• Ultrastructure of bacterial cell

• Permanent and Non-permanent

elements of bacterial cell
Bacterial Morphology and
Ultrastructure
• Only two types of cells are produced by all living
organisms on earth.
• Prokaryotes (pro. or primitive nucleus) do not have
a membrane bound nucleus
– eubacteria (true bacteria)
– archaebacteria (ancient bacteria)

• Eukaryotes (eu, or true nucleus) have a membrane

bound nucleus
– Algae
– fungi 
– protozoa
– plants
– animals
Prokaryotes
 Chemical Composition of
Bacteria
• Water - 70%
• Dry weight - 30% composed of:
– DNA - 5% MW 2,000,000,000
– RNA - 12%
– protein- 70% found in:
• Ribosomes(10,000) – RNA
• Protein particles - MW 3,000,000
• Enzymes
• Surface structures
– polysaccharides - 5%
– lipids - 6%
– phospholipids - 4%
 Prokaryote Structures:
1. Appendages- flagella, pili, fimbrae
2. Cell envelope- glycocalyx, cell wall , cell
membrane
3. Cytoplasm- ribosomes, granules,
nucleoid/chromosome.
 STRUCTURE OF BACTERIAL
CELL
ESSENTIAL STUCTURE NONESSENTIAL STUCTURE
Permanent elements: Non-permanent elements :
1) cell wall 1) capsule
2) cell membrane 2) flagella
3) ribosomes 3) fimbriae
4) cytoplasm 4) spore
5) nucleoid 5) intracytoplasmic inclusions
6) mesosomes 6) plasmides
 Cell wall
Peptidoglycan (polysaccharides +
protein),

• Support and shape of a bacterial cell.

The three primary shapes in
bacteria are:
• coccus (spherical),
• bacillus (rod-shaped)
• spirillum (spiral).
• Mycoplasma are bacteria
that have no cell wall and
therefore have no definite
shape.
 Cell wall
peptidoglycan (polysaccharides + protein)
Components of the peptidoglycan layer:
– Repeating glycan chains (N acetyl
glucosamine and N acetyl muramic acid)
– a set of identical tetrapeptide side
chains attached to N- acetylmuramic
acid
– a set of identical peptide cross bridges
 Differences in Cell Wall
Structure
• Basis of Gram Stain Reaction
– Hans Christian Gram- 1884
• Differential Stain
– Gram Positive vs Gram Negative Cells
• Gram Positive Cells-
– Thick peptidoglycan layer with embedded teichoic
acids
• Gram Negative Cells-
– Thin peptidoglycan layer, outer membrane of
lipopolysaccharide.
 Gram Stain Reaction
• Hans Christian Gram- 1880s
• Divides bacteria into 2 main groups-
– Gram positive
– Gram negative
• Also- gram variable
• Gram nonreactive
• Gram positive bacteria
– many layers of peptidoglycan and teichoic acids.
– form a crystal violet-iodine-teichoic acid complex
• Large complex, difficult to decolorize
Cell wall
 Gram Stain Reaction
• Gram negative bacteria
– Very thin peptidoglycan
– No teichoic acids
– Alcohol readily removes the crystal violet.
– Alcohol also dissolves the lipopolysaccharide of the cell wall.
• Gram variable cells
– Some cells retain crystal violet; some decolorize and take up the
safranin
– 4 factors-
• Genetics- variable amount of teichoic acid.
• Age of culture- older cultures have variable amount of teichoic acid
• Growth medium- necessary nutrients not available
• Technique-
– smear not thin or evenly made.
– Staining procedure not done correctly- decolorizer left on too long.
• Gram nonreactive cells
– Have peptidoglycan but have very waxy- thick
lipids –waterproof, dyes cannot enter either.
– Examples- Mycobacterium tuberculosis and
leprosy.
• Alternative staining- acid fast stain
 Cell wall deficient forms

• L- forms ( Lister Institute where discovered)

– Bacteria loses cell wall during the life cycle
• Result of a mutation in cell wall forming genes
• Induced by treating with lysozyme or penicillin which disrupts
the cell wall
– Protoplast-
• G + bacterium with no c. wall, only a c. membrane
• Fragile, easily lysed
– Spheroplast-
• G – bacterium loses peptidoglycan, but has outer membrane
• Less fragile but weakened.
 Surface structures
continued:
• Outer membrane
– This lipid bilayer is found in Gram negative
bacteria and is the source of
lipopolysaccharide (LPS) in these bacteria
– LPS is toxic and turns on the immune
system.
– Not found in Gram positive bacteria.
Lipopolysaccharide
 C. Cell membrane
• Located just under cell wall
• Very thin
• Lipid bilayer, similar to the plasma membrane of
other cells. Transport of ions, nutrients and
waste across the membrane
• Typical
– 30-40% phospholipids
– 60-70% proteins
• Exceptions-
– Mycoplasma- sterols
– Archaea- unique branched hydrocarbons
 Functions of Cell Membrane
• Carries out functions normally carried out by eukaryote
organelles.
• Site for energy functions
• Nutrient processing
• Synthesis
• Transport of nutrients and waste
• Selectively permeable
• Most enzymes of respiration and ATP synthesis
• Enzyme synthesis of structural macromolecules
– Cell envelope and appendages
• Secretion of toxins and enzymes into environment.
 Mesosome
Extension of cell membrane
– Folding into cytoplasm – internal pouch
– Increases surface area.
• Gram-positive bacteria-prominent
• Gram negative bacteria- smaller, harder to see.
• Functions-
– Cell wall synthesis
– Guides duplicated
chromosomes into
the daughter cells
in cell division.
 3. Cell cytoplasm
• Encased by cell membrane
• Dense, gelatinous
• Prominent site for biochemical and
synthetic activities
• 70-80% water- solvent
• Mixture of nutrients- sugar, amino acids,
salts
– Building blacks for cell synthesis and energy
A. Bacterial chromosome
• Singular circular strand of DNA
• Aggregated in a dense area- nucleiod
• Long molecule of DNA tightly coiled
around protein molecules.
• The nucleoid can be clearly visualized on
an electron micrograph at high magnification,
where, although its appearance may differ, it is
clearly visible against the cytosol. Sometimes
even strands of what is thought to be DNA are
visible. By staining with the Feulgen stain,
which specifically stains DNA, the nucleoid can
also be seen under a light microscope.
 B. Plasmids
– Nonessential pieces of DNA
• Often confer protection- resistance to drugs
– Tiny, circular
– Free or integrated
– Duplicate and are passed on to offspring
– Used in genetic engineering
 Types of plasmid
• Fertility-F-plasmids. They are capable of conjugation (transfer of
genetic material between bacteria which are touching).

• Resistance-(R)plasmids, which contain genes that can build a

resistance against antibiotics or poisons and help bacteria produce
pili.

• Col-plasmids, which contain genes that determine the production

of bacteriocins, proteins that can kill other bacteria.

• Degradative plasmids, which enable the digestion of unusual

substances, e.g., toluene or salicylic acid.

• Virulence plasmids, which turn the bacterium into a pathogen

(one that causes disease).
 C. Ribosomes
• Site of protein synthesis
• Thousands
– Occurs in chains –polysomes
• 70S
– 2 smaller subunits
– 30S and 50S
NONESSENTIAL STUCTURE
Non-permanent elements :

1) fimbriae
2) flagella
3) capsule
4) spore
5) intracytoplasmic inclusions
6) Plasmides
 Bacterial Appendages:
• Pili (pl), pilus (s)
– only found in gram negative bacteria
– tubulare, hairlike structures of protein larger
and more rare than fimbriae.
• 2 types of pili
- atacnement pilus - allow bacteria to attach to
other cells
- sex pilus,
pilus - transfer from one bacterial cell to
another- conjugation.
 Fimbriae
• fimbriae (pl) fimbria (s)
– Adhesion to cells and surfaces
– Responsible for biofilms.
– Pathogenesis of gonococcus and E.coli

Escherichia coli.
At the end of each fimbria are special proteins called
adhesins.
The specific type of adhesin varies by type of bacteria, but
regardless of the type, adhesin molecules allow bacteria
with fimbriae to adhere to host cells by docking, like a
lock and key, with receptor proteins on the surface of
host epithelial cells.
This ability of fimbriae to stick to epithilial cells leads to
many diseases transmitted via mucous membranes,
including gonorrhoeae, bacterial meningitis and
infections of internal medical devices and indwelling
catheters. 

 
 Flagella
– long appendages which rotate by means of a "motor" located just under the cytoplasmic membrane.
– bacteria may have one, a few, or many flagella in different positions on the cell.
•Advantages
- chemotaxis –
positive and negative.
- motility
•All spirilla, half of bacilli,
•rare cocci.
 Flagella
Three morphological regions
• Helical filament
– long outermost region; composes up to 90% of its length
– contains the globular (roughly spherical) protein flagellin
arranged in several chains and form a helix around a hollow core

• Hooked or curved area

– filament is attached; consists of a different protein

• Basal body
– terminal portion of the flagellum 
– fix the flagellum to the cell wall and plasma membrane
– composed of a central rod inserted into a series of rings
 
Gram negative - 2 pairs of rings
• Outer pair - fixed to the outer membrane and peptidoglycan layer
• Inner pair - fixed to the plasma membrane (SM ring)
Gram positive - only inner pair is present
 Motility
• Types of bacterial motility
– run or swim - when a bacterium moves in one direction
for a length of time
– tumbles - periodic, abrupt random changes in direction
– swarming - rapid wavelike growth across a solid culture
medium

• Mechanism of flagellar movement - relative

rotation of the rings in the basal body of the
flagellum

Antigenicity
– flagellar or H antigen - useful in the serological
identification of serotypes of Salmonella organisms
 Arrangements
• Flagella vary in number and arrangement.
• Polar arrangment
– Monotrichious - 1 flagellum at one end
• Fastest; Pseudomonas -example
– Lophotrichious - tuft at one end
– Amphitrichious- bipolar
– Peritrichious - multiple flagella; randomly
dispersed around the bacterial cell
• E. coli - example
 Axial filaments

• tuft of fibrils that arise at the ends

of the cell under the outer
membrane and spiral around the
cell

• rotation an opposing of the outer

membrane movement that propels
the spirochetes by causing them to
move like corkscrews

• Found in Spirochetes and are

similar to flagella, but are located
between the cell wall and an
outer membrane, and are
attached to one end of the
organism.
 Evidence of motility
Two ways by which motility can be demonstrated:
DIRECT - staining
• Indirect
– hanging drop preparation or wet mount preparation by dark field
mycroscope
– Distinguishes:
• Brownian movement - when the bacteria show molecular movement
• true motility - if a bacterium describes a rotatory, undulatory or
sinuous movement

• indirect
– Stab inoculation of the semisolid media
• nonmotile - growth is limited at the point of inoculation
• motile - growth is diffuse or moves away from the line of inoculation;
turbidity of the medium
Detection of Motility
Indirect
• DIRECT METHOD (to deter. Flagella)
• Purpose:  To determine the
presence/absence and location of flagella
on various microorganisms
• Principle:  Because bacterial flagella are
very thin and fragile a special stain
(flagella stain) is prepared that contains a
mordant.  This mordant allows piling of the
stain on the flagella, increasing the
thickness until they become visible. 
Various arrangements of flagella are seen
on different cells.
2. Bacterial Surface Structure
- cell envelope
A. Glycocalyx - some extracellular material
secreted by many bacterial cells in the form of:
– capsule - attached tightly to the bacterium and
has definite boundaries.
– slime layer - loosely associated with the bacterium
and can be easily washed off
Compositions:
- layer of polysaccharide
- proteins - sometimes
 Capsule
Functions Medical Importance

Rapid serological identification

• Protection
of:
• Identification
• Several groups of streptococci
• Vaccine preparation
• Meningococcus
• Tissue attachment
• Hemophilus influenzae
• Antibiotic barrier
• Klebsiella pneumoniae
• Some of the coliforms
 
• Yersinia and Bacillus specie
 Identification
Two simple methods to distinguish the capsule
India ink technique - most satisfactory method of
demonstrating the capsule by Burri-Gins technique

– Bacteria is suspended in diluted India ink

– Stain with fuxin
– Bacterial cells appear to lie in a lacunae
and red cytoplasme.
 
Quellung reaction - Homologous antibody is added to a
preparation of capsule.
– microprecipitation at the periphery of the capsule altering
its refractive index rendering the capsule to be visible
• Capsule Stain
• 1.Place a single drop of India ink on a clean
microscope slide, adjacent to the frosted adge.
• 2. Using a flamed loop and sterile technique, remove
some K. pneumoniae (or the organism you
• want to stain) from your tube or plate and mix it into
the drop of India ink. Be sure there are no
• large clumps of organism, but try to avoid spreading
the drop.
• 3. Place the end of another clean microscope slide at
an angle to the end of the slide containing the
• organism. Spread out the drop out into a film. This is
done by contacting the drop of India ink
• with the clean microscope slide and using the capillary
action of the dye/ slide to spread the
• India ink across the smear. Refer to the Negative
Stain portion of this handout for a diagram.
• 4. Allow the film to air dry. DO NOT heat or blot dry!!!!
Heat will melt the capsule!
• 5. Saturate the slide with crystal violet for 1 minute.
• 6. Rinse the slide gently with water.
• 7. Allow the slide to air dry. DO NOT heat or blot dry!!!!
Heat will melt the capsule!
• 8. Observe the slide under the microscope, using
proper microscope technique.
• The background will be dark.
• The bacterial cells will be stained purple.
• The capsule (if present) will appear clear
against the dark background.
Staining by Burri-Gins
 Neisseria meningitidis - Gram-
negative coccus, non-motile bacteria occur
as two cells (orange) in a capsule (yellow)
Haemophilus influenza bacteria in the
process of expressing polysaccharide capsules.
 D. Inclusions
• If nutrients abundant- stored intracellularly
• Granules
– Crystals of inorganic compounds not enclosed
by membranes
• Polyphosphate- corynebacterium
• Sulfur granules- photosynthetic
• Metachromatic- Mycobacterium
 • Principle of Albert Staining:
• Albert stain is basically made up of two
stains that is Toluidine blue’ O’ and Malachite
green both of which are basic dyes with high
affinity for acidic tissue components like
cytoplasm. The pH of Albert stain is adjusted
to 2.8 by using acetic acid which becomes
basic for volutine granules as pH of volutine
Granule is highly acidic.
Albert Staining
• Albert’s A solution consist of
• Toludine blue                      0.15 gm
• Malachite green                  0.20 gm
• Glacial acetic acid               1 ml
• Alcohol (95% ethanol)       2ml
• Albert’s B solution consist of
• Iodine                                    2gm
• Potassium iodide (KI)          3 gm
• The main procedure to identify
Corynebacterium diphtheriae is microscopic
observation of pathologic material taken with a
pharyngeal plug. In the same time it's carried
out a cultural test to
show methaphosphate methacromaticpartic
les contained in Corynebacterium and red
coloured if painted with the Methylene blue
• Staining of volutin granules with alkaline
methylene blue (by Loeffler's technique). 
• On a fixed smear pour alkaline methylene
blue to act for 3-5 min» wash with water, dry
with filter paper, and examine under the mi­
croscope. The cytoplasm of diphtheria
corynebacteria is stained light-blue, while
granules of volutin are dark-blue.

Volutin’s granules, Loeffler’s technique

• Neisser's staining. Staining of volutin granules by
this method in­cludes the following stages.
• 1. A fixed smear is stained with acetic-acidic
methylene blue for 1 min, then the dye is poured off,
and smear is washed "with water.
• 2. Pour in Lugol’s solution to act for 20-30 s.
• 3. Without washing with water, stain the preparation
with vesuvin for 1-3 min, then wash it with water and
dry.
•  
Volutin’s granules, Neisser’s technique
 Bacterial Internal
Structures
• Endospores
– inert, resting, cells produced by some G+ genera:
Clostridium, Bacillus and Sporosarcina
• have a 2-phase life cycle:
– vegetative cell – metabolically active and growing
– endospore – when exposed to adverse environmental conditions;
capable of high resistance and very long-term survival
» Features of spores- size, shape, location=identification
– sporulation -formation of endospores
• hardiest of all life forms
• Forms inside a cell- functions in survival
• not a means of reproduction
• withstands extremes in heat, drying, freezing, radiation and
chemicals
– germination- return to vegetative growth
 Endospores
• Resistance linked to high levels of calcium
and dipicolinic acid
• Dehydrated, metabolically inactive thick coat
• Longevity verges on immortality - 25,250
million years.
• Resistant to ordinary cleaning methods and
boiling
• Pressurized steam at 120oC for 20-30
minutes will destroy
Thank you for your attention