Part I

General Microbiology

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 MORPHOLOGY
OF
BACTERIA

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 MICROORGANISMS

EUKARYOTIC PROKARYOTIC
• Bacterial cell
• Parasites, fungi
• Do not contain
• Membrane enclosed organelles
organelles
• Cell wall,
• Cytoskeleton peptidoglycan

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 PROKARYOTE AND EUKARYOTE

Prokaryote and eukaryote cells

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 MORPHOLOGY OF BACTERIA

• SIZE - 0.25-1 µm width

• 1-3 µm length
• SHAPE – cocci
bacilli
coccobacilli
fusiform
spiral
• ARRANGEMENT

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 SHAPE OF BACTERIA

Shapes of bacteria: 1. coccus; 2. bacillus; 3. vibrio; 4. spirillum; 5. spirochete

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 ARRANGEMENT OF BACTERIA

Arrangement of curved bacteria: 1. vibrio; 2. spirilla; 3. spirochetes

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 MICROSCOPY

OPTICAL/LIGHT MICROSCOPE

Principle of bright-field (light) microscopy

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 MICROSCOPY

PHASE CONTRAST MICROSCOPE

• Improves contrast
• Makes evident structures within cells
• ‘Phase differences’ converted to differences
in intensity of light – producing light and
dark contrast in image

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 MICROSCOPY

FLUORESCENT MICROSCOPE
• Light of high intensity source excites a
fluorescent agent, which in turn emits a low
energy light of longer wavelength that
produces the image

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 FLUORESCENT MICROSCOPE

Fluorescent microscopy

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 FLUORESCENT MICROSCOPE APPLICATIONS

Principles of fluorochroming and immunofluoresence

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 MICROSCOPY
DARK GROUND MICROSCOPE
• Improves contrast
• Reflected light used instead of transmitted
light
• Dark field condenser
• Light rays falling on the object are reflected or
scattered on to objective lens
• Object self-luminous against dark background

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 DARK GROUND MICROSCOPE

Dark-field microscopy
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 MICROSCOPY

ELECTRON MICROSCOPE

• Beam of electrons instead of beam of light

• Electron beam focused by circular

electromagnets - analogous to lens in light
microscope
• Object held in the path of electron beam
and produces an image which is focused on
a screen

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 ELECTRON MICROSCOPE APPLICATIONS

• Shadow casting
• Negative staining – phosphotungstic acid
• Freeze etching

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 STAINING TECHNIQUES

• SIMPLE STAIN – Methylene blue or

basic fuchsin
• NEGATIVE STAINING – India ink or
nigrosin
• IMPREGNATION – Silver impregnation

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 STAINING TECHNIQUES

• Differential stains
GRAM STAIN – Christian Gram (1884)
• Principle: Gram positive – acidic protoplasm
- retain basic primary dye
• Peptidoglycan of Gram-positive bacteria
thick – retain the dye iodine complex
• High lipid content of Gram-negative
bacteria makes them permeable to
counterstain (secondary dye)

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 STAINING TECHNIQUES

ACID FAST STAIN - Ehrlich

• Modified – ZIEHL and NEELSEN
• Principle: High lipid content, variety of fatty
acids and lipids
• Mycolic acid – peculiar to acid fast bacilli
• Integrity of the cell wall

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 STAINING TECHNIQUES

• ALBERT STAIN - demonstrate

metachromatic granules – C.diphtheriae
• Neisser’s stain
• Ponder’s stain

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 BACTERIAL CELL

Diagram of an idealised bacterial cell

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 BACTERIAL CELL

• Cell envelope and appendages

• Cell interior
• Cell structures function as a complex
integrated unit

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 BACTERIAL CELL

CELL ENVELOPE
• Outer membrane (GNB)
• Cell wall – peptidoglycan
• Periplasm (GNB)
• Cytoplasmic membrane

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 OUTER MEMBRANE

• Bilayered
• Composed of lipopolysaccharide (LPS)
• Gives GNB - net negative charge
• Scattered throughout LPS - porins
• Porins - control passage of nutrients and
antibiotics

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 CELL WALL

• Composed of disaccharide-pentapeptide
• Disaccharide-N-acetyl glucosamine
N-acetyl muramic acid
• Amino acid only linked to N-acetyl muramic
acid
• Polymers-crosslink-via peptide bridges to
form peptidoglycan sheets

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 CHEMICAL STUCTURE OF BACTERIAL CELL WALL

Chemical structure of bacterial cell wall

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 CELL WALL

• Notable difference - Gram positive and

Gram negative

• Gram positive-Peptidoglycan thick

Teichoic acid
Glycerol
Ribitol phosphate
Mycolic acid

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 CELL WALL

Gram-positive call wall

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 CELL WALL

Gram-negative cell wall

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 CELL WALL

• Cannot be seen by light microscope

• Cannot stain with simple stain
• Demonstration
- Plasmolysis - hypertonic solution
bacterial ghost
- Micro dissection
- Reaction with specific antibody
- Differential staining
- Electron microscope

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 PERIPLASMIC SPACE

• Found in Gram-negative bacilli

• Inner surface of outer membrane
• Contains the peptidoglycan layer
• Helps secure nutrients
• Has enzymes that degrade macromolecules
and detoxify antibiotics

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 CYTOPLASMIC MEMBRANE

• Present in Gram-positive and Gram-

negative bacteria
• Deepest layer of cell envelope
• Heavily laced with proteins and
enzymes vital to cell metabolism
• Cytoplasmic membrane functionally
similar to eukaryotic cell organelles
(Mitochondria, Golgi, Lysosomes)

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 CYTOPLASMIC MEMBRANE FUNCTIONS

•Transport of solutes
•Enzymes involved in cell synthesis
•Generation of chemical energy
•Cell motility
•Chromosomal segregation

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 CELLULAR APPENDAGES

• Capsule

• Fimbria-pili

• Flagella

• Role in causing infection

• Help identification in laboratory

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 CAPSULE
• Immediately exterior to peptidoglycan
• Glycocalyx/Slime
• Protect bacteria from attack of cells of
human defense mechanism
• Facilitates and maintains bacterial
colonisation of biological surface
Example: teeth, prosthetic heart valve

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 CAPSULE

SIGNIFICANCE
•Virulence - Inhibit phagocytosis
- Protect cell from lysozyme
•Permit adherence - Cell surface
Example: implant, catheters
•Prevents cell from drying
•Toxicity to host cell
•Protects cell from bacteriophage

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 CAPSULE

• Covalently linked to cell wall

• No net charge - so do not bind to dyes
• Gram stain – clear halo around bacteria
• Demonstrated by negative staining
- India ink
- Nigrosin
- Congo red
• Demonstrated immunologically - Quellung
reaction

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 CAPSULE

S.pneumoniae capsule seen by India ink staining

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 CAPSULE APPLICATIONS

• Serological typing - capsular antigen

• Detection of antigen - CSF, blood
Example: S.pneumoniae - CSF
Neisseria meningitidis
• Vaccine - capsular polysaccharide food
antigen

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 FIMBRIAE/PILI
• Short, hair-like structures
• Proteinaceous-antigenic
• Protrude through cell wall
• Two types: common pili-adhesins, sex
pili

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 FIMBRIAE/PILI

SIGNIFICANCE
• Act as adhesins - bacteria colonise
• Receptor for bacteriophage
• Streptococcus pyogenes - M protein
• Virulence factor
• Some fimbriae - agglutinate RBCs

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 FLAGELLA

• Complex structure
• Composed of protein flagellin
• Embedded in cell envelope
• Motility - survival

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 FLAGELLA

• Non-contractile
• Single protein subunit - flagellin
• Anchored to bacterial cytoplasmic
membrane by disc-like structure

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 FLAGELLA

• Three parts: filament, hook, basal body

• Flagella attached to cell body by hook and
basal body
• Hook and basal body embedded in envelope
• Basal body -1 set of rings-Gram positive
2 sets of rings-Gram negative

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 FLAGELLA - STRUCTURE

General structure of the flagellum of a Gram-negative bacterium

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 ARRANGEMENT OF FLAGELLA

Types of flagellar arrangement: 1. monotrichous; 2. lophotrichous;

3. amphitrichous; 4. amphilophotrichous; 5. peritrichous fl agella

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 FLAGELLA

• Detection of motility-Direct-hanging drop

Phase contrast
Dark ground
Motility media
• Demonstration of flagella-Flagella stain
Electron microscope

Immunology

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 SPORE

• Adverse physical and chemical conditions

• Nutrients scarce
• Metabolic and structural change
• Bacterial spores – endospores
• Favorable conditions endospore germinates
• Spores killed – autoclave, formaldehyde

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 BACTERIAL SPORE

Diagrammatic representation of a bacterial spore: 1. germinal groove;

2. outer cortical layer; 3. cortex; 4. internal spore coat; 5. subcoat material; 6.
outer spore coat; 7. cytoplasmic membrane; 8. cell wall primordium.
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 TYPES OF BACTERIAL SPORES

Types of bacterial spores: 1. central,

bulging; 2. subterminal, bulging;
3. terminal, spherical; 4. central, not
bulging; 5. subterminal, not
bulging; 6. terminal, oval

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 PLEOMORPHISM

• Pleomorphism – defective cell wall synthesis

• Involution forms – activity of autolytic
enzymes
• L forms – Kleineberger and Nobel - aberrant
morphological forms
• Lister Institute, London

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