Exercise 4 - Differential Staining

BACTERIAL SMEAR

It is the primary material for morphology screening or evaluation

• SIMPLE STAIN

• DIFFERENTIAL STAIN

• SPECIAL STAIN

3 Kinds of Stains:

SIMPLE STAIN

A kind of stain that uses one stain

SIMPLE STAIN

A kind of stain that cannot differentiate different colors

DIFFERENTIAL STAIN

A kind of stain that routinely used

DIFFERENTIAL STAIN

A kind of stain that uses 2 or more stains

DIFFERENTIAL STAIN

A kind of stain that can differentiate different colors

SPECIAL STAIN

A kind of stain that not routinely used

SPECIAL STAIN

A kind of stain that used to demonstrate structures

GRAM STAINING

Other term of Differential Staining

Hucker’s Method

Other term of Gram’s Staining

• CRYSTAL VIOLET

• GRAM’S IODINE

• ACETONE

• SAFRANIN

Differential Staining:

GENTIAN VIOLET

What is the alternative of Crystal violet

HEXAMETHYL-PARA-ROSANILINE CHLORIDE

Chemical name of Crystal Violet

CRYSTAL VIOLET

What is the primary stain used in Gram staining?

GRAM’S IODINE

What is the mordant used in Gram staining?

ACETONE ALCOHOL

What is the decolorizer used in Gram staining?

SAFRANIN

What is the counterstain used in Gram staining?

VIOLET/PURPLE-COLORED SOLUTION

Colored solution of Crystal violet

BROWN-COLORED SOLUTION

Colored solution of Gram’s Iodine

COLORLESS SOLUTION

Colored solution of Acetone

RED-COLORED SOLUTION

Colored solution of Safranin

• PRIMARY STAINING

• MORDANT

• DECOLORIZER/ DIFFERENTIATION

• COUNTER STAINING/ SECONDARY STAINING

Gram Staining Steps:

PRIMARY STAINING

Dip smear in crystal violet solution for 1 minute

MORDANT

Dip smear in iodine for 1 minute

DECOLORIZATION/ DIFFERENTIATION

Dip smear in acetone alcohol for 5 seconds

COUNTER STAINING/ SECONDARY STAINING

After dipping in acetone, counterstain with safranin for 1 minute

THICK PEPTIDOGLYCAN LAYER W/ TEICHOIC ACID

Why do Gram-positive bacteria retain the crystal violet stain?

THIN PEPTIDOGLYCAN LAYER W/ LIPOPOLYSACCHARIDES

Why do Gram-negative bacteria not retain the crystal violet stain?

DISACCHARIDES & AMINO ACIDS

Teichoic acid is composed of

LIPIDS & POLYSACCHARIDES

Lipopolysaccharide is composed of

PRIMARY STAINING

Both gram (+) and gram (-) organisms appear violet because it is the only stain employed yet

DECOLORIZATION/ DIFFERENTIATION

Gram (+): Cell wall will remain intact and will never get destroyed because of teichoic acid and the crystal violet color is retained

Gram (-): Cell wall is destroyed and the crystal violet is decolorized, making them colorless

COUNTER STAINING/ SECONDARY STAINING

Gram (+): Will no longer take up the red color and retain their purple color

Gram (-): Will take up the safranin and red color, appearing red

VIOLET/ PURPLE

Gram (+) will appear what color

RED/ PINK

Gram (-) will appear what color

HEAT FIXING

simultaneously kills and allows the smear to adhere to the slide and create pores or openings in its cell wall which makes the crystal violet easily enter the cell walls of the gram (+) microorganism

CRYSTAL VIOLET

is smaller than the pores and openings in the cell wall

IODINE MOLECULES

also easily penetrate via the pores or openings

SPECIAL STAINS

used to color / stain and isolate specific parts of the organism that are responsible for the virulence of the microorganism

CARBOL FUSCHIN

What is the primary stain used in acid-fast staining?

ACID ALCOHOL

What is the decolorizer used in acid-fast staining?

LOEFFLER’S METHYLENE BLUE

What is the counterstain used in acid-fast staining?

RED/ PINK

What color do acid-fast positive bacteria appear after staining?

BLUE/ GREEN

What color do non-acid-fast bacteria appear after staining?

HEATING/ STEAMING

Bring flame of alcohol lamp to non-frosted side of the glass slide until steam comes off of the slide

ZIEHL-NEELSEN METHOD

A hot acid-fast staining technique that uses heat to drive the stain into the cell wall

KINYOUN METHOD

A cold acid-fast staining technique that does not require heat but uses a higher concentration of phenol