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.
• Stains are salts composed of a positive and a negative ion,
• The colored ion is known as the chromophore.
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
• 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
• 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 – 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 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 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.
• 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 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
• 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
• 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
• Morphological stains help in identifying the cell size, shape and structure
• Simple stains color the cells
• Negative staining color the background
• Gram staining differentiates between gram positive and gram negative cells
• The difference in gram staining is due to difference in cell wall composition
• Stages of gram staining –
• Primary stain
• Acid fast staining is used to identify bacteria containing mycolic acid in cell wall – belonging to genus Mycobacterium and Nocardia
• It differentiates between acid fast and non-acid fast organisms
• Special structural stains like capsule spore and flagella staining, help in visualization of these bacterial structures
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