York College - CUNY Bio 265L Lab Week #3

Overview of Laboratory Techniques: Gram Stain and Acid-Fast Stain

1. Gram Stain

  • Definition: The Gram stain is a differential staining technique that classifies bacteria as either Gram-positive or Gram-negative based on the structure of their cell walls.

  • Method: The procedure involves several steps:

    • Crystal Violet: Primary stain that penetrates all cells, giving them a violet color.

    • Iodine: Mordant that forms a complex with crystal violet, enhancing retention.

    • Alcohol/Acetone Decolorization: Key step where Gram-negative bacteria lose the primary dye due to the structure of their cell walls (thin peptidoglycan layer).

    • Safranin: Counterstain that imparts a pink color to the decolorized Gram-negative bacteria while leaving the violet Gram-positive bacteria unchanged.

  • Results Explanation:

    • Gram-Positive Bacteria: Retain crystal violet due to a thick peptidoglycan layer that traps the dye.

    • Example: Staphylococcus epidermidis

    • Gram-Negative Bacteria: Lose crystal violet when decolorized and take up safranin, appearing pink.

    • Example: Escherichia coli

  • Clinical Application: Gram staining useful in identifying bacterial infections from clinical specimens.

    • Examples of applications include identifying urinary tract infections (UTIs) or bacterial pneumonia.

Comparison of Gram Reaction, Morphology, and Cellular Arrangement

  1. Escherichia coli

    • Gram Reaction: Negative

    • Morphology: Rod-shaped (bacillus)

    • Cellular Arrangement: Single or in pairs

  2. Bacillus subtilis

    • Gram Reaction: Positive

    • Morphology: Rod-shaped (bacillus)

    • Cellular Arrangement: Chains or singles

  3. Neisseria sicca or N. subflava

    • Gram Reaction: Negative

    • Morphology: Cocci (spherical)

    • Cellular Arrangement: Diplococci (pairs)

  4. Staphylococcus epidermidis

    • Gram Reaction: Positive

    • Morphology: Cocci (spherical)

    • Cellular Arrangement: Clusters (like grapes)

2. Acid-Fast Stain

  • Definition: Acid-fast staining is a method used to detect specific bacteria that retain the color of the primary dye even after being washed with a decolorizing agent due to their waxy cell wall.

Comparisons

  • Mycobacterium smegmatis vs. Staphylococcus epidermidis

    • Mycobacterium smegmatis: Acid-fast, retains bright red stain due to mycolic acids in the cell wall.

    • Staphylococcus epidermidis: Non-acid-fast, does not retain the stain, appears blue after applying methylene blue as a counterstain.

Characteristics of Mycobacteria and Nocardia

  • Reason for Acid-Fast Stain: Mycobacteria have a unique lipid-rich cell wall that resists decolorization by acids, necessitating the acid-fast staining technique for their diagnosis.

Clinical Significance

  • Detection of Mycobacterium tuberculosis:

    • Importance: Acid-fast staining is crucial for identifying the presence of Mycobacterium tuberculosis in clinical specimens such as sputum.

    • Application in Active/Reactivated TB: Positive acid-fast stains indicate active tuberculosis disease.

Methodology of Acid-Fast Stain

  • Procedure: Involves the application of a primary stain (carbol fuchsin) followed by heat to enhance penetration, decolorization using acid-alcohol, and a counterstaining step (methylene blue).

Analysis of Results

  • Definitions:

    • Acid-Fast Bacterial Cells (Mycobacteria): Stain red under the microscope, indicating they resist decolorization.

    • Non-Acid Fast Bacterial Cells: Stain blue, indicating they do not resist decolorization and can be washed away.

Safety Note

  • All bacterial species discussed are classified as BSL-1 (Biosafety Level 1), indicating they pose minimal risk to healthy adults and are suitable for teaching laboratories.

  • Mycobacterium smegmatis ATCC19420: Classified as BSL-1, making it safe to handle in a laboratory setting.