Biology 9: Living vs Dead Cells

Distinguishing Living from Dead Cells

• Detroff Hausser Counting Chamber allows counting microorganisms directly with a microscope, but visual identification does not confirm liveliness.
• Fluorescence Microscopy distinguishes living from dead cells using dyes:
  • Propidium Iodide stains dead cells red by penetrating only energized membranes.
  • Syto 9 stains both live and dead cells green.
  • Combined application results in living cells appearing green and dead cells appearing red, yellow, or orange.

Flow Cytometry

• FACS (Fluorescence-Activated Cell Sorting) counts and separates cells based on fluorescence.
• Cells flow single-file past a laser for counting and fluorescence intensity measurement, allowing for separation.
• Useful for differentiating bacterial cells with varying fluorescent protein synthesis capabilities.

Viable Counts

• Viable Cells replicate and form colonies (e.g., 100 colonies from 100 ml of 10³ dilution corresponds to 1000 cells/ml).
• Optical Density measures turbidity in liquid cultures via spectrophotometer, indicating cell density.

Bacterial Growth

• Standard growth through binary fission; one parent cell divides to produce two offspring.
• Growth cycle includes nucleoid expansion as DNA replicates.
• Bacteria utilize nitrogen in various transformations: nitrogen fixation (N₂ to NH₄⁺), nitrification (NH₃ to NO₃⁻), and denitrification (NO₃⁻ to N₂).

Nitrogen Cycle and UTIs

• Nitrate Reduction: Some bacteria (e.g., E. coli) convert nitrates (NO₃⁻) in urine to nitrites (NO₂⁻), indicating potential infections.
• Presence of nitrite in urine suggests UTI but requires confirmation since other causes may exist.

Nitrite Test in UTIs

• Sensitivity and Specificity of nitrite tests varies—sensitivity at 25% and specificity at 94%-100%.
• A positive nitrite result usually indicates bacterial presence, especially if lacking in normal urine.

Treatment Considerations

• Infections typically warrant treatment to prevent complications, particularly for asymptomatic bacteriuria or in pregnant patients.
• Potential risk of sepsis and preterm birth linked to untreated infections.

Extremophiles

• Extremophiles thrive in harsh conditions; classified by temperature, pH, salinity, and pressure.
• Thermophiles grow at high temperatures (>55°C), while psychrophiles thrive at low temperatures (as low as -10°C).
• Bacteria employ unique proteins to function under extreme conditions.

Oxygen Utilization

• Strict Aerobes require oxygen for growth and metabolize reactive oxygen species (ROS).
• Strict Anaerobes cannot use oxygen and are often harmed by ROS.
• Facultative Anaerobes can grow with or without oxygen, while Microaerophiles prefer lower oxygen environments.

Catalase Test

• Detects the presence of catalase, an enzyme that decomposes hydrogen peroxide into water and oxygen.
• Catalase-positive bacteria form bubbles upon exposure to hydrogen peroxide; key for differentiating bacterial types.

Biofilms and Quorum Sensing

• Biofilms consist of masses of bacteria adhering to surfaces, offering resistance to treatment.
• Quorum Sensing enables communication and coordinated behavior among bacteria in biofilms.
• Biofilms pose significant challenges in infections, such as in cystic fibrosis-related respiratory illnesses.

Case History Insight

• Cystic fibrosis is linked to compromised chloride transport, leading to severe respiratory conditions.
• The persistence of Pseudomonas aeruginosa in infected patients demonstrates the challenges in treating biofilm-associated infections.