Chapter 7: The Control of Microbial Growth – Comprehensive Study Notes

The Control of Microbial Growth

Terminology of Microbial Control

Understanding the terminology associated with microbial control is essential for interpreting laboratory and clinical practices. These terms describe various methods and outcomes in reducing or eliminating microorganisms.

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

• Asepsis: The absence of significant contamination; aseptic techniques are used in surgery to prevent infection.

• Sterilization: The process of removing and destroying all microbial life, including endospores.

• Commercial Sterilization: Specifically targets Clostridium botulinum endospores in canned goods.

• Disinfection: Destruction of harmful microorganisms on non-living surfaces.

• Antisepsis: Destruction of harmful microorganisms from living tissue.

• Degerming: Mechanical removal of microbes from a limited area (e.g., hand washing).

• Sanitization: Lowering microbial counts on eating utensils to safe levels.

• Biocide (Germicide): Treatments that kill microbes.

• Bacteriostasis: Inhibition, not killing, of microbes.

Example: Joseph Lister's use of aseptic techniques in surgery revolutionized infection control.

Patterns and Rate of Microbial Death

Microbial death occurs at a logarithmic rate when exposed to control agents. The effectiveness of treatment depends on several factors, including the number of microbes, environmental conditions, exposure time, and microbial characteristics.

• Logarithmic Death Rate: Microbial populations decrease by a constant percentage per unit time.

• Factors Affecting Death Rate: Number of microbes, environment (organic matter, temperature, biofilms), time of exposure, and microbial characteristics.

Example: The death curve shows that killing 90% of a population (one log decrease) results in a straight line when plotted logarithmically.

Actions of Microbial Control Agents

Microbial control agents act by targeting essential cellular structures, leading to cell death or inhibition.

• Alteration of Membrane Permeability: Disrupts transport and homeostasis.

• Damage to Proteins (Enzymes): Denaturation impairs metabolic functions.

• Damage to Nucleic Acids: Prevents replication and transcription.

Physical Methods of Microbial Control

Heat

Heat is a widely used method for microbial control, primarily through protein denaturation.

• Thermal Death Point (TDP): Lowest temperature at which all cells in a liquid culture are killed in 10 minutes.

• Thermal Death Time (TDT): Minimal time for all bacteria in a liquid culture to be killed at a particular temperature.

• Decimal Reduction Time (DRT): Minutes to kill 90% of a population at a given temperature.

Moist Heat Sterilization

• Boiling: Kills most vegetative cells.

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

• Pasteurization: Reduces spoilage organisms and pathogens. Equivalent treatments include 63°C for 30 min, HTST (72°C for 15 sec), and UHT (140°C for 4 sec).

• Thermoduric Organisms: Survive pasteurization.

Dry Heat Sterilization

• Flaming, Incineration, Hot-Air Sterilization: Kills by oxidation.

Filtration

Filtration is used for heat-sensitive materials, allowing the passage of substances through a screenlike material.

• HEPA Filters: Remove microbes >0.3 µm.

• Membrane Filters: Remove microbes >0.22 µm.

Other Physical Methods

• Low Temperature: Bacteriostatic effect (refrigeration, deep-freezing, lyophilization).

• High Pressure: Denatures proteins.

• Desiccation: Absence of water prevents metabolism.

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

Radiation

Radiation is used to kill or inhibit microorganisms by damaging their DNA.

• Ionizing Radiation: (X rays, gamma rays, electron beams) ionizes water to create reactive hydroxyl radicals, causing lethal mutations.

• Nonionizing Radiation: (UV, 260 nm) creates thymine dimers in DNA.

• Microwaves: Kill by heat, not especially antimicrobial.

Chemical Methods of Microbial Control

Principles of Effective Disinfection

Several factors influence the effectiveness of disinfectants:

• Concentration of Disinfectant

• Presence of Organic Matter

• pH

• Time of Exposure

The Disk-Diffusion Method

This method evaluates the efficacy of chemical agents by measuring the zone of inhibition around filter paper disks soaked in chemicals and placed on a culture.

Phenol and Phenolics

Phenol and its derivatives injure lipids of plasma membranes, causing leakage.

• Bisphenols: Contain two phenol groups connected by a bridge (e.g., hexachlorophene, triclosan).

• Mode of Action: Disrupt plasma membranes.

Alcohols

Alcohols denature proteins and dissolve lipids, but have no effect on endospores and nonenveloped viruses. Ethanol and isopropanol require water for optimal activity.

• Why 70% Ethanol? Water supports protein denaturation, making 70% ethanol more effective than 95%.

Biocidal Action of Ethanol

The effectiveness of ethanol as a biocidal agent depends on its concentration and exposure time.

Concentration of Ethanol (%)	Time of Exposure (sec)	Growth (G) or No Growth (NG)
100	10-50	G
95	10-50	NG
90	10-50	NG
80	10-50	NG
70	10-50	NG
60	10-50	NG
50	10-50	NG
40	10-50	G

Heavy Metals and Their Compounds

Heavy metals exert antimicrobial activity through oligodynamic action, denaturing proteins.

• Silver Nitrate: Prevents ophthalmia neonatorum.

• Mercuric Chloride: Prevents mildew in paint.

• Copper Sulfate: Used as an algicide.

• Zinc Chloride: Found in mouthwash.

Surface-Active Agents

• Soap: Degerming and emulsification.

• Acid-Anionic Sanitizers: Anions react with plasma membrane.

• Quaternary Ammonium Compounds (Quats): Cations are bactericidal, denature proteins, and disrupt plasma membrane.

Chemical Food Preservatives

• Sulfur Dioxide: Prevents wine spoilage.

• Organic Acids: Inhibit metabolism (e.g., sorbic acid, benzoic acid, calcium propionate).

• Nitrites and Nitrates: Prevent endospore germination.

Antibiotics

Bacteriocins are proteins produced by one bacterium that inhibit another. Nisin and natamycin prevent spoilage of cheese.

Aldehydes

Aldehydes inactivate proteins by cross-linking with functional groups. They are used for preserving specimens and medical equipment. Glutaraldehyde is a liquid chemical sterilizing agent.

Chemical Sterilization

Gaseous sterilants cause alkylation, cross-linking nucleic acids and proteins. Used for heat-sensitive materials (e.g., ethylene oxide).

Plasma

Plasma is the fourth state of matter, consisting of electrically excited gas. Free radicals destroy microbes and are used for tubular instruments.

Supercritical Fluids

Supercritical CO2 has both gaseous and liquid properties and is used for sterilizing medical implants.

Peroxygens and Other Forms of Oxygen

Peroxygens are oxidizing agents used for contaminated surfaces and food packaging. Examples include ozone (O3), hydrogen peroxide (H2O2), and peracetic acid.