LABCONCEPT 5.1 plasma Prokaryotic Features Cell cytoplasm Cytoplasmic not present on all cells or cell membrane

pili

fimbriae 2 types of Bacterial Cell Walls LABFACT 5.1

Gram-positive Gram-negative bacteria

glycocalyx glycocalyx

What is ?

(1) Gram-positive cell wall (2) Gram-negative cell wall Figure 3.13 Possible structure of peptidoglycan. Sugar peptidoglycan backbone

Tetrapeptide (amino acid) crossbridge

Connecting chain of amino acids LABFACT 5.1

Gram-positive bacteria

Gram-negative bacteria Gram-positive bacterial cell wall LABFACT 5.1

Appear purple following Gram Peptidoglycan layer (cell wall) staining Cytoplasmic membrane procedure

Lipoteichoic acid

Teichoic acid

Integral protein

Cell wall = thick layer of peptidoglycan Cell wall contains unique polyalcohols called teichoic acids Gram-Negative Bacterial Cell Walls LABFACT 5.1

Porin

Outer Porin membrane (sectioned) of cell wall Appear pink Peptidoglycan following Gram layer of cell wall Periplasmic space

Cytoplasmic staining membrane procedure

Phospholipid layers (LPS) layer, containing Integral lipid A proteins Cell wall = a thin layer of peptidoglycan + Outer membrane or OM

OM is a phospholipid bilayer membrane outside the peptidoglycan contains phospholipids, proteins, and lipopolysaccharide (LPS) OM is protective - may impede the treatment of disease Lipid A portion of LPS can cause fever, vasodilation, inflammation, shock, and blood clotting Gram-Positive vs Gram-Negative Bacterial LABFACT 5.1 Cell Walls

Gram-Positive Gram-Negative Peptidoglycan PRS – THICK PRS – THIN

Outer Membrane PRS Periplasmic Space PRS - ONE PRS - TWO LPS ABS PRS (lipopolysaccharide) Porin ABS PRS Techoic Acid PRS ABS

PRS = PRESENT ABS= ABSENT How to perform Gram Staining Reaction ? LABFACT 5.2 purple Gram-positive bacteria pink Gram-negative bacteria Mechanism behind Gram Staining Reaction LABFACT 5.3

Thick peptidoglycan prevents decolorization

Thick peptidoglycan Thin peptidoglycan in cell wall in cell wall Gram Staining Method LABFACT 5.3

Primary Stain Step

Crystal Violet

Gram positive cell wall

Gram negative cell wall Gram Staining Method

Mordant Step

Gram’s Iodine Gram Staining Method

Decolorizing Step

Alcohol 25 sec Gram Staining Method

Counter Stain Step

Safranin Gram Staining

Simple/Differential Stain ? Acid Fast Bacterial Cell Wall LABCONCEPT 6.1 LABFACT 6.1

Peptidoglycan layer (cell wall)

Cytoplasmic membrane

Gram-positive cell wall Lipoteichoic acid

Teichoic acid

Integral Some gram positive bacteria have up to 60% protein mycolic acid (waxy lipids) which helps cells survive desiccation – called Acid Fast bacteria

Acid Fast bacteria cell wall Acid Fast Bacterial Cell Wall LABCONCEPT 6.1

Some Gram positive bacteria cannot be stained by LABFACT 6.1 Gram staining procedure

They have a waxy lipid called mycolic acid in their cell wall (60 %) mycolic acid which prevents water-soluble stains from penetrating the cell wall

Acid Fast bacteria cell wall How to perform Acid-Fast Staining Method ? Acid-Fast Staining Method Ziehl-Neelsen Method Primary Stain Step LABCONCEPT 6.2 HEAT Carbolfuchsin

Mechanism behind Acid-Fast Staining Method Acid-Fast Staining Method

Decolorizing Step Acid-Fast

Acid Alcohol

Non Acid-Fast Acid Fast Staining Method

Counter Stain Step

Methylene Blue

LABCONCEPT 7.1 LABFACT 7.1 prs in Cytoplasm of some Bacteria

• Unique structures produced by some bacteria • Those which make endospores also make endotoxins • E.g. bacteria causing anthrax, tetanus, gangrene • Defensive strategy against unfavorable conditions • Vegetative cells transform into endospores when multiple nutrients are limited • Resistant to extreme conditions such as heat, radiation, chemicals Why are making bacteria dangerous ? In canning industry heat is used to kill microorganisms inside cans – but endospore containing bacteria can survive heat LABFACT 7.2 Vegetative Bacterial Cell Endospore Free spore

Environmental Challenges Heat Chemical Starvation

Spore germinates Vegetative Bacterial Cell Endospore Free spore 1 1

Spore germinates

Spores are not part of bacterial reproductive cycle bacterial reproduction is 1 bacteria = 2 bacteria

1 Bacteria Endospore 1 Bacteria LABFACT 7.3

Spores are metabolically inactive with tough special cell walls

Location of endospores unique for different spore-forming bacteria Schaeffer-Fulton Staining Method for Endospores

Primary Stain Step LABFACT 7.4 Endospore containing HEAT Malachite Green

Vegetative Bacterial Cell Schaeffer Fulton Staining Method for Endospores

Decolorizing Step Endospore containing NO HEAT X Water

Vegetative Bacterial Cell Schaeffer Fulton Staining Method for Endospores

Counter Stain Step Endospore containing Endospore containing

Safranin

Vegetative Bacterial Cell Vegetative Bacterial Cell

Crystal Violet Staining LABFACT 7.5 External Structures of Bacterial Cells LABCONCEPT 8.1

glycocalyx cell wall

cell membrane bacteria

• Glycocalyces • Structure - Gelatinous, sticky substance surrounding the outside of the cell. Composed of polysaccharides or polypeptides, or both

• Function 1) avoid dessication 2) adhesion, 3) protect from host defense LABFACT 8.1 A Type of Glycocalyces (1) Capsule • Firmly attached to cell surface • Prevent bacteria from being recognized by host – avoid phagocytosis by immune cells

Capsule enhances the ability of bacteria to cause disease Figure 14.8 Relative virulence of some microbial pathogens. LABCONCEPT 8.2

Virulence

Degree of pathogenicity – not about severity of the disease it causes

But how easily it causes disease LABCONCEPT 8.3

Capsule enhances the ability of bacteria to cause disease – makes them more virulent Observe Capsule stained slide