Gram Staining Simple/Differential Stain ? Figure 3.13 Possible structure of peptidoglycan. Sugar Composed of peptidoglycan backbone Tetrapeptide (amino acid) crossbridge Connecting chain of amino acids Gram-positive bacterial cell wall 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 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 Staining Procedure and Mechanism Gram Staining Method 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 Gram Staining Method Counter Stain Step Safranin Gram Staining Simple/Differential Stain ? Some Gram positive bacteria cannot be stained by Gram staining procedure They have a waxy lipid called mycolic acid in their cell wall (60 %) which prevents water-soluble stains from penetrating the cell wall Acid-Fast Staining Method Ziehl-Neelsen Method Primary Stain Step HEAT Carbolfuchsin Acid-Fast Staining Method Decolorizing Step Acid-Fast Acid Alcohol Non Acid-Fast Acid Fast Staining Method Counter Stain Step Methylene Blue 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 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 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 Figure 3.2 Typical prokaryotic cell. Inclusions Nucleoid Gelatinous, sticky substance surrounding theRibosome outside of the cell. Composed of Flagellum polysaccharides, Glycocalyx Cell wall polypeptides, or both Two Types of Glycocalyces • Capsule • Firmly attached to cell surface • Function - prevent bacteria from being phagocytosed by immune cells - protect from host defense • Slime layer • Loosely attached to cell surface - water soluble • Function – adhesion - sticky layer allows prokaryotes to attach to surfaces Figure 14.8 Relative virulence of some microbial pathogens. Virulence Degree of pathogenicity – not about severity of the disease it causes But how easily it causes disease Capsule enhances the ability of bacteria to cause disease – makes them more virulent .