Micro Lecture 2

Cell Structure
Classification and Identification
Methods
Growth, Survival and Death of
Microorganism
 Characteristics of prokaryotic and eukaryotic
cells
Characteristic Prokaryotic Eukaryotic Human
Bacterial Cells Cells
DNA within a No Yes
nuclear membrane
Mitotic division No Yes
DNA associated No Yes
with histones
Chromosome One More than one
number
 Characteristics of prokaryotic and eukaryotic
cells
Characteristic Prokaryotic Eukaryotic Human
Bacterial Cells Cells
Membrane-bound No Yes
organelles such as
mitochondria and
lysosomes
Size of ribosomes 70S 80S
Cell wall containing Yes No
peptidoglycan
Structure of Bacterial Cells

Classification by shape
Cocci – sphericals (lancet-shaped, kidney shaped)
Bacilli - rods
Spirilla - spiral

Some bacteria are variable in shape and are said to be

pleomorphic (many shaped). The shape is
determined by its rigid cell wall.
 Size
0.20 to 2.0 um in diameter
2 to 8 um in length
micrometers

Basic shapes
Cocci (Coccus)
Bacilli (Bacillus)
Spirilla (Spirillum)
Structure of Bacterial Cells

The microscopic appearance of a bacterium is one of

the most important criteria used in its identification.

The arrangements of bacteria are determined by the

orientation and degree of attachment of the bacteria
at the time of cell division.
 Cocci –spherical (berries)

Usually round but can be oval, elongated,

flattened on one side
When cocci divide to reproduce, the cells
remain attached to one another
Multiple planes & form grapelike
clusters on broad sheets
staphylococci

Two planes & remain in

groups of four - tetrads
Three planes & remain attached in
cubelike groups of eight
sarcinae

Those that remain in pairs after

dividing - diplococci

Those that divide & remain

attached in chainlike patterns
streptococci
 Bacilli – rod shape (little staffs)

Bacilli divide only across their short axis, so there

are fewer groupings of bacilli than cocci

Single rods In pairs -diplobacilli

In chains - streptobacilli
 Some bacilli look like straw, others have square ends or
rounded ends or tapered ends like cigars & termed fusiform
or they may align themselves side by side (palisading)

Others are oval & look so much

like cocci - coccobacilli
Spirilla – Spiral shape

Spiral bacterial have one or

more twists

Look like curved rods (vibrios)

Spirilla – helical shape, like a

corkscrew & fairly rigid bodies

Spirochetes – helical & flexible

 Star-shaped cells (genus Stella)
Square & flat cells (genus Haloarcula)
Triangular cells

Shape – determined by heredity

Genetically, most bacteria are monomorphic

Number of environmental conditions can alter the shape

Pleomorphic – they can have many shapes

Ex. Rhizobium & Corynebacterium
Bacterial Cytology
1. Cell wall
rigid structure that maintains the shape of the cell
prevents bursting of the cell (high osmotic pressure)

Gram positive cell wall

Peptidoglycan (antibiotics, lysozyme)

Glycan chains – NAG & NAM

Teichoic acid ( peptidoglycan)
Lipoteichoic acid (plasma membrane)
antigenic
Gram negative cell wall
Inner peptidoglycan – thinner than the
Gram positive cell walls
Outer membrane:
proteins, lipids & LPS
LPS – antigenic O-specific polysaccharide
core polysaccharide
inner lipid A ( endotoxin) – fever & shock
conditions (esp. hypotension)
Functions:
barrier to hydrophobic compounds
acts as a sieve (through porins)
attachment sites
Periplasmic space
gel-like matrix
nutrient –binding proteins
degradative & detoxifying enzymes
 Physical
Chemical
Gram positive Gram negative
L-forms
Media supplemented w/ serum or sugar
to prevent osmotic rupture
Capable of reverting to normal form
(residual peptidoglycan)
Protoplast Spheroplast

Major portion of the cell wall

material will remain
 Acid Fast cell wall

Contains a small amount of peptidoglycan

Waxy layer & fatty acid (Mycolic acid)

Absence of Cell Wall

Mycoplasma & Ureaplasma

Lack cell wall but they have
sterols
 Comparison of cell walls of Gram positive and
Gram negative bacteria

Component Gram positive Gram negative

cells cells
Peptidoglycan Thicker; multilayer Thinner; single layer
Teichoic acids Yes No
Lipopolysaccharide No Yes
(endotoxin)
 Gram positive Gram negative

Gram reaction Purple color Red color

Peptidoglycan layer thick thin

Teichoic acid present absent

LPS content absent present

Periplasmic space absent present

Outer membrane absent present

Lipid & lipoprotein low high

content
Flagellar structure 2 rings in basal 4 rings in
body basal body
Toxins produced exotoxin endotoxin
Resistance to physical high low
disruption
Cell wall disruption by high low
lysozyme
Susceptibility to high low
penicillin &
sulfonamide
Susceptibility to low high
streptomycin,
chlorampenicol &
tetracycline
Inhibition by basic dyes high low
Susceptibility to high low
anionic detergents
Resistance to sodium high low
azide
Resistance to drying high low
Structure of Bacterial cell

Cytoplasmic membrane
1. Composed of phospholipid bilayer..

2. Four important functions

a. active transport of molecules into the cell
b. energy generation by oxidative
phosphorylation
c. synthesis of the precursors of the cell wall
d. secretion of enzymes and toxins
Structure of Bacterial cell

Mesosome
1. This invagination of the cytoplasmic membrane is
important during cell division when it functions as
the origin of the transverse septum that divides the
cell in half.

2. Binding site of the DNA which will become the

genetic material of each daughter cell.
Structure of Bacterial cell

Cytoplasm
Has two distinct areas:
1. An amorphous matrix that contains ribosomes,
nutrient granules, metabolites and plasmids.

2. An inner nucleoid region composed of DNA.

Structure of Bacterial cell

Nucleoid
1. The area in the cytoplasm in which DNA is located.

2. The DNA of prokaryotes is a single, circular

molecule, containing 2000 genes.

3. No nuclear membrane, no nucleolus, no mitotic

apparatus, no histones, no introns.
Structure of Bacterial cell

Ribosomes
1. Site of protein synthesis as in eukaryotic cells, but
they differ in size and chemical composition.

2. Bacterial ribosomes are 70S in size, with 50S and

30S subunits.

3. The differences in both the ribosomal RNAs and

proteins constitute the basis of the selective action
of several antibiotics.
Structure of Bacterial cell

Granules
1. Serve as storage areas for nutrients and stain
characteristically with certain dyes.

2. For example, volutin is a reserve of high energy

stored in the form of polymerized metaphosphate. It
appears as a “metachromatic”
granule since it stains red with
methylene blue instead of blue.
Structure of Bacterial cell

Plasmids
1. Extrachromosomal, double-stranded, circular DNA
molecules that are capable of replicating
independently of the bacterial chromosome.

2. They can be integrated into the bacterial

chromosome.
Structure of Bacterial cell

a. Transmissible plasmids can be transferred from cell to

cell by conjugation. They are large, containing a dozen
genes responsible for the synthesis of the sex pilus,
antibiotic resistance and for the enzymes required for
transfer.

b. Nontransmissible plasmids are small, since they do not

contain the transfer genes.

c. Occurs in both Gram positive & Gram negative bacteria

Structure of Bacterial cell

Transposon
1. Pieces of DNA that move readily from one site to
another, either within or between the DNAs of bacteria,
plasmids and bacteriophages. In view of their unusual
ability to move, they are nicknamed “jumping genes.”

2. They can code for drug resistance enzymes, toxins or

a variety of metabolic enzymes and they can either
cause mutations in the gene into which they insert or
alter the expression of nearby genes.
Specialized Structures of
Bacterial cell
A. Capsule
1. Gelatinous layer covering the entire bacterium,
composed of a polysaccharide except in anthrax
bacillus, which has a capsule of polymerized D-
glutamic acid.

2. The sugar component of the polysaccharide vary

from one species of bacteria to another and
frequently determine the serologic type within a
species.
Specialized Structures of
Bacterial cell
3. A determinant of virulence of
many bacteria, since it limits the ability of
phagocytes to engulf the bacteria. Variants of
encapsulated bacteria that have lost the ability to
produce a capsule are usually nonpathogenic.

4. Specific identification of an organism can be

made using antiserum against the capsular
polysaccharide. In the presence of homologous
antibody, the capsule will swell greatly.
Specialized Structures of
Bacterial cell
5. Capsular polysaccharides are
used as the antigens in certain vaccines since they
are capable of eliciting protective antibodies.

6. Play a role in the adherence of bacteria to human

tissues, an important initial step in causing
infection.
Specialized Structures of
Bacterial cell
B. Glycocalyx
1. A polysaccharide coating that is secreted by many
bacteria.

2. Covers surfaces like a film and allows the bacteria

to adhere firmly to various structures, e.g. skin,
heart valves and catheters.
Specialized Structures of
Bacterial cell
3. Also mediates adherence of certain bacteria such
as S. mutans to the surface of the teeth

4. Plays an important role in the formation of

plaque, the precursor of dental caries.
Specialized Structures of
Bacterial cell
C. Flagella
1. Long, whiplike appendages that move the
bacteria toward nutrients and other attractants. A
process called chemotaxis.

2. Acts like a propeller, composed of many subunits

of a single protein, flagellin arranged in several
intertwined chains.
Specialized Structures of
Bacterial cell
3. Bacterial species vary in their possession of
flagella from none (atrichous) to many flagella that
extend from one end of the bacterium (polar)

4. Flagellated bacteria have a characteristic number

and location. They may be monotrichous (single),
ampitrichous (one flagellum at both ends),
lophotrichous (tuft of flagella at one end) and
peritrichous (around the cell).
monotrichous lophotrichous
Specialized Structures of
Bacterial cell
5. Only certain bacteria have flagella, many rods do
but most cocci do not have flagella and therefore
nonmotile.

6. Spirochetes move by using a flagellum like

structure called the axial filament, which wraps
around the spiral-shaped cell to produce an
undulating motion.
Specialized Structures of
Bacterial cell
Medically important
1. Some species of motile bacteria like E. coli and
Proteus species, are common causes of UTIs.
Flagella may play a role in pathogenesis by
propelling the bacteria up the urethra into the
bladder.

2. Some species of bacteria , e.g. Salmonella species

are identified by the use of specific antibodies
against flagellar proteins.
Specialized Structures of
Bacterial cell
D. Pili (Fimbriae)
1. Hairlike filaments that extend from the cell
surfaces.

2. Shorter and straighter than flagella composed of

a protein , pilin, arranged in helical strands.

3. Found mainly on Gram negative organisms

Specialized Structures of
Bacterial cell
Important roles
a. Mediate attachment of
bacteria to specific receptors on
the human cell surface.

b. Sex pilus forms the attach-

ment between male (donor)
and female (recipient) bacteria during conjugation.
Specialized Structures of
Bacterial cell
E. Spores
1. Highly resistant structures formed in response to
adverse conditions by two genera of medically
important gram positive rods: Bacillus (central) and
Clostridium (terminal).
Specialized Structures of
Bacterial cell
2. Spore formation (sporulation) occurs when
nutrients, such as sources of carbon and nitrogen
are depleted.

3. The spore forms inside the cell and contains

bacterial DNA , a small amount of cytoplasm, cell
membrane, peptidoglycan, very little water and
most importantly a thick keratin like coat that is
responsible for the remarkable resistance of the
spore to heat, dehydration, radiation and chemicals.
Specialized Structures of
Bacterial cell
4. The resistance may be mediated by dipicolinic
acid, a calcium ion chelator found on spores.

5. The medical importance of spores lies in their

extraordinary resistance to heat and chemicals.

6. As a result of their resistance to heat, sterilization

cannot be achieved by boiling. Autoclaving at 121oC
for 30 minutes is recommended.
 Bacterial Structures
Structure Chemical Function
Composition
Cell wall
Peptidoglycan Sugar backbone with Gives rigid support,
peptide side chains protects against
that are crosslinked osmotic pressure; is
the site of action of
penicillins and
cephalosphorins and is
degraded by lysozyme
Surface fibers of Gram Teichoic acid Major surface antigen
positive bacteria but rarely used in
diagnosis
 Bacterial Structures
Structure Chemical Function
Composition
Outer membrane of Lipid A Toxic component of
Gram negative endotoxin
bacteria Polysaccharide Major surface antigen
used frequently in
diagnosis
Cytoplasmic Lipoprotein bilayer Site of oxidative and
membrane without sterols transport enzymes
Ribosome RNA and protein in 50S Protein synthesis, site
and 30S of action of
aminoglycosides,
erythromycin,
tetracycline & chloram
 Bacterial Structures
Structure Chemical Function
Composition
Nucleoid DNA Genetic material
Mesosome Invagination of plasma Participates in cell
membrane division and secretion
Periplasm Space between plasma Contains many
membrane and outer hydrolytic enzymes
membrane including ß-lactamases
Capsule Polysaccharide Protects against
phagocytosis
Flagellum Protein Motility
Spore Keratinlike coat, Provides resistance to
dipicolinic acid dehydration, heat &
chemicals
 Bacterial Structures
Structure Chemical Function
Composition
Pilus or fimbria Glycoprotein Two types:
1) Mediates attachment to cell
surfaces
2) Sex pilus mediates
attachment of two bacteria
during conjugation
Plasmid DNA Contains a variety of genes for
antibiotic resistance and toxins
Granule Glycogen lipids, Site of nutrients in cytoplasm
polyphosphates
Glycocalyx Polysaccharide Mediates adherence to surfaces
 Identification of Bacteria
• Phenotypic
1. Microscopic morphology
(staining characteristics)

2. Macroscopic morphology
 Identification of Bacteria
3. Nutritional requirements – utilization of various
carbon and nitrogen sources

4. Environmental requirements – temperatures,

oxygen & other gases, pH levels, presence of ions
and salts

5. Resistance profile – inherent resistance to

antibiotics, heavy metals or toxins
 Identification of Bacteria

6. Antigenic properties – antigenic structures -

antibodies

7. Subcellular properties – typical molecular

constituents of the cell
 Identification of Bacteria
• Genotypic
1. DNA base composition ratio
the extent to which the DNA from two
organisms is made up of cytosine & guanine

2. Nucleic acid (DNA & RNA) base sequence

analysis
Assoc. Prof. Ma. Jennifer R. Tiburcio, MSMT
Chair, Department of Med Tech
UST Faculty of Pharmacy
 Growth

Increase in number Binary fission

 Time required for a single cell
to divide

Nutrients
Physical conditions Generation per hour
 Phase of Growth Growth Rate

A Lag phase Zero

Accelerating Increasing

B Log or Exponential Constant

Retardation Decreasing

C Maximum Stationary Zero

D Death or Period of Negative

Decline
 Lag Phase

Enzymes & Intermediates are formed

Cells are metabolizing

Lag in cell division

 Exponential Phase
(Log Phase)

Dividing steadily at a constant rate

Cellular Reproduction – most active

Susceptible to adverse conditions

Studies of metabolism
 Steady state or balanced growth

Maintenance – continuous culture

Supply of fresh nutrients

Removal of toxic wastes & excess microorganisms

Chemostat or turbidostat
 Maximum Stationary Phase

Exhaustion of nutrients

Growth Ceased

Production of toxic products

 Death Phase

Bacteria may die faster

Depletion of nutrients
Accumulation of toxic products

Inability to form colonies

Empiric Test of Death
Solid medium
 Reasearch

Synchronous
growth
Cell growth
Organization & differentiation

Cells dividing at the

same time
 Measurement of growth

Directly by microscopy or electronic

Cell count
particle counter or indirectly by a colony count

Directly by weighing or by determining nitrogen

Cell mass
content or indirectly by turbidity

Indirectly by relating the degree of biochemical

Cell Activity activity to the size of the population