Chapter 13

Introduction to Control of Microbial Growth

• Chapter focuses on controlling microbial growth, discussing:

  • Biosafety levels

  • Terminology related to microbial control

  • Physical and chemical means for controlling microbes

  • Measuring effectiveness of antiseptics and disinfectants

Biosafety Levels

• Biosafety Levels (BSLs): Defined by CDC and NIH to categorize microbes based on risk and required safety precautions.

• Organized like a pyramid, increasing risk from Level 1 (low) to Level 4 (high).

  • Biosafety Level 1:

  • Non-infectious microorganisms, e.g., nonpathogenic E. coli, Bacillus subtilis.

  • Very few precautions, basic PPE required.

  • Biosafety Level 2:

  • Moderate risk; e.g., Salmonella, Staphylococcus aureus, hepatitis viruses.

  • Requires PPE, restricted lab access, biological safety cabinets, eyewash stations, and autoclave for sterilization.

  • Biosafety Level 3:

  • Potentially lethal organisms; e.g., Mycobacterium tuberculosis, HIV.

  • Includes all precautions of Level 2 plus medical surveillance, potential respirator use, and negative pressure rooms.

  • Biosafety Level 4:

  • Most dangerous, e.g., Ebola, Marburg virus.

  • All Level 3 precautions plus full-body suits, changing clothes, extensive decontamination practices.

Key Terminology for Controlling Microbial Growth

• Disinfectant: Chemical used on inanimate surfaces to destroy/inhibit microorganisms.

• Antiseptic: Chemical applied to external tissues for microbial control.

• Antibiotic: Treats bacterial infections, differentiated into true, synthetic, and semi-synthetic.

• Bacteriostatic vs. Bactericidal:

  • Bacteriostatic: Inhibits growth without killing.

  • Bactericidal: Kills microorganisms.

• Sterilization: Complete elimination of all microorganisms, including spores.

• Commercial Sterilization: Heat treatment to kill specific pathogens (e.g., Clostridium botulinum) without complete sterilization.

Mechanisms of Microbial Damage

• Damage to bacterial cells can occur through:

  • Cell membrane disruption: Leads to loss of cellular contents.

  • Transport and protein function disruption: Denaturation affects metabolic processes.

  • Nucleic acid disruption: Impairs replication and transcription.

Physical Methods of Microbial Control

• Heat:

  • Dry Heat: Use of incinerators for sterilization (e.g., sterilizing loops).

  • Moist Heat: Includes boiling (not fully effective), autoclaving (most effective).

  • Autoclave functions under pressure, killing almost all microorganisms (excluding prions unless prolonged).

• Pasteurization: Method to reduce pathogens in food, involves:

  • High Temperature Short Time (HTST): 72°C for 15 seconds.

  • Ultra High Temperature (UHT): 138°C for 2 seconds.

  • Holding Method: 63°C for 30 minutes (classic)

• Refrigeration and Freezing: Slows growth; freezing halts growth but does not kill.

• Desiccation: Removal of moisture which can kill some bacteria, but not endospores.

• Radiation: Includes UV (non-ionizing) disrupting DNA; Ionizing (gamma rays) damages DNA directly.

• Filtration: Removal of microbes from liquids/gases, doesn't kill but traps.

  • HEPA filters and membrane filters effectively filter out microorganisms.

Chemical Methods of Microbial Control

• Phenols: Denature proteins, e.g., Lysol, used as disinfectants.

• Heavy Metals: Denature proteins; e.g., silver (antibacterial), mercury (toxic), copper (algaecide).

• Halogens: Impair protein synthesis; e.g., chlorine, iodine, bromine used in antiseptics and disinfectants.

• Alcohols: Effective at 70% concentrations; denature proteins, act as lipid solvents.

• Quaternary Ammonium Compounds (Quats): Detergents disrupt membranes; used in cleaning and mouthwashes.

• Alkylating Agents: (e.g., glutaraldehyde, formaldehyde) used for sterilization.

• Hydrogen Peroxide (H2O2): Good disinfectant, can slow wound healing due to catalase breakdown.

• Ozone and Benzoyl Peroxide: Used for water treatment and acne treatment respectively.

Measuring Effectiveness of Antiseptics/Disinfectants

• Disc Diffusion Test (Kirby Bauer): Assess the effectiveness through zones of inhibition on agar plates; larger zones indicate greater effectiveness.

• Use Dilution Test: Involves dipping materials in bacterial solutions and testing various disinfectant concentrations for turbidity to find the most effective concentration.

Summary

• Understanding biosafety levels, microbial terminology, physical and chemical control methods is crucial for laboratory safety and effective microbial control.

• Always consider specific context and microbes when choosing effective methods of control.