glycocalyx LABCONCEPT 5.1 cell wall plasma Prokaryotic Cell membrane 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 bacteria Gram-negative bacteria glycocalyx glycocalyx What is peptidoglycan ? (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 Lipopolysaccharide (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 mycolic acid (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 Endospores 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 endospore 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.