Gram Staining

Simple/Differential Stain ?

Figure 3.13 Possible structure of . Sugar Composed of peptidoglycan backbone

Tetrapeptide (amino acid) crossbridge

Connecting chain of amino acids Gram-positive bacterial

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 (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 cannot be stained by Gram staining procedure

They have a waxy lipid called 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 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 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, 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

• • Loosely attached to cell surface - water soluble • Function – adhesion - sticky layer allows 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