The Control of Microbial Growth

Introduction

The control of microbial growth is a fundamental aspect of microbiology, essential for preventing infection, ensuring food safety, and maintaining sterile environments in medical and laboratory settings. This chapter explores the terminology, mechanisms, and methods used to control microbial populations.

Terminology of Microbial Control

Key Terms and Definitions

• Sepsis: Refers to bacterial contamination, often associated with infection.

• Asepsis: The absence of significant contamination; critical in surgical procedures.

• Aseptic Techniques: Methods used to prevent microbial contamination of wounds during surgery.

• Sterilization: Removal and destruction of all microbial life, including endospores.

• Disinfection: Destruction of harmful microorganisms; does not kill endospores.

• Antisepsis: Destruction of harmful microorganisms on living tissue (e.g., applying antibiotic cream).

• Degerming: Mechanical removal of microbes from a limited area (e.g., alcohol swab, handwashing).

• Sanitization: Lowering microbial counts on surfaces or utensils to safe public health levels.

• Germicide (Biocide): Treatments that kill microbes (except endospores).

• Bacteriostasis: Inhibiting, not killing, microbes to stall or prevent growth.

The Rate of Microbial Death

Factors Affecting Microbial Death

Microbial death occurs at a constant rate and can be plotted as a straight line on a log scale. The effectiveness of microbial control treatments depends on several factors:

• Number of Microbes: More cells require longer time to kill.

• Environment: Organic matter, temperature, and biofilms can inhibit contact between antimicrobial agents and microbes.

• Time of Exposure: Minimum contact time is required for effectiveness.

• Microbial Characteristics: Cell structure, life history, and reproductive methods affect susceptibility.

Actions of Microbial Control Agents

Mechanisms of Action

• Alteration of Membrane Permeability: Disrupts cell function and integrity.

• Damage to Proteins/Enzymes: Denaturation leads to loss of function.

• Damage to Nucleic Acids: Prevents replication and transcription.

Physical Methods of Microbial Control

Overview

Physical methods are often used on non-living materials and include heat, filtration, cold, desiccation, osmotic pressure, and radiation.

1. Heat

• Denatures Enzymes: Breaks hydrogen bonds, collapsing protein structure.

• Moist Heat: More effective due to rapid heat transfer. Includes boiling, free-flowing steam, and autoclaving.

• Autoclave: Steam under pressure (121°C at 15 psi for 15 min) kills all organisms and endospores. Steam must contact the item’s surface.

• Pasteurization: Mild heating to reduce spoilage organisms and pathogens. Methods include 63°C for 30 min, HTST (72°C for 15 sec), and UHT (140°C for 4 sec).

• Dry Heat: Kills by oxidation. Includes flaming, incineration, and hot-air sterilization (170°C for 2 hours).

2. Filtration

• Mechanism: Passage of liquid or gas through a filter with pores small enough to trap microbes.

• Applications: Used for heat-sensitive materials (e.g., culture media, enzymes, vaccines).

• HEPA Filters: Remove microbes > 0.3 µm.

• Membrane Filters: Trap microbes as small as 0.01 µm, sufficient to capture viruses.

3. Cold

• Bacteriostatic Effect: Low temperature prevents growth and toxin production.

• Refrigeration: Slows metabolism of most microbes (0-7°C).

• Freezing: Causes ice crystal formation, disrupting cellular structures.

• Deep-Freezing: Suspended in liquid and frozen at -50°C to -95°C.

• Lyophilization: Freeze-drying by removing water and rapidly freezing at -54°C to -72°C.

4. Desiccation

• Mechanism: Removal of water prevents metabolism and reproduction.

• Viability: Many cells remain viable for years and resume growth when exposed to water.

5. Osmotic Pressure

• Mechanism: Uses salts and sugars to create a hypertonic environment, causing plasmolysis.

• Applications: Common in food storage; effective for some bacteria, less so for molds and yeasts.

6. Radiation

• Ionizing Radiation: (X rays, gamma rays, electron beams) causes ionization of water, forming hydroxyl radicals that damage DNA.

• Nonionizing Radiation: (Ultraviolet light) causes thymine dimers in DNA, disrupting replication. Most effective at ~260 nm.

• Microwaves: Kill by heat, not especially antimicrobial.

Principles of Effective Disinfection

Factors Influencing Disinfectant Effectiveness

• Microbial Susceptibility: Gram-positive bacteria are more susceptible than gram-negative. Pseudomonads and mycobacteria are resistant.

• Endospores, Cysts, Viruses: Not affected by most chemical agents.

• Environmental Factors: Organic matter, temperature, pH, and contact time affect effectiveness.

Testing Disinfectant Effectiveness

• Use-Dilution Tests: Metal cylinders dipped in bacteria, placed in disinfectant, then transferred to culture media to check survival.

• Disk-Diffusion Method: Filter paper disks soaked in chemicals placed on agar plates; zone of inhibition indicates effectiveness.

Chemical Methods of Microbial Control

Overview

Chemical agents are used to control microbial growth on living tissue and inanimate objects. The effectiveness depends on the type of agent, concentration, and environmental conditions.

1. Phenol and Phenolics

• Mechanism: Injure lipids of plasma membranes, causing leakage and denature proteins.

• Applications: Effective in presence of organic matter; used for cleaning body fluids.

• Examples: Hexachlorophene, Lysol, triclosan.

2. Biguanides

• Chlorhexidine: Used in surgical hand scrubs and preoperative skin preparation.

• Mechanism: Binds to cell wall components, disrupts plasma membranes, resulting in cell death.

• Limitations: Not effective against some spores and viruses.

3. Halogens

• Iodine: Used as tincture (aqueous alcohol) or iodophor (organic molecules).

• Chlorine: Used as bleach (hypochlorous acid), gaseous form for water treatment, and chloramine for slow release.

• Mechanism: Oxidizing agents destroy amino acids and disrupt plasma membranes.

4. Alcohols

• Mechanism: Denature proteins and dissolve membrane lipids.

• Applications: Effective against bacteria, enveloped viruses, and fungi; not effective against endospores and nonenveloped viruses.

• Common Types: Ethanol and isopropanol; 70% alcohol is most effective.

5. Heavy Metals

• Mechanism: Heavy metal ions denature proteins.

• Examples: Silver nitrate (wound dressings), mercuric chloride (paint), copper sulfate (algicide), zinc chloride (mouthwash).

6. Surface-Active Agents

• Mechanism: Decrease surface tension, aiding degerming.

• Examples: Soaps and detergents; some contain triclosan for antiseptic action.

7. Quarternary Ammonium Compounds (Quats)

• Mechanism: Detergents with positively charged ammonium ions attracted to bacterial cell walls.

• Effectiveness: More effective against gram-positive bacteria; not effective against endospores or in presence of organic matter.

• Examples: Zephiran, Cepacol.

8. Chemical Food Preservatives

• Mechanism: Organic acids and salts inhibit metabolism and prevent growth.

• Examples: Sorbic acid, benzoic acid, calcium propionate (prevent molds); nitrites and nitrates (prevent endospore germination).

9. Aldehydes

• Mechanism: Inactivate proteins by cross-linking with functional groups.

• Applications: Used for preserving specimens and disinfecting medical equipment.

• Examples: Formaldehyde, glutaraldehyde.

10. Chemical Sterilization

• Mechanism: Gaseous sterilants cause alkylation, cross-linking nucleic acids and proteins.

• Applications: Used for heat-sensitive materials; ethylene oxide is common.

11. Peroxygens (Oxidizing Agents)

• Mechanism: Disrupt multiple cell components; not useful as wound disinfectant due to catalase in human cells.

• Applications: Used for contaminated surfaces and food packaging.

• Examples: Benzoyl peroxide (wounds), hydrogen peroxide (inanimate objects), peracetic acid (viruses and endospores).