GRAM and ACID FAST STAINS Differential Stains Gram

GRAM and ACID FAST STAINS

Differential Stains • Gram Stains and Acid Fast Stains are differential stains (use more than one color) – Color should be on opposite sides of the color wheel to make visualization easier

What is a gram stain? • One of the most important biological staining processes in microbiology • Differential stain • Used to separate all known bacteria into 2 groups – Gram positive – Gram negative • Dependent upon the construction of the bacteria's cell wall – The cell will either retain or lose the first dye

Cell Wall Of Gram Pos & Neg

The Gram Stain Procedure Step 1 - Prepare a Smear Prepare the smear as we did last week. Use a mix of either: • Staph and Pseudomonas • Staph and Bacillus • Bacillus and Pseudomonas Watch what happens to the “Bacteria” at each step “Bacteria”

The Gram Stain Procedure Step 2 - Apply the Primary Stain Flood the Smear with Crystal Violet. Crystal violet is a basic dye, stains all the cells purple The dye is positively charged and is attracted to the negative charge of the bacterial cell wall Allow to stand for 1 min, rinse with water to remove excess stain

The Gram Stain Procedure Step 3 - Apply the Mordant Flood the Smear with Iodine solution The iodine forms an insoluble complex with the crystal violet to anchor it into the cell wall Allow to stand 1 min

The Gram Stain Procedure Step 4 - Rinse with water to remove excess Iodine

The Gram Stain Procedure Step 5 - Decolorizer Drip Decolorizer (80% Methanol +20% Acetone) across the slide about 2 -3 sec This removes the outer membrane of the gram negative bacteria, leaches the dye-mordant complex out of gram negative cells

The Gram Stain Procedure Step 6 - Rinse immediately with water to remove excess alcohol

The Gram Stain Procedure Step 7 - Counterstain Flood the slide with Safranin solution Let stand for 1 minute The gram negative cell wall will pick up the dye.

The Gram Stain Step 8 - Rinse, Dry and Observe Rinse with water to remove excess stain Blot dry Observe under Oil Immersion Gram-Positive Gram-Negative

The Acid Fast Stain • AFB Stain used to detect: – Mycobacterium • Mycobacterium tuberculosis • Mycobacterium leprae – Nocardia

Bacterial Cell Walls

The Acid Fast Stain Procedure Step 1 - Prepare a Smear Prepare the smear as we did last week. Use a mix of either: • Staph and Mycobacterium phlei. . be sure to mix really, really well Watch what happens to the “Bacteria” at each step “Bacteria”

The Acid Fast Stain Procedure Step 2 - Apply the Primary Stain Flood the Smear with Carbolfuchsin The dye is positively charged and is attracted to the negative charge of the bacterial cell wall Allow to stand for 5 min, rinse with water to remove excess stain

The Acid Fast Stain Procedure Step 5 - Decolorizer Flood slide with Decolorizer (ACID-alcohol) across the slide about 30 sec This removes the peptidoglycan layers of the gram positive and negative bacteria. . but the mycolic acid of the acid fast positive bacteria remains

The Acid Fast Stain Procedure Step 7 - Counterstain Flood the slide with Methylene Blue solution Let stand for 30 seconds

The Acid Fast Stain Step 8 - Rinse, Dry and Observe Rinse with water to remove excess stain Blot dry Observe under Oil Immersion No Acid Fast Bacilli Seen