KK

Lab 12

Clean 2

  • Microscopes
  • Slides: $4
  • AF (-)
  • AF (+)
  • Morton Publishing Company

Ziehl-Neelsen Method For Acid Fast Staining

  • Publisher: Morton Publishing Company

Bacterial Cell Walls

  • Differences in bacterial cell walls:
    • Gram Negative
    • Characterized by a thin peptidoglycan layer
    • Contains lipopolysaccharides
    • Gram Positive
    • Characterized by a thick peptidoglycan layer
    • Acid Fast
    • Characterized by a thin peptidoglycan layer
    • Contains a thick mycolic acid layer

Mycolic Acid

  • Characteristics
    • Mycolic acid makes staining tricky
    • It is a waxy, sticky lipid that repels aqueous dyes
    • Appears as red squiggly lines in microscopic images
    • Contributes to cell clumping
    • Provides resistance to decolorization using ethyl alcohol
  • Staining Solutions
    • Carbol Fuchsin (CF) is used as the primary dye
    • Contains phenol as a base instead of water
    • Phenol is lipid-soluble and penetrates mycolic acid more easily
  • Heat Application
    • Steaming with Bunsen burner enhances dye penetration
    • Heat melts the waxy mycolic acid like a candle, mixing with CF
    • Upon cooling, the solidified mycolic acid traps the CF inside

Further Characteristics of Mycolic Acid

  • Hydrochloric acid mixed with ethyl alcohol is used for decolorization
    • Mycolic acid resists decolorization with this mixture
    • Gram positive cells, while resistant to simple ethyl alcohol, are not resistant to acid alcohol
    • This allows them to take on the secondary dye instead of the primary
    • Acid Fast cells are named for their resistance to decolorization by acid

Importance of Acid Fast Staining

  • Key Pathogenic Organisms
    • Mycobacterium tuberculosis
    • Causative agent of tuberculosis
    • Highly contagious with known antibiotic-resistant strains
    • Mycobacterium leprae
    • Causative agent of leprosy
    • Cryptosporidium oocysts
    • Causes diarrheal disease
    • Isospora oocysts
    • Causes diarrheal disease
    • Nocardia
    • Commonly infects the lungs, causing pneumonia
    • Can spread to the brain and cause skin infections

Rationale for Performing Acid Fast Stains

  • Very few organisms are acid fast
  • AF stains are performed when infection from an acid-fast organism is suspected
  • The accuracy of AF stains is over 90% in diagnosing Mycobacterium tuberculosis from a sputum sample
  • The slow-growing nature of Mycobacterium tuberculosis can delay diagnosis via culture, necessitating rapid testing
  • Patients cannot wait for bacterial culture as they may spread the disease during this time

Acid Fast Pathogens: Key Species

  • Mycobacterium leprae
    • Causative agent of leprosy (lepromatous and tuberculoid forms)
    • Attacks and damages nerves causing numbness and potential loss of function
  • Mycobacterium tuberculosis
    • Causative agent of tuberculosis
    • Leads to symptoms like bloody sputum and violent coughing
    • Transmittable via airborne respiratory droplets
    • Many medical professionals test positive for latent TB due to exposure
    • Ghon complex can form in lung tissue where it is sheltered from the immune response
    • If the immune system weakens, the infection can reactivate

Ziehl-Neelsen Method Procedure

  • No serum will be used during this procedure
  • Steps:
    1. Prepare a heat-fixed smear, breaking up bacterial clumps
    2. Place filter paper on the slide
    3. Apply Carbol Fuchsin dye
    4. Heat the dye with a flame until steam arises (5 minutes)
    • Avoid letting dye desiccate, boil, or cool
    1. Remove the filter paper (do not dispose of in the sink), rinse slide with water
    2. Decolorize with acid alcohol until runoff is clear
    3. Rinse with water
    4. Apply secondary dye (methylene blue) for at least 5 minutes
    5. Rinse off the secondary dye completely, then rinse, blot, and view using oil immersion lens

Results of Acid Fast Staining

  • Before staining: Cells are transparent
  • After staining with carbolfuchsin: Cells appear reddish-purple
    • Steaming enhances the dye's entry
    • Acid-fast negative cells lose the stain during decolorization with acid alcohol
  • Counterstaining: Methylene blue or brilliant green is used for acid-fast negative cells
  • Critical Thinking:
    • Acid-fast cells would appear gram-positive due to the thick peptidoglycan layer
    • The mycolic acid layer makes them resistant to staining without steaming, resulting in a purpleish-reddish appearance

Endospore Staining Overview

  • Endospore Formation Steps:
    1. Start as vegetative cells that are metabolically active
    2. Vegetative cells deposit dipicolinic acid and form a pocket with DNA
    3. Layering peptidoglycan and keratin proteins form the spore coat
    4. Vegetative cell dies, leaving a free endospore that is metabolically inactive
    5. Germination occurs when environmental conditions are favorable, making spores permeable to water and nutrients, returning them to vegetative form

Endospore Characteristics

  • Definition: Endospores are a dormant form of bacteria that ensure survival in hostile environments
  • After sporulation, cell metabolism is significantly reduced until conditions are right for germination
  • Environmental factors such as desiccation, starvation, and radiation can trigger sporulation
  • Endospores resist heat and chemicals due to a tough keratin outer coat, complicating staining

Endospore Staining Techniques

  • Primary Dye: Malachite green, which is steamed for dye penetration
    • This expands the spore coat to allow entry of malachite green
    • Removal of heat causes contraction of the spore coat, trapping the dye
  • Decolorization: Water is used to remove stain from vegetative cells; spores retain the dye
  • Secondary Dye: Vegetative cells take on secondary dye colors while endospores appear green

Identifying Endospore Producers

  • Genera that produce endospores include Clostridium and Bacillus
  • Identifying characteristics based on:
    • Shape: Spherical, oval (elliptical)
    • Size: Small, medium, large, bulging
    • Location: Terminal, central, or subterminal
  • Spore presence and characteristics play an important role in diagnostic criteria for identification

Pathogenic Sporulating Bacteria

  • Clostridium botulinum
    • Anaerobic, gram-positive bacillus
    • Causative agent of botulism: causes flaccid paralysis due to a toxin
    • Commonly linked to contaminated canned foods; endospores found in honey pose risk for infants
  • Clostridium tetani
    • Anaerobic, gram-positive bacillus causing tetanus
    • Results in spasmodic paralysis and overstimulation of muscles, potential for breaks from spasms
    • Found in soil and important to consider due to injury risks
  • Clostridium difficile
    • Anaerobic, gram-positive bacillus causing severe gastrointestinal infections
    • Highly antibiotic-resistant, often necessitating fecal transplants for treatment
  • Bacillus anthracis
    • Causes three forms of anthrax (cutaneous, respiratory, gastrointestinal)
    • Notable for its sporulation in outbreaks, such as the 2001 bioterrorism events

Endospore Staining Process

  • Steps:
    1. Heat malachite green dye until steam arises (5 minutes)
    2. Rinse the slide and remove the contaminated paper
    3. Apply secondary dye (safranin) for 1-3 minutes
    4. Rinse, blot, and view under an oil immersion lens

Capsule Staining Overview

  • Capsule Composition: Composed of mucoid polysaccharides or polypeptides, resistant to normal staining
  • High water content leads to evaporation during heat fixation
  • Capsules help in adhesion, immune evasion, and resistance to phagocytosis by leukocytes

Challenges in Capsule Staining

  • Due to the capsule's properties, a unique blend of acidic and basic dyes is used
    • The acidic dye stains the background while the basic dye stains the cell, leaving the capsule clear

Capsule Staining Procedure

  1. Prepare a clean glass slide with a drop of sheep serum and Congo red stain
  2. Aseptically add organisms and emulsify
  3. Use a second slide to spread the mixture across the first slide
  4. Air-dry and do not heat-fix
  5. Cover with Maneval's stain for 1 minute, then rinse with distilled water
  6. Let air-dry, and observe under oil immersion lens

Important Notes on Capsule Staining

  • Due to the complexity, demo slides may be provided for visual learning

Identification of Acid Fast Stain and Endospore Stain for Students

  • For Acid Fast Stain:
    • Use the provided “AF negative mix” as a water base
    • Mix Mycobacterium smegmatis culture into the broth mixture
    • Record details of AF reaction, morphology, and arrangement
  • For Endospore Stain:
    • Create a combined sample from Bacillus subtilis and Bacillus terminalis
    • Record morphology, arrangement, and endospore characteristics
    • Clean and label slides accordingly