TOPIC 3: DIFFERENTIAL STAINING

BACTERIAL SMEAR PREPARATION

1. Heat the glass slide to remove grease, then place it flame-side up on the table.

2. Sterilize the wire loop until it becomes red-hot.

3. If the specimen source is solid media:

   • Place a loopful of sterile distilled water on the slide's center.

   • Flame the mouth of the tube first (aseptic technique). If the source is liquid media:

   • Directly place it on the slide. Use a loopful of bacteria ½ inch from the slide.

4. Air dry to preserve the specimen's morphology.

5. Fix the smear with heat by passing the slide over a flame 3-5 times. This allows the smear to adhere and prevents it from washing off during decolorizing.

6. Stain with the desired stain.

7. Wash off with water.

8. Blot dry with filter paper.

9. Examine under an Oil Immersion Objective (OIO).

Gentian Violet

This is used as the primary stain of this kind of staining, a basic dye that forms complex with the peptidoglycan, it initially stains the cell wall of the bacteria.

Gram’s Iodine

This serves as the mordant that chemically binds with the cell wall of bacteria and increases the affinity of the Crystal Violet (primary stain).

Acetone Alcohol

This is the decolorizing agent, chemical that distinguishes Gram (+) bacteria from Gram (-) and is considered to be the most important stage. Gram (-) contains lipids that are soluble and will dissolve in the alcohol, this is why the Gram (-) organism will appear as clear.

Safranin

The red dye that makes the Gram (-) organism appear pink or red after the staining

blue/violet

Gram (+) color

red/pink

Gram (-) color

Teichoic acid

Gram-positive cell wall has thick peptidoglycan layer with _______ _______. This is insoluble in alcohol, and this is made up of disaccharides and amino acids. Being amino acids means it is rich in protein. o This can’t be destroyed by alcohol.

Lipopolysaccharides

• Gram-negative cell wall is composed of thin peptidoglycan layer with __________are soluble in alcohol. Made up of lipids and polysaccharides.

DIFFERENTIAL STAINS

are used to demonstrate the physio chemical differences among bacteria that aid in their description and classification

Setup for Gram’s Staining

glass slide, filter paper, slide holder, wire loop (1 uL), burner, broth, Gentian violet, Gram’s Iodine, Acetone Alcohol, Safranin

HUCKER’S METHOD

• Gram-Positive (+) Organisms: Have thick peptidoglycan and lipoteichoic acid cross-links.

• Gram-Negative (-) Organisms: Have thin peptidoglycan and lipids.

Issues with Staining:

• Gram (+) appears to be Gram (-):

  • Removal of messenger RNA

  • Precipitation with various salts

  • Aging or old specimen

  • Drying

  • Using acidic stain

  • Technical error (complete decolorization of the smear)

• Gram (-) appears to be Gram (+):

  • Incomplete decolorization

  • Thick smear

GRAM’S RULE:

• All COCCI are Gram (+) except for Neisseria, Moraxella, Veillonella, Branhamella.

• All BACILLI are Gram (-) except for Mycobacterium, Corynebacterium, Bacillus, Erysipelothrix, Lactobacillus, Listeria, & Nocardia.

• All SPIRALS are Gram (-).

ACID-FAST STAINING / Ziehl-Neelsen

• Developed by Franz Ziehl and Friedrich Neelsen.

• Acid-Fast Bacteria: Organisms not decolorized by acid once stained, due to high lipid content (mycolic acid) in their cell wall.

• N-acetyl-L-cysteine: Used to digest mucus of all specimens. Some sources describe its method as "The Lipid-barrier Principle."

Staining Process:

1. Carbol Fuchsin: Primary stain containing phenol that penetrates the Mycobacterium cell wall.

2. Steaming: Softens mycolic acid, making it penetrable by the stain.

3. Acid Alcohol: Decolorizer containing 95% ethanol in concentrated HCl.

4. Loeffler’s Methylene Blue: Counterstain.

Results:

• Mycobacterium: Red or green background.

• Non-acid-fast: Blue or green.

• Macrophage: Green/blue.

Alternative Techniques to Avoid Steaming:

• Increase the concentration of phenol or dye.

• Prolong the application of dye.

• Add wetting agents like Tergitol.

Set-up: Glass slide, wire loop, burner, formalin-fixed sputum, carbol fuchsin, acid alcohol, methylene blue.

mycolic acid

• Acid-Fast Bacteria: Organisms not decolorized by acid once stained, due to high lipid content ( ______ _______) in their cell wall.

N-acetyl-L-cysteine

Used to digest mucus of all specimens. Some sources describe its method as "The Lipid-barrier Principle."

• Carbol Fuchsin

• Steaming

• Acid Alcohol

• Loeffler’s Methylene Blue

Staining process of Acid-fast Staining

• Mycobacterium: Red or green background.

• Non-acid-fast: Blue or green.

• Macrophage: Green/blue.

Results/ color of acid-fast staining:

• Mycobacterium:

• Non-acid-fast:

• Macrophage:

Alternative Techniques to Avoid Steaming

• Increase the concentration of phenol or dye.

• Prolong the application of dye.

• Add wetting agents like Tergitol.

Set-up for acid fast staining

Glass slide, wire loop, burner, formalin-fixed sputum, carbol fuchsin, acid alcohol, methylene blue.

MODIFIED ACID-FAST STAINING / Kinyoun Method

• Developed by Joseph Kinyoun.

• Cold Method: No heating or steaming required. Uses an increased concentration of phenol to penetrate the bacterial cell wall.

Clinical Specimens

: Samples taken from the patient's body, such as blood or any body fluid.

Three Phases of Workflow in the Clinical Laboratory

• Pre-Analytical Phase:

  • Guidance on sample collection.

  • Ensuring the integrity of the specimen.

  • Most critical phase; errors affect subsequent phases.

• Analytical Phase:

  • Examination of the specimen.

• Post-Analytical Phase:

  • Laboratory sends the report to the physician for interpretation.

• Size

• Color

• Shape

• Arrangement

Morphology of the Microorganism:

Preparation of Smears

• Specimen Collection: Specimens from cotton swabs are collected using an applicator stick with cotton at the tip, which is rolled onto the specimen.

• Sterilization: Cotton swabs are autoclaved to ensure they are sterile.

• Transfer to Slide: Absorbed material from the cotton swab is transferred to a clean microscope slide for easier microscopic examination.

• Frosted End: The frosted end of the glass slide is marked with pencil to indicate where patient details should be placed. The non-frosted side is used for the smear.

• Patient Details to Include on Frosted End:

  • Patient’s name (family and first name)

  • Birth date

  • Sex

  • Date and time of specimen collection

  • Specimen source

• Transferring Material:

  • Roll the cotton swab over the surface of the glass slide to transfer the material.

  • Reminder: Do not rub the cotton swab back and forth; it may not transfer the entire specimen.

• Consistency Check:

  • Prepare two different bacterial smears from the same patient and specimen source to ensure consistency in results.

SMEARS FROM THICK LIQUIDS OR SEMISOLIDS

• Specimen Collection: _________ ____ like _______ or _______ are collected by the patient in a sterile container and sent to the laboratory.

• Preparation:

  • Immerse a cotton swab in the container to absorb the material.

  • Roll the cotton swab over a glass slide to transfer the absorbed material.

SMEARS FROM THICK, GRANULAR, OR MUCOID MATERIALS

• Specimen Characteristics: Granular or mucoid materials are similar to sputum but with visible granules.

• Preparation Steps:

  1. Remove Granules: Fish out granules from the container and place them on a labeled glass slide.

  2. Create Smear: Place a second slide, frosted side down, over the material.

  3. Flatten and Rotate: Press and rotate the slides to spread the material and create two different smears for consistent results.

SMEARS FROM THIN FLUIDS

• Specimen Characteristics: Thin fluids have low protein content and cellular count, making them hard to visualize after staining.

• Preparation Steps:

  1. Drop Placement: Place a drop of the thin fluid in the pre-marked center of a glass slide using a wax pencil.

  2. Air Dry and Stain: Air dry the specimen before staining.

• Cytocentrifuge Preparation:

  1. Centrifugation: Place the thin fluid in a test tube and centrifuge to concentrate cellular elements at the bottom.

  2. Slide Preparation: Discard the liquid portion and transfer the concentrated sediment to a clean, labeled glass slide.

  3. Thin Smear: Emulsify the thick material with a wire loop and create a thin smear.

AIR DRYING

Importance: Ensure the smear is completely air-dried to preserve morphology. Premature drying can result in washed-out areas, leading to potential loss of important findings.

HEAT FIX

• Process: Expose the non-frosted side of the glass slide to the flame of an alcohol lamp several times. This step kills microorganisms and adheres the smear to the slide, preventing it from washing out during staining.

STAINING OF SMEARS

Purpose: Coloring microorganisms with a dye to enhance visibility.

Staining Principles

• Stains consist of positive and negative ions.

• Chromophore: The ion that colors the microorganism.

• Positive Ions: Carry the color of basic dyes and attract to the slightly negative cell wall.

• Negative Ions: Carry the color of acidic dyes, which repel from the cell wall and color the background.

Types of Staining Techniques

• Simple Stain: Uses a single dye to color microorganisms. Not effective for detailed morphology.

• Differential Stain: Uses multiple stains to differentiate organisms based on color.

  • Gram Stain: Differentiates between Gram-positive and Gram-negative bacteria.

    • Steps:

      1. Application of crystal violet (primary stain).

      2. Application of iodine (mordant).

      3. Alcohol wash (decolorization).

      4. Application of safranin (counterstain).

• Special Stains: Used to stain specific structures of microorganisms.

  • Examples:

    • Wirtz method for spores/endospores

    • Maneval’s staining for capsules

    • Leifson’s method for flagella

    • Albert stain, Loeffler’s methylene blue, or Gram stain for metachromatic granules

Chromophore

The ion that colors the microorganism.

Positive Ions

Carry the color of basic dyes and attract to the slightly negative cell wall.

Negative Ions

Carry the color of acidic dyes, which repel from the cell wall and color the background.

Simple Stain

Uses a single dye to color microorganisms. Not effective for detailed morphology.

Differential Stain

Uses multiple stains to differentiate organisms based on color.

• Gram Stain: Differentiates between Gram-positive and Gram-negative bacteria.

  • Steps:

    1. Application of crystal violet (primary stain).

    2. Application of iodine (mordant).

    3. Alcohol wash (decolorization).

    4. Application of safranin (counterstain).

1. Crystal Violet (primary stain)

2. Mordant (iodine)

3. Decolorization (alcohol wash)

4. Safranin (counterstain)

Gram Stain steps

Wirtz method

Maneval’s method

Leifsons’s method

Loeffler’s methylene blue

Special Stains: Used to stain specific structures of microorganisms.

• ____ _____ for spores/endospores

• ______ staining for capsules

• _______ method for flagella

• _____ ___ for metachromatic granules

Centrifugation

Method for thin fluids to stain

Place the thin fluid in a test tube and ______ to concentrate cellular elements at the bottom.

hexamethyl-para-rosanaline chloride; gentian violet

Chemical name for Crystal violet

• Alternative Name: ______ ______when Crystal Violet is unavailable)

Gram staining procedure and its purpose

1. Primary Stain:

• Stain Used: Crystal Violet (hexamethyl-para-rosanaline chloride)

• Alternative Name: Gentian violet (when Crystal Violet is unavailable)

• Application Time: 60 seconds (1 minute)

• Effect: Both Gram-positive and Gram-negative organisms appear violet.

2. Mordant:

• Reagent Used: Gram’s Iodine (brown-colored reagent)

• Purpose: Intensifies the color of the primary stain by forming a complex with crystal violet.

• Effect:

  • Gram-Positive Organisms: Thick peptidoglycan and teichoic acids remain intact and retain violet color.

  • Gram-Negative Organisms: Lipopolysaccharides are destroyed, leading to decolorization of crystal violet.

3. Decolorization:

• Reagent Used: Acetone Alcohol (combination of acetone and alcohol)

• Purpose: Differentiates Gram-positive (remains violet) from Gram-negative organisms (becomes colorless).

• Effect:

  • Gram-Positive Organisms: Retain crystal violet-iodine complex; remain violet.

  • Gram-Negative Organisms: Cell wall destroyed, crystal violet is removed, leaving the organism colorless.

4. Counterstain:

• Stain Used: Safranin (red color)

• Purpose: Stains Gram-negative organisms red.

• Effect:

  • Gram-Positive Organisms: Remain purple/violet.

  • Gram-Negative Organisms: Take up the red stain and appear pink/red.

Principle of Staining:

• Gram-Positive Organisms: Thick polypeptide layer with teichoic acids; crystal violet-iodine complex is trapped due to protein coagulation.

• Gram-Negative Organisms: Thin peptidoglycan layer with lipopolysaccharides; cell wall is destroyed, allowing safranin to stain.

polypeptide layer with teichoic acids

Gram-Positive Organisms: Thick ________ _______ with ______ _____; crystal violet-iodine complex is trapped due to protein coagulation.

peptidoglycan layer with lipopolysaccharides

• Gram-Negative Organisms: Thin _______ _____ ______ _____ ______; cell wall is destroyed, allowing safranin to stain.

ACID-FAST STAINING procedure and its purpose

. Ziehl-Neelsen Method (Hot Method):

• Primary Stain: Carbol Fuchsin (red color)

• Purpose: Stains acid-fast organisms red.

• Process:

  • Heat Fixing: Opens pores in the cell wall.

  • Steaming: Heat application causes mycolic acid to relax, allowing carbol fuchsin to penetrate.

  • Cooling: Closes pores, trapping carbol fuchsin in acid-fast organisms.

  • Decolorizer: Acid Alcohol (removes carbol fuchsin from non-acid-fast organisms).

2. Kinyoun Method (Cold Method):

• Primary Stain: Carbol Fuchsin (red color)

• Purpose: Stains acid-fast organisms red without heating.

• Process:

  • Increased Phenol Concentration: Enhances staining effectiveness.

  • Decolorizer: Acid Alcohol (removes carbol fuchsin from non-acid-fast organisms).

Principle of Staining:

• Acid-Fast Organisms: Cell wall composed of mycolic acid (fatty acids); resists decolorization due to its slippery nature.

• Non-Acid Fast Organisms: No mycolic acid; carbol fuchsin is easily removed by acid alcohol and stained blue with LAMB (Loeffler’s Alkaline Methylene Blue).

mycolic acid

• Acid-Fast Organisms: Cell wall composed of _____ ____ (fatty acids); resists decolorization due to its slippery nature.

Methylene blue

• Non-Acid Fast Organisms: No mycolic acid; carbol fuchsin is easily removed by acid alcohol and stained blue with LAMB (Loeffler’s Alkaline ______ _____).

1. Carbol Fuchsin

2. Steaming

3. Acid Alcohol:

4. Loeffler’s Methylene Blue

Acid-fast staining steps