MORPHOLOGY AND

PHYSIOLOGY OF BACTERIA
Competency Covered: MI 1.1
Learning objectives

 At the end of the session, the students will be able to understand:

 Classification of bacteria depending on their morphology and
Gram staining property
 Morphology of bacterial cell
 Physiology of bacteria
Intorduction

 Microorganisms such as bacteria, fungi, and protozoa are

included under a separate Kingdom – Protista
 Based on the differences in cellular organisation and
biochemistry the Kingdom Protista has been divided into two
groups
 Prokaryotes and Eukaryotes
 Bacteria are prokaryotes posses a simpler type of cell structure
 Fungi and protozoa are eukaryotes have advanced type of cells
Characteristics Prokaryotes Eukaryotes

Nucleus
Nuclear membrane Absent Present
Nucleolus Absent Present
DNA Single and circular Many and linear

Chromosome number One More than one

Mitotic division Absent Present
Cytoplasm
Cytoplasmic streaming Absent Present
Oraganelles such as mitochondria, Absent Present
ER & Lysosomes, golgi
apparatus.
Size of ribosome 70s 80s
Cell wall containing Peptidoglycan Present Absent
Unit of microscopic measurement

 The unit of measurement in microbiology is micron or

micrometer
 1 micron (µ) = 1/1000 of mm or 10-3 mm
 Mostly used to measure the size of bacteria, fungi, protozoan
parasites and parasitic eggs
 1 millimicron or nanometer = 1/1000 of micron or 10-6 mm
 1 Angstrom unit A˚ = 1/10 of a nm
 These units are used to measure the size of viruses and ultra
microscopic organelles of microorganisms
Shape of Bacteria

 Not all bacteria look alike, depending on their shape bacteria are
classified as
 Cocci - spherical
 Bacilli - rod shaped
 Vibrios - comma shaped
 Spirocheates - flexible spirals
 Spirillum - rigid spirals
 Actinomycetes – branched and filamentous
Shape and arrangements of bacteria
Arrangement of cocci

 In clusters- E.g.: Staphylococcus

 In chains- E.g.: Streptococcus
 In pairs- Diplococci
 E.g.: Meningococci/Pneumococci
 In tetrads- E.g.: Micrococcus
 In groups of eight - E.g.: Sarcina
Morphology/structure/ anatomy of
Bacteria or Bacterial cell:
Diagram of basic bacterial cell
Parts of Bacterial cell structure

 Bacterial cell structure can be studied under following headings

 I) Protoplasm – consists of cytoplasm, cytoplasmic inclusions,
granules and nuclear body
 The cell envelope – consists of cell membrane cell, wall and
additional layer capsule
 III) Surface appendages – flagella and pili
 IV) Spores
Cytoplasm/Cytoplasmic matrix

 Is a colloidal system consisting of a variety of organic and

inorganic solutes in a viscous water solution
 Differ from eukaryotic protoplasm by not showing Golgi
complex, mitochondria, endoplasmic reticulum and cytoplasmic
streaming
 Packed with ribosomes, storage granules - inclusions and cell
membrane invaginations - mesosomes.
 Demonstration: Stains easily by basic dyes
Cytoplasmic inclusions

 RIBOSOMES -Site of protein synthesis

 Bacterial ribosome’s are 70s in size with 50s and 30s subunits.
 Composed of a ribosomal protein and a ribosomal RNA.
 MESOSOMES - Invagination of cytoplasmic membrane in the
shape of vesicles or tubules
 Principle centers of respiratory enzymes,
 Cell wall formation
 Chromosome replication
Intracytoplasmic granules

 Cytoplasm contains several different types of granules that serve

as storage areas for nutrients & stain characteristically with
certain dyes.
 1. Metachromatic granules
 2. Polysaccharide granules
 3. Sulfur granules
 4. Lipid inclusions
Metachromatic granules of
C.diphtheriae in Albert’s
staining
Nuclear body/Nucleoid

 Incomplete, no nucleolus and nuclear membrane hence called

“Nucleoid” which appear as oval or elongated body generally one
per cell
 It consists of a single molecule of highly coiled double stranded
DNA occur in the form of a closed circle known as bacterial
chromosome
 Replicates by simple fission
 It governs the entire function of the cell
 Demonstration: Electron microscope, Feulgen staining
Plasmids:

 Some bacteria may possess extra nuclear genetic material

consisting of circular double stranded DNA – called plasmids
 Not essential for the life of the cell but may code for special
properties like drug resistance, toxigenicity, sex pili etc.
The cell envelope

 The layers that surround the prokaryotic cell are collectively

termed as the cell envelope
 Depending upon the structure and organisation of the cell
envelope , bacteria are generally divided in to 2 types – Gram
Positive and Gram negative bacteria
 Cytoplasmic membrane
 Cell wall
 Capsule (Variable)
Cytoplasmic membrane

 Also called plasma membrane

 Innermost layer of the cell membrane which surrounds the
cytoplasm of the cell.
 In gram negative bacteria it is called ‘inner membrane’
 Structurally it is an ‘unit membrane’ composed of a phospholipid
bilayer in combination with protein
Cytoplasmic membrane
Functions of Cytoplasmic membrane

 Maintains osmotic pressure within the cell

 Semi permeable membrane -Transport system -nutrient uptake,
and waste excretion
 Site for metabolic processes - Biosynthetic functions - Electron
transport and oxidative phosphorylation
 Secretion of hydrolytic enzymes
Antibiotics acting on the cytoplasmic
membrane

 Polymyxin
 Novobiocin
 Nalidixic acid
 Nitrofurantoin.
Cell wall

 Tough and rigid structure - surrounding the cell membrane.

 15–25 nm thickness
 Basically made up of multilayered structure composed of a
macromolecular network called Peptidoglycan /Mucopeptide/
Murein - back bone
Gram positive - Cell wall

 Peptidoglycan: Thicker (50–100 layers, 16–80 nm) than gram-

negative cell wall
 Each layer - mucopeptide (murein) chain - alternate units of N-
acetyl muramic acid (NAM) and N-acetyl glucosamine (NAG)
molecules - cross linked via tetrapeptide side chains and
pentaglycine bridges
 Tetrapeptide side chain - from NAM molecule - composed of L-
alanine-D-glutamine - L-lysine - D-alanine
 Teichoic Acid:
 Polymers of glycerol or ribitol - joined by phosphate groups.
 Two types: (i) Cell wall teichoic acid and (ii) Lipoteichoic acid.
 Absent in gram-negative bacteria.
 Function - cell shape determination, regulation of cell division,
and other fundamental aspects of gram-positive bacterial
physiology.
Peptidoglycan layer of gram-positive cell wall
Gram-negative Cell Wall

 Gram negative cell wall consists of thin layer of peptidoglycan

present within periplasmic space.
 Peptidoglycan layer: Very thin (1–2 layer, 2 nm thick) -
composed of a mucopeptide chain - similar to gram-positive cell
wall.
 Consists - alternate NAM and NAG molecules
Gram-negative Cell Wall

 Outer Membrane:
 Phospholipid layer - lies outside the thin peptidoglycan layer
 Serves as - protective barrier to the cell
 Outer membrane proteins (OMP) or porin proteins - specialized
proteins
Gram-negative Cell Wall

 Lipopolysaccharide (LPS): Extra cell wall component

 Consists of three parts:
 Lipid A (or the endotoxin)
 Core polysaccharide
 O side chain (O or somatic antigen)
 Periplasmic Space - Space between the inner cell membrane and
outer membrane. It encompasses the peptidoglycan layer.
 Meso-diaminopimelic acid - present at third position of the
tetrapeptide side chain .
 Pentaglycine bridge is absent
CHARACTERS GRAM POSITIVE GRAM NEGATIVE
Peptidoglycan layer Thick(multilayered) Thin
Teichoic acids Present Absent
Outer membrane Absent Present
Lipopolysaccharide Absent Present
Lipid and lipoprotein Low High due to the presence of
content outer membrane.
Susceptibility to penicillin high low
and sulfonamides

Gram reaction Retain crystal violet and Takes the color of counter
stain purple stain and appear pink
Differences between gram-positive and
gram negative cell wall
Functions of cell wall

 Rigid structure hence accounts for the shape of bacteria cell

 Provide rigidity and ductility - Protection and strength -presence
of peptidoglycan layer in the cell wall
 Protection against osmotic damage and from adverse condition
 Protect cell from toxic substances
 Site of action for several antibiotics
Antimicrobial agents acting on cell wall

 Includes enzymes and antibiotics.

 1.Enzymes - a. Lysozymes, b. Autolysins
 2. Antibiotics :-
 a. Beta lactam antibiotics like penicillin and cephalosporins.
 b. Glycopeptides- Vancomycin, Teicoplanin
 c. Bacitracin
 d. Cycloserine
Demonstration
 By electron microscopy
 Plasmolysis (The shrinking of protoplasm away from the cell
wall of a bacterium due to water loss from osmosis, thereby
resulting in gaps between the cell wall and cell membrane
BACTERIA WITH DEFECTIVE CELL WALL

 Mycoplasma:- naturally occurring cell wall lacking bacteria.

 Protoplast:- Gram positive bacteria when treated with lysozymes
in hypertonic solution.
 Spheroplast: - Gram negative bacteria when treated with
lysozymes in hypertonic solution.
 L- forms :- Cell wall deficient bacteria that are able to grow and
divide.
 Significance - Recurrent infection and difficulty in treatment
Capsule and Slime Layer

 Layer of amorphous viscid material lying outside the cell wall called
glycocalyx.
 Capsule -well organized and not easily washed off
 Slime layer - diffuse, unorganized loose material that can be removed easily
The Capsule

 Examples for capsulated bacteria

 Streptococcus pneumoniae, Haemophilus influenzae, Neisseria
meningitides, Klebsiella pneumoniae
 Capsule producing bacteria form mucoid colonies with a stringy
consistency on agar plate
Properties

 Polysaccharide in nature (Exception Bacillus anthracis

polypeptide)
 Heatlabile, get destroyed when heated at 100oC for 30
minutes
 Antigenicand capsular antigen is known as ‘K’ antigen
(German word kapsel = capsule)
FUNCTIONS OF CAPSULE

 1. Enhances bacterial virulence by inhibiting phagocytosis

 2. Act as protective covering against bacteriophage, lytic
enzymes and drugs
 3. Helps in adherence of bacteria to various surfaces.
 Biofilm Formation - Biofilm - living ecosystem made of millions
of adherent bacterial cells embedded within a self-produced
matrix of extracellular polymeric substance
 Capable of adherence to damaged tissues and plastic surfaces.
CLINICAL SIGNIFICANCE AND USES

 Capsular ‘K’ antigen specific for bacteria and can be used for
identification and epidemiological typing of bacteria.
 Capsules as vaccine:
 Capsular vaccines are available for bacteria such as
Pneumococcus, Meningococcus and Haemophilus influenzae
serotype-b.
DEMONSTRATION OF CAPSULES

 1. DIRECT METHODS
 (A) Unstained preparation - Electron microscopy ,Phase contrast
microscopy
 (B) Stained preparations
 Positive staining – Welch’s method
 Negative staining - India ink/Nigrosin
 2. INDIRECT METHODS :-
 (a) Immunofluorescence and latex agglutination –Ag detection
 (b) Serological method- Quellung reaction.
WET INDIA INK PREPARATION QUELLUNG REACTION
III. Cell appendages/surface appendages

 Two types
 Flagella
 Pili
Flagella (Flagellum)

 Definition – Are long filamentous , semi rigid ,helical structure

extends from the cell membrane, which help in the locomotion of
bacteria
 Properties
 3-10 micron long and 20 nm in diameter
 Structure – has 3 components, basal body, hook and filament
 Chemical nature - composed of a protein – flagellin
 Antigenic in nature – ‘H’ Ag, (Hauche = flagellum in German)
 Destroyed by heat at 100o C
PARTS OF FLAGELLA

(a) FILAMENT

(b) HOOK :-

(c) BASAL BODY :-

FUNCTIONS OF FLAGELLA

 It is the organ of locomotion. Motility helps in :-

 (a) Movement of bacteria towards favorable environment where
there is higher concentration of nutrients.
 (b) Penetration and spread of infections.
 SIGNIFICANCE OF FLAGELLA
 1. Highly antigenic(H- antigen). Specific antibodies produced are
useful in serodiagnosis of infections
 2. Preparation of flagellar antigen
 3. Helps in identification of organisms.
DEMONSTRATION OF FLAGELLA
 1. DIRECT METHODS :-
 (a) Electron microscopy
 (b) Special staining techniques – in which thickness of flagella
increased by superficial deposition of stain - Silver impregnation
method e.g. Leifson’s stain, Fontana’s Stain
 2. INDIRECT METHODS :- includes demonstration of flagella
by motility.
 (a) Hanging drop method
 (b) by dark ground microscopy
 (c) By semi solid agar medium.
Flagella in Leifson’s staining method
Hanging drop preparation
Semisolid agar
Fimbriae/Pili (Fimbria/Pilus)

 Definition – they are very fine , rigid hair like surface appendages of
certain bacteria
 Properties
 0.5 micron long and less than 10 nm in diameter
 Shorter and thinner than flagella and originate from the cell membrane
as straight filaments
 Made up of protein subunit - called pilin .
 Antigenic in nature – pili are antigenically distinct for each species and
elicit formation of antibodies by the host which can protect the host by
preventing attachment by that bacterial species
Types of pili

 Based on function 2 types

 Common pili/ ordinary pili – mainly responsible for adhesion of
the bacteria to various host cells
 Further classified into 4 types as Type I to IV based on
haemagglutination and mannose sensitivity
 Sex pili – certain bacteria possess specialised fimbriae/pili which
are longer and thinner than common type and present in smaller
numbers.
 These are involved in formation of conjugation bridges between
donor and recipient bacteria during ‘conjugation’ – a process of
transfer of genetic material from one bacterium to another
bacterium
 Presence of sex pili is coded by a plasmid ‘F’ plasmid
Functions/clinical significance of
Fimbriae
 Common pili - Mediate the attachment/adhesion of bacteria to
specific receptors on the host cell surface, which is a necessary
step in the initiation of infection for some organisms
 Sex pili -Transfer of genetic material from donor to recipient cell
by a process called conjugation
 transfer of drug resistance between bacteria – known as plasmid
mediated (transferable ) drug resistance
DEMONSTRATION OF FIMBRIAE

 1. DIRECT METHOD :-
 (a). Electron microscopy
 2. INDIRECT METHODS :-
 (a). Pellicle formation
 (b). Haemagglutination using
Guinea pig RBC’s
Bacterial spore

 Definition – It is a resting or dormant stage of some bacteria,

highly resistant to desiccation, heat and chemical agents
 The spore is formed inside the parent vegetative cell hence the
name ‘Endospore’
 Examples for Spore forming/bearing bacteria
 Members of the genus Bacillus - aerobic
 Members of the genus Clostridium -anaerobic
 Spore formation is not involved in multiplication and not a
method of reproduction unlike in other microorganisms
 Sporulation
 This process occurs when environmental conditions become
unfavourable for the growth of bacteria such as
 Depletion/exhaustion of exogenous nutritional sources/
accumulation of toxic products/extremes of temperatures etc.
Properties of the spore

 The high resistance of the spore towards desiccation , heat and chemical
agents is due to -
 Low water content of the spore
 Presence of calcium dipicolinate which helps in stabilisation of the spore
enzymes
 Presence of thick layer
 Spores are destroyed by
 Autoclaving at 121 o C for 18 minutes
 Hot air sterilisation – 180 o C for 1 hour
 Chemical sterilisation – ethylene oxide
Spore structure (ultramicroscopic)

1. Core
2. Spore wall (Germ cell wall)
3. Spore cortex
4. Spore coat
5. Exosporium
GERMINATION OF SPORE

 Occurs when external conditions become favourable to growth

and one spore will give rise to one vegetative cell
 Process by which a spore is converted into a vegetative cell.
 FUNCTIONS
 1. Helps to survive in adverse condition
 2. Spores may be the infective form in some bacteria like
Clostridium tetani , Bacillus anthracis
Practical uses

 1. Used as the biological indicator for sterilization.

 a. Dry heat - spores of Bacillus subtilis
 b. Moist heat – spores of Bacillus stearothermophilus
 2. Shape and arrangement of spores helps in the identification of
bacteria
DEMONSTRATION

 1. UNSTAINED METHODS :-
 (a) By phase contrast microscopy
 (b) Electron microscopy

 2. STAINED METHODS :-
 (a) Modified Ziehl- Neelsen stain.
 (b) Special staining like Dorner’s stain.
Gram staining Modified Z-N Staining
PHYSIOLOGY OF
BACTERIA
Bacterial Growth Requirement

 Water constitutes - 80% of total bacterial cell.

 Minimum nutritional requirements - essential for growth and
multiplication of bacteria include - sources of carbon, nitrogen,
hydrogen, oxygen and some inorganic
Bacterial Cell Division

 Bacteria divide by a relatively simple form of cell division, i.e. by

binary fission
 Nuclear division
 Cytoplasmic division
Rate of Multiplication in Bacteria

 Generation time is the time required for a bacterium to give rise

to two daughter cells under optimum condition.
 Escherichia coli and most of the other pathogenic bacteria -20
minutes;
 Mycobacterium tuberculosis - 20 hours
 Mycobacterium leprae - 20 days
Bacterial Count

 Total count: Indicates total number of bacteria (live or dead) in

the specimen. This is done by counting the bacteria under
microscope using counting chamber.
 Viable count: Measures the number of living (viable) cells in the
given specimen. Viable count may be obtained by a technique
called as pour plate method.
Bacterial Growth Curve

 When a bacterium is inoculated into a suitable liquid culture

medium and incubated, its growth follows a definite course.
 When bacterial count of such culture is determined at different
intervals and plotted in relation to time, a bacterial growth curve
is obtained.
Bacterial Growth Curve (Cont..)

 Bacterial growth curve comprises of four phases.

 Lag phase
 Log phase
 Stationary phase
 Phase of decline
Bacterial Growth Curve
Lag phase

 Initial period in the life of a bacterial population when cells are

adjusting to a new environment before starting exponential
growth.
 Period between inoculation and beginning of multiplication of
bacteria.
 Bacteria increase in size due to accumulation of enzymes and
metabolites.
 Bacteria reach their maximum size at the end of lag phase.
Log phase

 Bacteria divide exponentially so that the growth curve takes a

shape of straight line.
 At this stage, the bacterium is:
 Smaller in size
 Biochemically active
 Uniformly stained
Stationary phase

 Number of viable cells remain stationary as there is a balance

between the dying cells and the newly formed cells. But the total
count keeps raising.
 In this phase:
 Bacterium becomes gram-variable
 More storage granules are formed
 Sporulation occurs in this phase
 Bacteria produce exotoxins, antibiotics and bacteriocins
Decline phase

 Bacteria stop dividing completely; while the cell death continues

due to exhaustion of nutrients, and accumulation of toxic
products.
 Involution forms are seen.
Bacterial Growth Curve (Cont..)
Factors Affecting Growth of Bacteria

 There are several environmental factors that affect the growth of

the bacteria.
 Oxygen
 Carbon dioxide
 Temperature
 pH ,Light ,Osmotic Effect
 ­Mechanical and Sonic Stresses
 Moisture and Desiccation
Oxygen

 Depending on the oxygen requirements, microorganisms can be

classified as
 Obligate aerobes – can grow only in the presence of oxygen -
Pseudomonas,
 Facultative anaerobes (can grow in the presence or absence of
O2) – most of the pathogenic bacteria – E.coli, S.aureus
 Obligate anaerobic (growth in the absence of O2) - Clostridium
 Microaerophilic (require slightly decreased O2 tension) –
Campylobacter, Helicobacter.
Carbon dioxide

 Organisms that require higher amounts of carbon dioxide (5–

10%) for growth are called capnophilic bacteria.
 Examples - Brucella abortus, Streptococcus pneumoniae, etc
Temperature

 Temperature - Psychrophiles: Grow at temperatures below 20°C;

example, most of the saprophytes, e.g. Pseudomonas
 Mesophiles: Grow within a temperature range 25°C - 40°C;
example, most of the pathogenic bacteria
 Thermophiles: Grow at a high temperature range of 55°C–80°C,
e.g. Geobacillus stearothermophilus.
pH

 Most pathogenic bacteria grow between pH 7.2 -7.6.

 Very few bacteria (e.g. lactobacilli) can grow at acidic pH below pH 4
 Vibrio cholerae are capable of growing at alkaline pH (8.2–8.9).
 Osmotic Effect
 Bacteria are able to withstand a wide range of external osmotic variation
because of the mechanical strength of the cell wall.
 Sudden exposure to hypertonic saline may cause cell shrinkage (plasmolysis)
 Exposure to distilled water may cause cell swelling and rupture
(plasmoptysis).
Moisture & Desiccation

 Moisture is an essential requirement for the growth of bacteria

because 80% of the bacterial cell consists of water. However, the
drying has varying effects on different organisms.
 Some organisms like Treponema pallidum and N. gonorrhoeae
die quickly after drying, while M. tuberculosis and S. aureus may
survive drying for several weeks.
 Drying in cold and vacuum (lyophilization) is used for
preservation of microorganisms..