Ultrastructure of

bacterial cell.

Form and Function.

10.02.16
Structure of a Prokaryotic 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 :
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
Peptidoglycan
Cell wall
 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
Gram positive bacteria
Gram negative bacteria
 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 stain technique
Gram stain
Gram stain
 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
Acid-fast
 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.
 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).
Cell division in Prokaryotes
Prokaryotes use a
relatively simple form of
cell division - binary
fission.
The diagram at 1.shows a
bacterial cell.
The cell wall and membrane
are in red,
the bacterial chromosome in
blue,
the cytoplasm in light green,
the yellow dot represents a
point of attachment of the
chromosome to the cell
membrane.
 C. Ribosomes
Site of protein synthesis
Thousands
Occurs in chains polysomes
70S
2 smaller subunits
30S and 50S
 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, - 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.
 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
Flagella (pl), flagellum(s)
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.

 Structure of flagella
allows for 360 degree filament rotation
 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
 Flagellar arrangements
A. Monotrichous
B. Lophotrichous
C. Amphitrichous
D. Peritrichous
E. Atrichous
Axial filaments
 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

Presence mobile bacteria

 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:
a. capsule - attached tightly to the bacterium and has
definite boundaries.
b. slime layer - loosely associated with the bacterium
and can be easily washed off

Compositions:
- layer of polysaccharide
- proteins - sometimes
 Functions of the Capsule

Protection
Identification
Vaccine preparation
Tissue attachment
Antibiotic barrier
 Medical Importance -
rapid serological identification of:
Several groups of streptococci
Meningococcus
Hemophilus influenzae
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
 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 microscope. The cytoplasm of diphtheria
corynebacteria is stained light-blue, while
granules of volutin are dark-blue.

Volutins granules, Loefflers 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 Lugols 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.

Volutins granules, Neissers 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