Bacterial Staining Techniques

Objectives of Staining

• Staining is used to determine bacterial morphology, differentiate bacterial groups, and identify organisms with special structures.

Kinds of Ionizable Dyes Used in Staining Bacteria

• Basic dyes are cationic dyes with positively charged groups that bind to negatively charged molecules like nucleic acids and proteins.
  • Examples include methylene blue, basic fuchsin, crystal violet, safranin, and malachite green.
• Acidic dyes are anionic dyes with negatively charged groups that bind to positively charged cell structures.
  • Examples include eosin, rose Bengal, and acid fuchsin.

Staining Techniques

• Simple staining uses a single stain to color all cells, revealing their shape and forms.
  • Example: methylene blue.
• Differential staining divides bacteria into groups based on staining properties.
  • Examples: Gram staining and acid-fast staining.
  • Steps involved in differential staining:
    • Application of the primary stain.
    • Application of the mordant.
    • Application of the decolorizing agent.
    • Application of the secondary stain/counterstain.
• Negative staining uses stains repelled by negatively charged bacterial surfaces, leaving the bacteria light-colored against a dark background.
  • Useful for studying bacterial capsules, gas vacuoles, and viral morphology.
  • Examples: India ink or Nigrosin dye.

Gram Stain

• The most widely used differential stain in clinical microbiology.
• Uses crystal violet as the primary stain and safranin as the secondary stain.
• Iodine acts as the mordant, and acetone-alcohol mixture as the decolorizing agent.
• Decolorization step is the key to differentiating bacteria.
• Gram-positive bacteria have thick cell walls containing teichoic acid, retaining the crystal violet-iodine complex and appearing purple.
• Gram-negative bacteria have thinner cell walls containing lipopolysaccharides, losing the dye complex and appearing pink or red.
• Precautions during Gram staining:
  • Avoid excessive rinsing of crystal violet before iodine application to prevent unstained Gram-negative bacteria.
  • Prolonged decolorization may falsely result in Gram-negative appearance of Gram-positive bacteria.
  • Insufficient decolorization may lead to false Gram-positive appearance.
• General rules for Gram staining:
  • Most cocci are Gram-positive except for Neisseria, Veilonella, and Branhamella (Moraxella).
  • Most bacilli are Gram-negative except for Actinomadura, Arcanobacterium, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Gordonia, Kuthria, Listeria, Mycobacterium, Nocardia, Rhodococcus, Streptomyces, Tropheryma whipplei, and Tsukamurella.
• Reasons for Gram-positive bacteria becoming Gram-negative:
  • Aged, dying, or autolyzing cells.
  • The use of acidic iodine during staining.
  • Technical errors or incorrect use of stains.

Exceptions in Gram Staining

• Organisms residing mostly within host cells (Chlamydia).
• Organisms lacking cell walls (Mycoplasma and Ureaplasma).
• Organisms too small to be resolved by light microscopy (Spirochetes).

Acid-Fast Staining

• Used to stain bacteria with high lipid content in their cell walls.
• Uses carbol fuchsin as the primary stain and methylene blue or malachite green as the secondary stain.
• Acid-fast bacteria resist decolorization with acid-alcohol, retaining the primary stain and appearing pink or red.
• Non-acid-fast bacteria are decolorized and stained blue or green by the secondary stain.
• Methods of acid-fast staining:
  • Ziehl-Neelsen/Hot staining method.
  • Kinyoun's/Cold staining method.
  • Pappenheim method (differentiates Mycobacterium smegmatis from Mycobacterium tuberculosis).
  • Baumgarten method (differentiates Mycobacterium leprae from Mycobacterium tuberculosis).
  • Auramine-rhodamine method (selective for AFB cell walls).

Ways to Facilitate Acid-Fast Staining

• Heating or steaming for 5-7 minutes to remove mycolic acid.
• Increasing the concentration of dye and phenol in the staining reagent.
• Prolonged contact of the specimen with the primary stain.
• Adding a wetting agent like tergitol.

Modified Acid-Fast Staining Method

• Used for identifying intestinal coccidian oocysts, especially Cryptosporidia and Cyclospora in stool specimens.
• Uses the same reagents as conventional acid-fast staining except for the concentration of acid alcohol (1% H₂SO₄).
• Oocysts appear magenta-stained against a blue background.

Notes to Remember about Acid-Fast Staining

• Acid-fastness can be affected by colonial age, growth medium, and UV light.
• Ziehl-Neelsen method is suitable for concentrated smears and partially acid-fast bacilli like Nocardia species.
• Acid-alcohol decolorizing agent is a mixture of hydrochloric acid and ethanol.

Microbial Nutrition

• Microorganisms require specific nutrients for growth and multiplication, including oxygen, carbon, hydrogen, nitrogen, sulfur, and other elements.
• Temperature, pH, and moisture are also essential for their growth and survival.

Physiologic Requirements of Bacteria

• Oxygen Requirement:

  • Aerobes: Require oxygen and grow well in room air (15-21% oxygen, 1% CO₂).
    • Examples: Bordetella, Brucella, Mycobacteria, and Pseudomonas.
  • Anaerobes: Do not require oxygen for growth.
    • Obligate anaerobes: Die after prolonged exposure to oxygen.
      • Examples: Clostridium and Bacteroides.
    • Facultative anaerobes: Grow in the presence or absence of oxygen; considered aerobes that can grow anaerobically.
      • Examples: Enterobacteriaceae.
    • Aerotolerant anaerobes: Can survive in the presence of oxygen but cannot perform metabolic processes without an anaerobic environment.
      • Example: Propionibacterium acnes.
  • Microaerophiles: Require 2-10% oxygen for growth.
    • Examples: Campylobacter and Treponema pallidum.

• Carbon Dioxide Requirement:

  • Capnophiles: Require increased CO₂ (5-10%).
    • Examples: Haemophilus influenzae, Neisseria gonorrhoeae, and Streptococcus pneumoniae.
  • Most aerobic and facultative aerobic bacteria need 0.03% CO₂.

• Nutritional Requirement:

  • Carbon source:
    • Autotrophs: Use CO₂ as their sole carbon source.
    • Heterotrophs: Use reduced, preformed organic molecules from other bacteria.
  • Electron source:
    • Lithotrophs: Reduce inorganic molecules for biosynthesis or energy conservation.
  • Energy source:
    • Phototrophs: Use light as their energy source.
    • Chemotrophs: Utilize energy produced by organic or inorganic compound oxidation.
  • Other requirements:
    • Fastidious bacteria: Depend on additional substances, such as vitamins, purines, pyrimidines, and hemoglobin for growth.
    • Saprophytes: Require dead organic matter.
    • Parasites: Obtain organic substances from living tissues.

• Temperature Requirement:

  • Psychrophiles/Cryophiles: Grow well at 0°C to 20°C.
    • Examples: Listeria monocytogenes and Yersinia enterocolitica.
  • Mesophiles: Grow between 20°C and 45°C.
    • Most commonly encountered pathogenic bacteria in clinical labs.
  • Thermophiles/Hyperthermophiles: Grow between 50°C and 125°C.
    • Examples: Bacillus stearothermophilus, Sulfolobus, Pyrococcus, Pyrodictium, and Thermus aquaticus.
  • Extremophiles: Prokaryotes surviving in unusual conditions (e.g., lack of oxygen, increased temperatures, living below the earth's surface).
    • Example: Bacillus infernus.

• Thermal Death Time: Minimum time required to kill organisms at a constant temperature.

• Thermal Death Point: Lowest temperature required to kill organisms in a constant time.

• pH:

  • Acidophiles: Grow between pH 0 and 5.5.
    • Examples: Sulfolubus, Picrophilus, and Aconitium.
  • Neutrophiles: Grow between pH 5.5 and 8.0.
    • Most clinically significant bacteria.
  • Alkalophiles: Grow between pH 8.5 and 11.5.
    • Examples: Bacillus alcalophilus and Natronobacterium.

• Notes to Remember:

  • The optimal pH for most pathogenic bacteria is 6.5 to 7.5.
  • Diagnostic laboratory culture media are usually prepared with a final pH between 7.0 and 7.5.