Microbiology Laboratory Oral Exam Reviewer BIOL 105 2ND Semester

Flashcards about Gram Staining

Gram's Iodine (Mordant)

Attaches to negatively charged parts of the bacterial cell wall via electrostatic attraction and helps retain the stain inside Gram-positive cells.

GRAM STAINING

A staining technique used to classify bacterial species into large groups: Gram-positive bacteria and Gram-negative bacteria, based on the structure of their cell walls.

Purpose of Gram Staining

To identify and classify bacteria based on cell wall composition; to guide treatment decisions; to help diagnose bacterial infections quickly in clinical settings

Gram-positive bacteria

Bacteria with a thick peptidoglycan wall, which retains the crystal violet stain after decolorization.

Gram-negative bacteria

Bacteria with a thin peptidoglycan wall and an outer lipid membrane, which does not retain the primary stain and takes up the counterstain instead.

Crystal Violet (Primary Stain)

Stains all cells purple at first and allows for differentiation in later steps.

Gram’s Iodine (Mordant)

Forms a larger crystal violet–iodine complex.

95% Ethanol (Decolorizer)

In Gram-negative bacteria, it dissolves the outer membrane and washes out crystal violet-iodine complex; in Gram-positive bacteria, it dehydrates the cell wall, trapping the complex inside.

Safranin (Counterstain)

Stains decolorized Gram-negative cells pink/red to make them visible after they’ve lost the primary stain.

Function of 95% Ethanol in Gram Staining

Removes stain from Gram-negative cells and locks stain in Gram-positive cells.

Function of Safranin in Gram Staining

Stains Gram-negative bacteria pink/red.

Why Decolorization Step is Differential

Gram-positive cell walls shrink and trap the stain, while the outer membrane of Gram-negative cells is dissolved, making them lose the stain and appear colorless before safranin is added.

Why Gram-negative bacteria are important in medicine

They often show more antibiotic resistance and need special treatment.

Effect of Over-decolorization

Gram-positive may appear Gram-negative.

Effect of Under-decolorization

Gram-negative may appear Gram-positive.