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Third lecture for microbiology Third lecture for microbiology manualmanual

Smear preparation and staining

Design by: Abed Al Rahman HamadDesign by: Abed Al Rahman Hamad

M.sc. Biological science M.sc. Biological science

2008

Preparation of Specimens for Light Microscopy

• A thin film of a solution of microbes on a slide is a smear.

• A smear is usually fixed to attach the microbes to the slide and to kill the microbes.

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• Live or unstained cells have little contrast with the surrounding medium. However, researchers do make discoveries about cell behavior looking at live specimens.

Preparing Smears for Staining

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• Stains consist of a positive and negative ion.

• In a basic dye, the chromophore is a cation.

• In an acidic dye, the chromophore is an anion.

• Staining the background instead of the cell is called negative staining.

Preparing Smears for Staining

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• Use of a single basic dye is called a simple stain.

• A mordant may be used to hold the stain or coat the specimen to enlarge it.

Simple Stains

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• Named after Christian Gram, Danish scientist and physician, 1853–1938.

• The Gram stain classifies bacteria into gram-positive and gram-negative.

• Gram-positive bacteria

• Gram-negative bacteria.

• The Gram stain technique is most reliable when performed on 24 hrs to 48 hrs or on sample directly taken from patient.

Differential Stains: Gram Stain

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Differential Stains: Gram Stain

Color of

Gram + cells

Color of

Gram – cells

Primary stain:

Crystal violet

Purple Purple

Mordant Iodine

Make complex with crystal violet and increase retention of stain

Purple Purple

Decolorizing agent:

Alcohol-acetone

Purple Colorless

Counterstain:

Safranin

Purple Red

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Differential Stains: Gram Stain

Figure 3.10b

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• Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast.

• Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with a different color basic stain) to see them.

• T.B. cell wall consist of wax D and mycolic acid.

Differential Stains: Acid-Fast Stain

Figure 3.11

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Procedure of acid fast stain:

Color of

Non acid fast bacteria

Color of

Acid fast bacteria

Primary stain:

Carbol fuchsin

Red Red

Heating Red Red

Decolorizing agent:

H2SO4

Colorless Red

Counterstain:

Methylene blue

Blue Red

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The kinyoun procedure

• Kinyoun’s procedure is often referred to as cold staining

the concentration of carbol fuchsin and phenol in the stain

has been increase and stain prepared with Tergitol No. 7

(heat is not necessary).

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The metachromatic granules stain

• Metachromatic granules or volutin are storage crystals produced by some bacteria when there excess phosphate and carbohydrate in their growth medium. These crystals are deposits of cellular phosphate and are large enough to see when stained.

• Corynebacterium diphtheriae is an example for this stain.

• There are two procedure for this stain:

• 1. Albert’s procedure

• 2. Loeffler’s procedure

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• Negative staining is useful for capsules (india ink stain).

• Malachite green is the stain used for examine endospore formation

• Heat is required to drive a stain into endospores.

• Flagella staining requires a mordant to make the flagella wide enough to see.

Special Stains

Figure 3.12a-c