COTA-ROBLESDivision of Life Sciences, University of
California, Riverside, California
Received for publication 30 August 1962
ABSTRACT
COTA-ROBLES, EUGENE H. (University ofCalifornia, Riverside).
Electron microscopy ofplasmolysis in Esche7ichia coli. J.
Bacteriol.85:499-503. 1963.-Escherichia coli cells plasmo-lyzed in
0.35 M sucrose reveal plasmolysis at onetip of a cell or in the
center of dividing cells inwhich protoplast partition has been
complete.Central plasmolysis reveals that protol)lastseparation can
be completed before the in-vagination of the cell wall is complete.
Thesestudies support the concept that these cellsdivide by
constriction. The strength of the unionbetween cell wall and
cytoplasm is not uniformaround the entire cell. It is strongest
along thesides of these rod-shaped cells and weakest atone til) of
the single cell. Thus, a single cellgenerally forms one cup-shaped
vacuole inwhich the cytoplasm has collapsed away fromone tip of the
cell.
Robinow's (1960) recent review of bacterialstructure succinctly
summarizes our currentunderstanding of the anatomy of plasmolysis
ofbacterial cells. He clearly points out that theunion between the
cell wall and the cytoplasmicmembrane may be so strong that the
separationof these two structures may be incompleteduring
plasmolysis. In the observations de-scribed below, this point is
underscored andelaborated upon by the examination of
ultrathinsections of plasmolyzed cells of Escherichia coliB by
electron microscopy.
MIATERIALS AND MNIETHODS
E. coli B was routinely cultured aerobically inFraser and
Jerrel's (1953) glycerol medium at37 C for 16 hr. The culture was
diluted 100-foldinto fresh medium and further incubated
aerobi-cally at 37 C for 2 hr. The cells at this time are inthe
early log phase of growth. The cells werethen harvested from the
growth medium bycentrifugation and suspended in 0.35 M sucrose
to effect plasmolysis. To maintain the plasmo-lyzed state of the
cells for any appreciable time,it was necessary to subject the
cells to at leastone wash with distilled water prior to
suspensionin the plasmolyzing medium. Plasmolyzed cellswere
maintained at room temperature for 5 to 20min prior to addition of
fixative.
Electron microscopy. Several fixative procedureswere used
throughout these investigations. Themost successful one in our
hands has been acombination of formalin fixation with
Kellen-berger, Ryter, and Sechaud's (1958) procedureof OS04
fixation in the presence of Tryptone(Difco). To facilitate fixation
with formalin, cellswere plasmolyzed in buffered 0.35 M sucrose.The
buffer contained Na2HP04, 7.0 g; KH2PO4,3.0 g; NaCl, 4.0 g; MgSO4,
0.2 g; water, 1,000ml; pH 6.8. Plasmolyzed cells were fixed in
10Cc/)formalin for 1 hr at 24 C. The cells were thencentrifuged,
and the pellet was resuspended in 1ml of 1 % buffered OS04
(Kellenberger et al.,1958) plus 0.1 ml of 1% Tryptone. This
mixturewas maintained at room temperature for 16 hr.The cells were
post-stained with 0.5 c0 uranylacetate for 2 hr. After uranyl
acetate staining,the cells were centrifuged, and the pellets
wereprepared for embedding in Epon 812 (Luft,1961) or Vestopal WV
(Kellenberger et al., 1958).
Ultrathin sections were cut on a Porter-Pllummicrotome by use of
glass knives. Occasionally,the sections were further stained with
leadhydroxide (Watson, 1958). Electron micrographswere taken with
an RCA EMU-3B electronmicroscope equipped with an objective
aperture.
RESULTS AND DISCUSSIONThe plasmolysis demonstrated by
actively
dividing cells of E. coli upon suspension in 0.35M sucrose after
removal from the growth mediumis short-lived but characteristic.
More long-lasting plasmolysis can be demonstrated after
apreliminary wash in distilled water prior tosuspension in 0.35 M
sucrose. I have not variedthe plasmolyzing conditions greatly,
since our
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COTA-ROBLES
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FIG. 1. Phase photomicrograph of cells of Escher-ichia coli B
suspended in 0.35 M sucrose buffer.Note cell with central
plasmolysis vacuole.
original observations of plasmolyzed cells weremade in
conjunction with the studies of D-amino acid-induced spheroplasts
of this organism(Cota-Robles and Duncan, 1962). Consequently,I
utilized 0.35 M sucrose in the observationsdescribed herein.The
pattern of plasmolysis in 0.35 M sucrose
is quite striking when viewed by phase micros-copy (Fig. 1).
Here, one can see that the separa-tion of the protoplast from the
cell wall is notuniform. One cell demonstrates a plasmolysisvacuole
at one tip of the cell. A cell with acentral vacuole can also be
seen in this figure.
This pattern is more clearly demonstrated inthe series of
electron micrographs containedherein. Figure 2, although not a
particularlynew, unusual, or technically refined electron
micrograph, shows a normal cell of E. coli Bthat is in the
process of cell division. Here, onecan discern that the cell wall
adheres closely tothe protoplast. Nuclear division appears
com-plete, even though the central invagination of thecell wall
appears to have just commenced. Otherobservations (Fig. 3) reveal
that nuclear divisionneed not be completed prior to invagination
ofthe cell wall. The dividing cell depicted in thisfigure is one
that was in the process of recoveryfrom plasmolysis. Hints of
separation of theprotoplast from the tips of the cell can be
ob-served.The central vacuole is well depicted in Fig. 4.
Here, separation of the daughter protoplasts iscomplete, and the
large central vacuole is bor-dered by a cell wall that is stretched
out. One ofthe newly formed protoplasts presents a concavesurface
toward the center; the cytoplasm can beseen adhering to the cell
wall to form a cup-shaped plasmolysis vacuole. The
plasmolysisvacuole may be both central and at the tip, asdescribed
in Fig. 5. This figure demonstratesthe beginning of an invagination
of the cell wall.However, an unusual feature is that the
invagina-tion appears to be spatially disoriented withrespect to
the separation of the protoplasts. Thesame protoplast that appears
to be out of placereveals a marked separation from the wall at
thetip of the cell. Again, a cup-shaped vacuole isformed. It is my
present belief that separation ofdaughter protoplasts occurs prior
to completeinvagination of the cell wall. The misplacedprotoplast
can be seen to possess a limiting mem-brane. This protoplast could
have been drawnout of place as a consequence of the act
ofplasmolysis. Figure 6 offers support to the con-tention that
protoplasts are capable of separating,under the influence of
plasmolyzing conditions,prior to complete invagination of cell
wall. Here,one can see that the central plasmolysis vacuoleis
completely formed, but that the separation ofdaughter protoplasts
is uneven. A well-definedvesicle can be seen within the vacuole. A
secondvesicle can also be discerned. However, thisstructure is
still joined to the protoplast. Thenature of these vesicles is
unknown, but theycould be portions of the cytoplasmic membranethat
have been pinched off.
Figure 7 reveals the marked separation of walland protoplast
that can occur at the tip of adividing cell in which the separation
of proto-
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VOL. 85, 1963 ELECTRON MICROSCOPY OF PLASMOLYSIS IN E. COJi
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FIG. 2. Electron micrograph of a thin section of normal cells of
Escherichia coli B. Fixed with osmiumtetroxide, embedded in Epon
812, and stained with lanthanum nitrate.
FIG. 3. Electron micrograph of a thin section of plasmolyzed
cells of Escherichia coli B. Fixed with osmiiumtetroxide, embedded
in Epon 812, and stained with lead hydroxide.
FIG. 4. Electron micrograph of plasmolyzed cells of Escherichia
coli B. Fixed with formalin and osmiiiumtetroxide, embedded in
Vestopal W, and stained with lead hydroxide. Note the cup-shaped
plasmolysis vacuole.
FIG. 5. Electron micrograph of plasmolyzed cells of Escherichia
coli B. Fixed with formalin and osmiumontetroxide, embedded in
Vestopal W, and stained with lead hydroxide. Central plasmolysis
vacuole is notcoordinated with the invaginating cell wall.
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FIG. 6. Electron micrograph of plasmolyzed cells of Escherichia
coli B. Fixed with formalin and osmiumtetroxide, embedded in
Vestopal W, and stained with lead hydroxide. Separation of
protoplasts almost com-pleted, permitting formation of central
plasmolysis vacuole.
FIG. 7. Electron micrograph of plasmolyzed cells of Escherichia
coli B. Fixed with formalin and osmiumtetroxide, embedded in
Vestopal W, and stained with lead hydroxide. Marked plasmolysis
vacuole retaininga small vesicle at the tip of the cell.
FIG. 8. Electron micrograph of plasmolyzed cells of Escherichia
coli B. Fixed with formalin and osmiumtetroxide, emibedded in
Vestopal W, and stained with lead hydroxide. Note that cytoplasm is
still conncctedto small vesicle within the plasmolysis vacuole.
FIG. 9. Electron micrograph of plasmolyzed cells of Escherichia
coli B. Fixed with formalin and osmiumtetroxide, embedded in
Vestopal W, and stained with lead hydroxide. The large cup-shaped
plasmolysisvacuole emtiphasizes seciure attachment between the wall
and the cytoplasm along the sides of the cell.
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VOL. 85, 1963 ELECTRON MICROSCOPY OF PLASMOLYSIS IN E. COLI
plasts has not progressed sufficiently to permit acentral
vacuole to form. The great separation atthe sides, I believe, is a
result of the preparativeprocedure which, in this instance,
involved rapiddehydration. Discrete bridges of cytoplasmicmaterial
can be seen joining the cell wall alongthe side of the cell. The
ill-defined vesicular struc-ture that appears in the plasmolysis
vacuole is ofunknown origin. It could be of a similar origin tothe
structure that can be seen within the plas-molysis vacuole of the
cell pictured in Fig. 8.Here, a portion of the protoplast remains
in thevacuole, but it is connected by a thin bridge ofcytoplasm to
the main mass of protoplasm. It canbe noted that the portion of
cytoplasm remainingwithin the vacuole is not homogeneous.
Theextreme upper part of the plasmolysis vacuole ofthis cell
contains a second structure whose originis vague but which may be
related to an ancillaryaspect of these investigations: namely, the
infec-tion of plasmolyzed cells of E. coli with coliphageT2.
Although the nuclear material is not as wellresolved in this figure
as in others, there seems tobe a clear indication that the nuclear
materialhas been completely partitioned prior to
extensiveinvagination of the cell wall. Figure 9 is a moreextreme
demonstration of the plasmolysisvacuole which presents a concave
surface to thetip of the cell. It appears as though the
separationof the cytoplasm from the wall at the tip hasbeen so
forceful that the cytoplasm has collapsed,leaving a cup-shaped
plasmolysis vacuole.From the foregoing observation, I believe
that
the protoplast of a growing cell of E. coli is notuniformly
bonded to the cell wall. In fact, itappears to be bonded tightly
along the sides of thecell, less tightly at one tip of the cell,
and poorly,if at all, at the center of dividing cells in
whichprotoplast separation has been completed.
If the cell has completed protoplast partition-ing, a
plasmolysis vacuole may occur centrally.It appears abundantly clear
that protoplastseparation may be complete prior to any exten-sive
invagination of the cell wall between daugh-ter protoplasts. From
these observations, itappears that E. coli divides by constriction,
i.e.,by the invagination of cell wall between partiallyor
completely partitioned protoplasts. There isno doubt that the
partitioning of nuclear mate-rial is completed prior to any
plasmolyticallysensitive separation of the daughter
protoplasts.These conclusions should be contrasted withthose of
Conti and Gettner's (1962) recentdescription of cell division in E.
coli.
One feature yet to explain is why the plas-molytic separation of
protoplast from cell wallshould be limited to one tip of the cell
(Fig. 1and 9). Aside from invoking uneven physicalstrains, the only
plausible explanation whichcomes to mind is that the association
betweenwall and membrane may become more securewith age. The two
tips of a single cell wereformed at different times; perhaps the
more re-cently formed tip has a more weakly bonded walland
cytoplasm, whereas the older tip has amore strongly joined wall and
cytoplasm. Thus,newly partitioned protoplasts in a dividing
cellwould show weak bonding between wall andcytoplasm, permitting
the formation of a markedcentral plasmolysis vacuole.
ACKNOWLEDGMENTS
The author gratefully acknowledges theskilled technical
assistance of Dawn Coffmanand Barbara Raymond. Thanks are also
duePaul Desjardins for use of the electron micro-scope.
This work was supported in part by researchgrant E-3525 of the
National Institutes ofHealth, U.S. Public Health Service.
LITERATURE CITED
CONTI, S. F., AND M. E. GETTNER. 1962. Electronmicroscopy of
cellular division in Escherichiacoli. J. Bacteriol. 83:544-550.
COTA-ROBLES, E. H., AND P. H. DUNCAN. 1962.The effect of
D-glutamic acid upon spheroplastformation in Escherichia coli B.
Exptl. CellRes. 28:342-349.
FRASER, D., AND E. A. JERREL. 1953. The aminoacid composition of
T3 bacteriophage. J. Biol.Chem. 205:291-295.
KELLENBERGER, E., A. RYTER, AND J. SE]CHAUD.1958. Electron
microscope study of DNA-con-taining plasms. II. Vegetative and
maturephage DNA as compared with normal bacterialnucleoids in
different physiological states. J.Biophys. Biochem. Cytol.
4:671-679.
LUFT, J. H. 1961. Improvements in epoxy resinembedding methods.
J. Biophys. Biochem.Cytol. 9:409-414.
ROBINOW, C. F. 1960. Outline of the visible organi-zation of
bacteria, p. 45-108. In J. Brachet andA. E. Mirsky [ed.], The cell,
vol. 4. AcademicPress, Inc., New York.
WATSON, M. L. 1958. Staining of tissue sections forelectron
microscopy with heavy metals.II. Application of solutions
containing leadand barium. J. Biophys. Biochem. Cytol.4
:727-730.
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