Microbiology Practical

Lab Practical 2
Morphological Staining Techniques Simple Stain- Uses 1 Stain
1. Acid Stains (- Charge)- Stains Background- Nigrosin, India Ink and Congo Red
2. Base Stains (+ Charge)- Stains Cell- Methylene Blue, Crystal Violet, Safranin
Differential Staining Techniques- Any Staining Technique using 2 or more stains is differential. It allows us to differentiate between parts.
1. Gram Stain- Two Stains, PLUS Reagents- Distinguishes Chemical Composition of Cell Wall PG only (+ Purple) or LPS and PG (- Pink) Primary Stain- Crystal Violet- Stains Cells Purple Binding Agent- Mordant (Iodine)- Fixes C.V. into Cell Wall of G+ cells Decolorizing Agent- Ethanol- Removes C.V. from G- Cells Counter Stain- Safranin (Reddish Pink)- Stains G- Cells Pink
2. Structural Staining Technique A. Endospore Stain- Differentiates between spores and vegetative cells Primary Stain- Malachite Green (shows spores) Counter Stain- Safranin (shows vegetative cells) B. Capsule Stain- Differentiated between capsule, cell and background Capsule is a thick structured layer that protects the cell from phagocytes. Primary Stain- Congo Red Counter Stain- Crystal Violet
Biochemical Tests- presence or absence of specific enzymes helps us to identify bacteria -used to evaluate metabolic capabilities of specific bacteria. 1. Catalase Test- Enzyme: Catalase Substrate: Hydrogen Peroxide Reagent: Hydrogen Peroxide Product: Oxygen Medium: TSA
Bubbles (+)- Catalase… No Bubbles (-)- No Catalase
2. Denitrification Test- Enzyme: Nitrate Reductase Substrate: Nitrate Reagent: None Product: Nitrogen Gas Medium: Nitrate Broth
Bubbles in Durham Tube (+)- Nitrate Reductase No Bubbles (-)- No Nitrate