Capsule Staining in Bacteria

Capsule Stain

• Glycocalyx is a layer of non-ionic polysaccharides located on the outer surface of the cell wall in some bacterial species.
• The cell wall of some species is made of amino acids such as D-glutamine.
• Capsule is a thick, dense layer of polysaccharides firmly attached to the cell wall.
• Slime layer is an unorganized layer of polysaccharides loosely attached to the cell wall.
• Bacteria with capsules have mucoid (mucus-like) smooth colonies.
• Examples of encapsulated bacteria include Klebsiella pneumoniae, Streptococcus pneumoniae, and Bacillus anthracis.
• Bacteria without capsules have rough colonies.

Capsule Stain-Pathogenicity

• Capsules are a major virulence factor that mediates attachment to host tissues.
• Capsules interfere with phagocytosis (anti-phagocytic).
• Some bacterial species have capsules made of polysaccharides similar to those found in host tissues, hence preventing immune system recognition.

Capsule Staining Methods

• Capsule staining methods are indirect: using a non-ionic dye (e.g., Congo Red (acidic)) to stain the background and a basic dye (e.g., Acid fuchsin) to stain the bacteria.
•  Maneval's capsule staining method is an indirect method.

Endospore Staining

• Endospores are metabolically inactive cells that provide protection from harsh environmental conditions.
• They are formed within the mother cell (sporulation).
• Endospores can remain viable for thousands of years.
• Endospores are formed in response to stress like lack of nutrients, inappropriate temperature, and pH changes.
• Bacteria form endospores to survive harsh environments.

Endospore Location

• Endospores can be located centrally, subterminally, or terminally within the mother cell.

Endospore Staining Techniques

• Endospores are resistant to regular staining procedures.
• Schaeffer-Fulton method employs Malachite green and subsequent counterstaining (e.g., Safranin or basic fuchsin) under gentle heating to stain endospores green and the cells red.

Bacterial Enumeration

• Microbial growth is the increase in the number of cells in a microbial population.
• Factors affecting bacterial growth include nutrients, oxygen concentration, pH, and temperature.
• Techniques for estimating bacterial cell counts include counting bacterial cells under light microscope or electronic particle counter, measuring the mass of bacterial cells (e.g, dry weight), measuring turbidity, and colony count.
• Dilution is essential prior to counting colonies since large populations cannot be accurately counted.
• Performing serial dilutions beforehand helps to accurately count bacteria and their densities.

Antibiotic Susceptibility Testing

• Antimicrobial agents are substances that kill or inhibit the growth of microorganisms without harming the host cells.
• Bacterial pathogens can develop resistance to antibiotics.
• Susceptibility testing methods include broth dilution, agar dilution, disk diffusion, antibiotic strips, and computerized systems.
• The minimum inhibitory concentration (MIC) is the lowest antibiotic concentration that prevents microbial growth.
•  The minimum bactericidal concentration (MBC) is the lowest concentration that kills the bacterium.