Chapter 7: The Control of Microbial Growth – Study Notes

The Control of Microbial Growth

7.1: The Terminology of Microbial Control

Understanding the terminology of microbial control is essential for effective communication and practice in microbiology and healthcare. These terms describe various methods and outcomes related to the reduction or elimination of microorganisms.

• Sepsis: Refers to bacterial contamination. Sepsis is a life-threatening condition caused by the body's response to infection, often by Staphylococcus aureus, Streptococcus pyogenes, Klebsiella spp., Escherichia coli, and Pseudomonas aeruginosa.

• Asepsis: The absence of significant contamination. Aseptic techniques are crucial in surgery to prevent microbial contamination of wounds.

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

• Commercial Sterilization: The process of killing Clostridium botulinum endospores in canned goods, but not necessarily achieving complete sterilization.

• Disinfection: The destruction of only harmful microorganisms, typically on inanimate objects.

• Antisepsis: The destruction of harmful microorganisms on living tissue.

• Degerming: Mechanical removal of microbes from a limited area, such as using an alcohol swab before injection.

• Sanitization: Lowering microbial counts on eating utensils to safe levels, often using high heat or disinfectants.

• Biocide (Germicide): Treatments that kill microbes; the suffix -cide means to kill.

• Bacteriostasis: Inhibiting, but not killing, microbes; the suffix -stat means to inhibit.

• Asepsis: The absence of significant contamination, especially important in surgical settings.

Example: Using an autoclave for sterilization of surgical instruments, disinfecting surfaces with chemical sprays, and applying antiseptics to skin before injections.

7.2: The Rate of Microbial Death

Microbial populations typically die at a constant rate when exposed to antimicrobial agents. Understanding the death curve is crucial for determining the effectiveness of sterilization and disinfection processes.

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

• Exponential Death Rate: Bacterial populations die logarithmically, meaning a constant percentage of the population is killed per unit time.

• Death Curve: Logarithmic plotting of microbial death shows a straight line, which is useful for understanding sterilization kinetics.

Example: In food preservation and medical sterilization, understanding the death curve helps determine the necessary exposure time to ensure safety.

Actions of Microbial Control Agents

Microbial control agents act by targeting essential cellular components:

• Alteration of Membrane Permeability: Damages the plasma membrane, leading to leakage of cellular contents.

• Damage to Proteins (Enzymes): Denaturation of proteins by breaking hydrogen and covalent bonds, inhibiting cellular function.

• Damage to Nucleic Acids: Interferes with DNA and RNA, preventing protein synthesis and cell replication.

7.4: Physical Methods of Microbial Control

Heat

Heat is one of the most common physical methods for controlling microbial growth. It works primarily by denaturing enzymes and other proteins.

• 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 given temperature.

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

Moist Heat Sterilization

Moist heat is effective at coagulating and denaturing proteins, leading to microbial death.

• Boiling: Kills vegetative forms of bacteria, viruses, and fungi within 10 minutes.

• Autoclaving: Uses steam under pressure (121°C at 15 psi for 15 minutes) to kill all organisms and endospores. Steam must contact the item’s surface for effective sterilization.

• Test Strips: Used to indicate sterility after autoclaving.

Example: Autoclaving surgical instruments and laboratory media.

Pasteurization

Pasteurization reduces spoilage organisms and pathogens in food and beverages without significantly affecting taste or quality.

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

• Ultra-High-Temperature (UHT): 140°C for 4 seconds, allowing storage at room temperature for months.

Dry Heat Sterilization

Dry heat kills by oxidation and is used for materials that can withstand high temperatures without moisture.

• Methods: Flaming, incineration, and hot-air sterilization.

Filtration

Filtration is used for sterilizing heat-sensitive materials by physically removing microbes from liquids or air.

• HEPA Filters: Remove microbes larger than 0.3 μm from air.

• Membrane Filters: Remove microbes from liquids; pore sizes as small as 0.01 μm are available.

Other Physical Methods

• Low Temperature: Bacteriostatic effect; includes refrigeration, deep-freezing, and lyophilization (freeze-drying).

• High Pressure: Denatures proteins; endospores are resistant.

• Desiccation: Absence of water prevents metabolism.

• Osmotic Pressure: High concentrations of salts and sugars create a hypertonic environment, causing plasmolysis.

Radiation

Radiation is used to kill microorganisms by damaging their DNA.

• Ionizing Radiation: (X-rays, gamma rays, electron beams) causes lethal mutations in DNA.

• Nonionizing Radiation: (Ultraviolet, 260 nm) creates thymine dimers in DNA, inhibiting replication.

• Microwaves: Kill by heat, not especially antimicrobial.

7.5: Chemical Methods of Microbial Control

Principles of Effective Disinfection

The effectiveness of chemical disinfectants depends on several factors:

• Concentration of disinfectant

• Presence of organic matter

• pH of the environment

• Time of exposure

Use-Dilution and Disk-Diffusion Tests

These tests evaluate the efficacy of disinfectants and antiseptics.

• Use-Dilution Test: Measures the ability of a disinfectant to kill microbes on metal or glass cylinders dipped in bacterial cultures.

• Disk-Diffusion Method: Filter paper disks soaked in chemical agents are placed on agar plates inoculated with bacteria. Zones of inhibition indicate effectiveness.

Major Types of Chemical Disinfectants

• Phenol and Phenolics: Injure plasma membranes, remain active in organic matter, and persist for long periods.

• Bisphenols: Derivatives of phenol with two phenol groups; effective against gram-positive bacteria.

• Biguanides: Broad-spectrum activity, especially against gram-positive bacteria; disrupt plasma membranes (e.g., chlorhexidine).

• Essential Oils: Plant-derived compounds with antimicrobial activity, mainly against gram-positive bacteria.

• Halogens: Iodine and chlorine are effective disinfectants and antiseptics. Iodine impairs protein synthesis; chlorine is an oxidizing agent.

• Alcohols: Denature proteins and dissolve lipids; ineffective against endospores and nonenveloped viruses. Ethanol and isopropanol require water for activity.

• Heavy Metals: Oligodynamic action; denature proteins. Examples include silver nitrate, mercuric chloride, copper sulfate, and zinc chloride.

• Surface-Active Agents: Soaps (degerming), acid-anionic sanitizers, and quaternary ammonium compounds (quats) disrupt membranes and denature proteins.

Chemical Food Preservatives

Certain chemicals are used to prevent spoilage and microbial growth in foods:

• Sulfur dioxide prevents wine spoilage.

• Organic acids (sorbic acid, benzoic acid, calcium propionate) inhibit metabolism and prevent molds in acidic foods.

• Nitrites and nitrates prevent endospore germination in meats.

Antibiotics as Preservatives

Bacteriocins are proteins produced by bacteria that inhibit other bacteria. Nisin and natamycin prevent spoilage of cheese.

Aldehydes and Chemical Sterilization

• Aldehydes: Inactivate proteins by cross-linking functional groups; used for preserving specimens and sterilizing medical equipment (e.g., glutaraldehyde, formaldehyde).

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

• Plasma Sterilization: Uses electrically excited gas (e.g., H2O2 plasma) to generate free radicals that destroy microbes, suitable for tubular instruments.

• Supercritical Fluids: Combine properties of gases and liquids; used for sterilizing medical implants.

• Peroxygens: Oxidizing agents (e.g., hydrogen peroxide, peracetic acid) used for contaminated surfaces and food packaging.

Microbial Characteristics and Microbial Control

Effectiveness of Chemical Antimicrobials against Endospores and Mycobacteria

The effectiveness of chemical agents varies depending on the type of microbe. Endospores and mycobacteria are particularly resistant to many chemical agents.

Chemical Agent	Effect against Endospores	Effect against Mycobacteria
Glutaraldehyde	Fair	Good
Chlorines	Fair	Fair
Alcohols	Poor	Good
Iodine	Poor	Good
Phenolics	Poor	Good
Chlorhexidine	None	Fair
Bisphenols	None	None
Quats	None	None
Silver	None	None

Additional info: Gram-negative bacteria are generally more resistant to chemical biocides than gram-positive bacteria due to their outer membrane, which acts as a barrier to many substances.