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Table of Contents

Identification of bacteria – Staining techniques

Intended learning objectives

At the end of this lecture, the student will be able to:

• Classify the staining techniques

• Explain the principle and procedure involved morphological and gram staining

• Outline the significance of staining in identification of bacteria

• Explain the principle and procedure of acid fast staining

• Explain the significance and principle of structural staining

Need for staining

• Most microorganisms appear almost colourless when viewed through a standard light microscope

• Hence must be fixed and stained to

– Increase visibility

– Accentuate specific morphological features

– Preserve them for future study

• Staining simply means colouring the microorganisms with a dye that emphasizes certain structures

Basic staining procedure

Step 1: Smear preparation

• A thin film of material containing the microorganisms is spread over the surface of the slide. This film, called a smear.

• It is allowed to air dry.

Step 2: Fixation

• By passing it through the flame of a bunsen burner several times, smear side up, or by covering the slide with methyl alcohol for one minute.

• Fixing simultaneously kills the microorganisms and fixes them to the slide.

• It also preserves various parts of microbes in their natural state with only minimal distortion

Step 3: Staining

• Stain is applied and then washed off with water

• Slide is blotted with absorbent paper.

• The stained microorganisms are now ready for microscopic examination.

Staining techniques

Stains

• Stains are salts composed of a positive and a negative ion,

• The colored ion is known as the chromophore.

Stain

Positive ion à Colored chromophore àBasic stain

Negative ion à Colored chromophore àAcidic stain

Bacteria are slightly negatively charged at pH 7.
Thus, the colored positive ion in a basic dye is attracted to the negatively charged bacterial cell.
Basic dyes include crystal violet, methylene blue, malachite green and safranin
Acidic dyes are not attracted to most types of bacteria
The dye’s negative ions are repelled by the negatively charged bacterial surface
The dye colors the background instead
Preparing colorless bacteria against a colored background is called negative staining.
Examples of acidic dyes are eosin, acid fuchsin, and nigrosin

Negative staining

• Valuable for observing overall cell shapes, sizes, and capsules

• The cells are made highly visible against a contrasting dark background

• Distortions of cell size and shape are minimized because fixing is not necessary and the cells do not pick up the stain

Simple staining

• A simple stain is an aqueous or alcohol solution of a single basic dye

• The primary purpose of a simple stain is to highlight the entire microorganism so that cellular shapes and basic structures are visible

• The stain is applied to the fixed smear for a certain length of lime and then washed off

• The slide is dried and examined

• Simple stains commonly used in the laboratory – methylene blue, carbolfuchsin, crystal violet, and safranin.

Mordant

• Mordant – A chemical that intensifies the stain

Functions of mordant

• To increase the affinity of a stain for a biological specimen

• To coat a structure (such as a flagellum) to make it thicker and easier to see after it is stained with a dye.

Differential stains

• Differential stains react differently with different kinds of bacteria

• Can be used to distinguish them

• The differential stains most frequently used for bacteria are the Gram stain and the acid-fast stain

Gram stain

• Gram stain was developed in 1884 by the Danish bacteriologist Hans Christian Gram

• Most useful staining procedures – classifies bacteria into two large groups: gram-positive and gram-negative.

Ø Step 1: Primary stain

Ø Step 2: Mordant

Ø Step 3: Decolorization

Ø Step 4: Counter stain

• The purple dye and the iodine combine in the cytoplasm of each bacterium and color it dark violet or purple.

• Bacteria that retain this color after the alcohol has attempted to decolorize them are classified as gram-positive

• Because gram-positive bacteria retain the original purple stain, they are not affected by the safranin counterstain

• Bacteria that lose the dark violet or purple color after decolorization are classified as gram negative

• Because gram-negative bacteria are colorless after the alcohol wash, they are no longer visible.

• This is why the basic dye safranin is applied; it turns the gram-negative bacteria pink.

• Stains such as safranin that have a contrasting color to the primary stain are called counterstains

Principle of Gram stain

• Different kinds of bacteria react differently to the Gram stain

• Structural differences in their cell walls affect the retention or escape of a combination of crystal violet and iodine, called the crystal violet- iodine (CV-I)complex

• Gram-positive bacteria have a thicker peptidoglycan cell wall than gram-negative bacteria

• Gram- negative bacteria contain a layer of lipopolysaccharide (lipids and polysaccharides) as part of their cell wall

Crystal violet + Iodine

• Enters Gram positive cell wall à Peptidoglycan layer retains CV-I during alcohol decolorization à Gram-positive cells retain the color of the crystal violet dye

• Enters Gram negative cell wall à Alcohol wash disrupts the outer lipopolysaccharide layer à CV- I complex is washed ou tthrough the thin layer of peptidoglycan à Gram negative Cells are colorless à Turn pink upon safranin staining

Clinical significance of Gram staining

• Gram reaction of a bacterium can provide valuable information for the treatment of disease.

• Gram-positive bacteria tend to be killed easily by penicillins and cephalosporins.

• Gram-negative bacteria are generally more resistant because the antibiotics cannot penetrate the lipopolysaccharide layer.

Acid-Fast Stain

• Used to identify all bacteria in the genus Mycobacterium and Nocardia

– Mycobacterium tuberculosis, the causative agent of tuberculosis

– Mycobacterium leprae the causative agent of leprosy

• Acid-fast stain binds strongly only to bacteria that have a waxy material in their cell walls

Acid fast staining Procedure

The red dye carbol fuchsin is applied to a fixed smear
The slide is gently heated for several minutes ((Heating enhances penetration and retention of the dye).

• The slide is cooled and washed with water.

• Smear is next treated with acid-alcohol, a decolorizer, which removes the red stain from bacteria that are not acid -fast.

• The acid-fast microorganisms retain the red color because the carbol fuchsin is more soluble in the cell wall lipids than in the acid-alcohol

Acid fast staining Principle

• The smear is then stained with a methylene blue counterstain.

• Non acid-fast cells appear blue after application of the counterstain

Special stains

• Special stains are used to color and isolate specific parts of microorganisms, such as endospores and flagella, and to reveal the presence of capsules.

Negative Staining for Capsules
Endospore (Spore) Staining
Flagella Staining

Negative Staining for Capsules

• Many microorganisms contain a gelatinous covering called a capsule

• Demonstrating the presence of a capsule is a means of determining the organism’s virulence, the degree to which a pathogen can cause disease.

• Step 1: Mix the bacteria in a solution containing a fine colloidal suspension of colored particles (usually India ink or nigrosin)

• Step 2: Provide a contrasting background and then stain the bacteria with a simple stain, such as safranin

• Due to their chemical composition, capsules do not accept most biological dyes, such as safranin, and thus appear as halos surrounding each stained bacterial cell.

Endospore (Spore) Staining

• Endospore is a special resistant, dormant structure formed within a cell

• Protects a bacterium from adverse environmental conditions

• Endospores cannot be stained by ordinary methods

• The dyes do not penetrate the wall of the endospore

Schaeffer- Fulton endospore stain

• Malachite green, the primary stain, is applied to a heat-fixed smear

• Heated to steaming for about 5 minutes (i.e, malachite green permeate the spore wall)

• Washed for about 30 seconds with water to remove the malachite green from all of the cells parts except the endospores.

• Safranin, a counterstain, is applied to the smear to stain portions of the cell other than endospores.

• The endospores appear green within red or pink cells

Acid-Fast Staining

• Note: In Gram Staining and AFB Staining we use Alcohol or Acid Alcohol or Acid as a decolorizer but in spore staining water is sufficient ( to be used as decolorizer) because:

Ø malachite green dye is water-soluble and does not adhere well to the cell wall

Ø vegetative cells have been disrupted by heat,

Ø because of these reasons, the malachite green rinses easily from the vegetative cells

Flagella staining

• Bacterial flagella (singular: flagellum) are structures of locomotion too small to be seen with a light microscope without staining.

• A tedious and delicate staining procedure uses a mordant and the stain carbol fuchsin to build up the diameters of the flagella until they become visible under the light microscope