1. Bacterial Cytology
Vishrut S. Ghare

2. Structure, chemical composition and functions of the
following components in bacterial cell:
1. Cell wall (Gram positive, Gram negative)
Concept of
Mycoplasma,
Spheroplast,
Protoplast,
L-form
2. Cell membrane

3. The cell wall of the bacterial cell is a complex, semi rigid structure
responsible for the shape of the cell.
Almost all prokaryotes have a cell wall that surrounds the underlying,
fragile plasma (cytoplasmic) membrane and protects it and the interior of
the cell from adverse changes in the outside environment.
Eubateria are classified into 3 groups depending upon the presence or
absence of cell wall and according to the type present:
i. Gram positive eubacteria
ii. Gram negative eubacteria
iii. Mycoplasma (Cell wall lacking group)
Composition and Characteristics:
The cell wall differs in thickness as well as in composition
The bacterial cell wall is composed of a macromolecular network
called peptidoglycan (also known as murein), which is present either
alone or in combination with other substances.

4. Principal component ofcell wall is Peptidoglycan which consists of a
repeating disaccharide connected by polypeptides to form a lattice that
surrounds and protects the entire cell.
The disaccharide portion is made up of monosaccharides called N-
acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) (from murus,
meaning wall), which are related to glucose.
A tetrapeptide made up of amino acids in which four amino acids
attached to NAMs in the backbone.
Parallel tetrapeptide side chains may be directly bonded to each other
or linked by a peptide cross-bridge, consisting of a short chain of amino
acids.
The composition of peptidoglycan changes from species to species.
The difference in cell wall composition lead to different response to
Gram stains and thus two classes are there as Gram positive and Gram
negative.
Cell wall of Gram negative bacteria are generally thinner (10 to 15 nm)
as compared to Gram positive bacteria (20 to 25 nm).

6. The cell walls of Bacillus subtilis and many other typical Gram-
positive bacteria consist of a single, 20- to 80-nm-thick homogeneous
layer of peptidoglycan (murein) lying outside the plasma membrane.
In contrast, the cell walls of E. coli and many other typical Gram-
negative bacteria have two distinct layers: a 2- to 7-nm-thick
peptidoglycan layer covered by a 7- to 8-nm-thick outer membrane.

7. Gram-Positive Cell Walls
In most Gram-positive bacteria, the cell wall consists of many layers of
peptidoglycan, forming a thick, rigid structure.
By contrast, Gram-negative cell walls contain only a thin layer of
peptidoglycan.
The space between the cell wall and plasma membrane of Gram-
positive bacteria is the periplasmic space and contains periplasm.
It contains the granular layer, which is composed of lipoteichoic acid.
In addition, the cell walls of Gram-positive bacteria contain teichoic
acids, which consist primarily of an alcohol (such as glycerol or ribitol)
and phosphate.
There are two classes of teichoic acids: lipoteichoic acid, which spans
the peptidoglycan layer and is linked to the plasma membrane, and wall
teichoic acid, which is linked to the peptidoglycan layer.

8. Because of their negative charge (from the phosphate groups),
teichoic acids may bind and regulate the movement of cations (positive
ions) into and out of the cell.
They protect the cell from harmful substances in the environment (e.g.,
antibiotics and host defense molecules).
They may also assume a role in cell growth, preventing extensive wall
breakdown and possible cell lysis, acts as receptor for many viruses.
It constitute about 50% of cell’s dry weight, and acts as major cell
surface antigen which is useful for grouping Gram positive cells.

11. Gram-Negative Cell Walls
Gram-negative cell walls are more complex than typical Gram-positive
walls.
One of the most striking differences is the paucity of peptidoglycan.
The peptidoglycan layer is very thin (2 to 7 nm, depending on the
bacterium) and sits within the periplasmic space.
The peptidoglycan is bonded to lipoproteins in the outer membrane
and is in the periplasm, a gel-like fluid in the periplasmic space of Gram
negative bacteria, the region between the outer membrane and the
plasma membrane.
The periplasm contains a high concentration of degradative enzymes
and transport proteins.
The periplasmic space is much larger than that of a typical Gram-
positive cell, ranging from about 30 to 70 nm wide.

12. Some bacteria have electron transport proteins in their periplasm (e.g.,
denitrifying bacteria, which convert nitrate to nitrogen gas).
Other periplasmic proteins are involved in peptidoglycan synthesis and
modification of toxic compounds that could harm the cell.
Gram-negative cell walls do not contain teichoic acids.
Because the cell walls of Gram-negative bacteria contain only a small
amount of peptidoglycan, they are more susceptible to mechanical
breakage.
The outer membrane lies outside the thin peptidoglycan layer.
It is linked to the cell by Braun’s lipoprotein, the most abundant protein
in the outer membrane.
This small lipoprotein is covalently joined to both the outer membrane
and the peptide chain of peptidoglycan.
The outer membrane of the gram-negative cell consists of
lipopolysaccharides (LPS), lipoproteins, and phospholipids

13. The outer membrane has several specialized functions.
Its strong negative charge is an important factor in evading
phagocytosis and the actions of complement (lyses cells and promotes
phagocytosis), two components of the defenses of the host.
The outer membrane also provides a barrier to detergents, heavy
metals, bile salts, certain dyes, antibiotics (for example, penicillin), and
digestive enzymes such as lysozyme.
permeability of the outer membrane is due to proteins in the
membrane, called porins, that form channels.
Porins permit the passage of molecules such as nucleotides,
disaccharides, peptides, amino acids, vitamin B12, and iron.
The external layer of the Gram-negative cell is comprised of
lipopolysaccharides (LPSs).
These large, complex molecules contain both lipid and carbohydrate,
and consist of three parts: (1) lipid A, (2) the core polysaccharide, and
(3) the O side chain.

14. Lipid A is the lipid portion of the LPS and is embedded in the top layer
of the outer membrane.
When Gram-negative bacteria die, they release lipid A, which functions
as an endotoxin.
Lipid A is responsible for the symptoms associated with infections by
gram-negative bacteria, such as fever, dilation of blood vessels, shock,
and blood clotting.
The core polysaccharide is attached to lipid A and contains unusual
sugars. Its role is structural—to provide stability.
The O polysaccharide extends outward from the core polysaccharide
and is composed of sugar molecules.
The O polysaccharide functions as an antigen and is useful for
distinguishing species of Gram-negative bacteria.
The O antigen consist of repeating units of carbohydrates arranged in
variety of combinations like glucose, galactose, mannose, rhamnose.

17. •Archaea may lack walls or may have unusual walls composed of
polysaccharides and proteins but not peptidoglycan.
•These walls do, however, contain a substance similar to peptidoglycan
called pseudomurein.
•Pseudomurein contains N-acetyltalosaminuronic acid instead of NAM
and lacks the d-amino acids found in bacterial cell walls.
•Archaea generally cannot be Gram-stained but appear Gram-negative
because they do not contain peptidoglycan.
Archae Cell wall

19. Functions of Cell wall
a) The main function of the bacterial cell wall is to provide overall strength
to the cell.
b) It helps maintain the cell shape, thereby helping the cell to grow,
reproduce, obtain nutrients and also move about.
c) Cell wall protects the cell from the osmotic lysis. The cell keeps moving
from one environment to other and moreover as water can freely move
from both the cell membrane and the cell wall, the cell is at risk of
osmotic imbalance thereby causing osmotic lysis of the cell.
d) The cell wall helps in keeping out certain molecules which may be toxic.
e) Bacterial cell wall contributes to the pathogenicity in other words,
disease causing ability of bacterial cells.
f) The O antigen of LPS determines the antigenic specificity of Gram
negative cell wall
g) The proteins found in cell wall acts as receptor sites for bacteriophages,
helps in cell division

20. Mycoplasma
Mycoplasma are pleomorphic smallest living organism, also known as
Pleuropneumonia like organism (PPLO).
These are the smallest free-livingorganisms, which lacks cell wall,
has no fixed shape or size.
Belongs to family Mycoplasmataceae.
Habitat:
Ubiquitous in nature
Plants, Insects and other animals serves as reservoir
Mycoplasma resides in mucosa of upper respiratory tract (URT)
and urogenital tract of human.
They are also found in oral cavity.
Medically important species are: Mycoplasma pneumoniae, M. hominis,
M. genitalium

21. Morphology:
Size: 0.2µm in diameter.
Shape varies, they may be coccoid or filamentous depending upon
species and growth conditions.
They lack cell wall so they are pleomorphic and do not stain with
conventional bacteriological stains, Giemsa stain is used, generally
Gram negative.
All members of mycoplasma contains cholesterol containing cell
membrane.
Electron microscopy revels M. pneumoniae and other some
pathogenic mycoplasma contains terminal structure bulbous
enlargement with a differentiated tip structure which helps in
attachment.
Mycoplasma is bridge between bacteria and virus. They differ
from virus is that they contains both DNA and RNA and can
reproduce in cell free media.
Genome is 580Kbp, cell divides by binary fission.

23. Cells do not possess fimbriae or flagella, spores are not produced.
Some species exhibit gliding motility
Cultural and biochemical characteristics:
Mycoplasma spp are facultative anaerobes but M. pneumoniae is
strict aerobe.
Optimum temperature requirement for growth is 35-37°.
They are fastidious and grow slowly in culture media.
All mycoplasma except Acholeplasma requires cholesterol or sterol
and Nucleic acid precursors for growth.
They grow on enrichment media with 20% human or horse serum.
Human or horse serum provides cholesterol and fatty acids which
is required for synthesis of cell membrane since these bacteria are
unable to synthesize the component of cell membrane by
themselves.

24. They gives small size Fried egg colony on semi solid enriched
media containing 20% horse serum, yeast extract and DNA after 7-
12 days of incubation in CO2 incubator.
Colony is typically biphasic with a fried egg appearance, consisting
of central opaque granular area of growth extending into the depth
of the medium, surrounded by a flat, translucent peripheral zone.
The growth slow in agar media than in broth culture
Surface glycoprotein of M. pneumoniae cross reacts with I-antigen
of human RBC at 4°
Cell wall inhibiting drugs such as Penicillin and Cephalosporin are
ineffective drugs because they lacks cell wall.
M. pneumoniae, M. genitalium, M. fermentans and M. penetrans ferment
glucose while M. hominis is glucose non fermenter.
M. hominis hydrolyses arginine
Mycoplasma pneumoniae is able to reduce 2,3,5-triphenyl-tetrazolium
chloride

28. Spheroplast
Spheroplast is a microbial cell from which the cell wall has
been almost completely removed by the action of lysozyme(Cell
wall degrading enzyme).
When lysozyme/penicillin acts on Gram negative bacterium it
breaks glycosidic linkage between NAG and NAM of
peptidoglycan and thus disturbs it.
Spheroplast are spherical in structure, lacking only
peptidoglycan layer.
It refers to the spherical shape assumed by the Gram negative
bacteria, in which after removal of cell wall it now possesses
two membranes ; cytoplasmic membrane and outer membrane.

29. Protoplast
•The cellular contents that remain surrounded by the plasma
membrane without the cell wall is called as protoplast.
•Protoplast is spherical in shape and still able to carry out
metabolism.
•The term protoplast refers to the spherical shape assumed by
Gram positive bacteria.
L-form
•Kleinberger in 1935 found pleuropneumonia like-forms in the
culture of Streptobacillus moniliformis and called them as L-form.
•This was first discovered in Lister institute, London.
•These are also known as cell wall deficient bacteria (CWDB), L-
phase or L-form.

30. •It was shown that many bacteria, either spontaneously or induced
by certain substances like penicillin (which inhibits cell wall
formation), lost part or all of their cell wall and develop L-forms.
•L forms can live and divide repeatedly or return to the walled
state.
•Some L-forms are stable while some are unstable.
•Unstable forms can be revert back to cell wall containing state
when inducing stimulus is removed.
•Stable forms do not revert back to normal state .

31. Cell membrane / Plasma membrane
The term 'Plasma membrane' was given by Pfeffer.
The term 'Plasma lemma' was given by Plower (1931).
Nageli and Cramer (1855) used the term 'Cell membrane'.
The plasma (cytoplasmic) membrane (or inner membrane) is a thin
structure lying inside the cell wall and enclosing the cytoplasm of the cell.
The plasma membrane of prokaryotes consists primarily of
phospholipids, which are the most abundant chemicals in the membrane,
and proteins.
Eukaryotic plasma membranes also contain carbohydrates and sterols,
such as cholesterol.
Because they lack sterols, prokaryotic plasma membranes are less
rigid than eukaryotic membranes.
One exception is the wall-less prokaryote Mycoplasma, which contains
membrane sterols.

32. A primary role of all plasma membranes is that they are selectively
permeable barriers: They allow particular ions and molecules to pass
either into or out of the cell, while preventing the movement of others.
Thus the plasma membrane prevents the loss of essential
components through leakage while allowing the movement of other
molecules.
Bacterial plasma membranes play additional critical roles.
They are the location of several crucial metabolic processes:
respiration, photosynthesis, and the synthesis of lipids and cell wall
constituents.

34. Fluid Mosaic Model of Membrane Structure
The most widely accepted model for membrane structure is the fluid
mosaic model of Singer and Nicholson, which proposes that
membranes are lipid bilayers within which proteins float.
Bacterial membranes have roughly equal amounts of lipids and
proteins. Cell membranes are very thin structures, about 2 to 3 nm
thick.
In electron micrographs, prokaryotic and eukaryotic plasma
membranes (and the outer membranes of Gram-negative bacteria) look
like two-layered structures; there are two dark lines with a light space
between the lines.
The phospholipid molecules are arranged in two parallel rows, called a
lipid bilayer.
Most membrane-associated lipids are amphipathic: They are
structurally asymmetric, with polar and nonpolar ends.

35. The polar ends interact with water and are hydrophilic; the nonpolar
hydrophobic ends are insoluble in water and tend to associate with
one another.
Most integral proteins penetrate the membrane completely and are
called transmembrane proteins.
Some integral proteins are channels that have a pore, or hole, through
which substances enter and exit the cell.
Many of the proteins and some of the lipids on the outer surface of the
plasma membrane have carbohydrates attached to them.
Proteins attached to carbohydrates are called glycoproteins, and lipids
attached to carbohydrates are called glycolipids.
Both glycoproteins and glycolipids help protect and lubricate the cell
and are involved in cell-to-cell interactions.
glycoproteins play a role in certain infectious diseases.
Studies have demonstrated that the phospholipid and protein molecules
in membranes are not static but move quite freely within the membrane
surface.

36. This dynamic arrangement of phospholipids and proteins is referred to as
the fluid mosaic model.
Functions of Cell membrane:
1) plasma membranes have selective permeability (sometimes called
semi-permeability) This term indicates that certain molecules and
ions are allowed to pass through the membrane but others are
stopped.
2) Plasma membranes are also important to the breakdown of nutrients
and the production of energy.
3) The plasma membranes of bacteria contain enzymes capable of
catalyzing the chemical reactions that break down nutrients and
produce ATP.
4) In some bacteria, pigments and enzymes involved in photosynthesis
are found in infoldings of the plasma membrane that extend into the
cytoplasm. These membranous structures are called
chromatophores.
5) Plasma membrane anchors the DNA during replication.