The Control of Microbial Growth

Terminology of Microbial Control

Understanding the terminology of microbial control is essential for microbiology students, as it forms the basis for discussing methods to prevent, inhibit, or destroy microorganisms in various environments.

• Sepsis: Refers to bacterial contamination. Pathogens such as Staphylococcus aureus, Streptococcus pyogenes, Klebsiella spp., Escherichia coli, and Pseudomonas aeruginosa can cause sepsis, a life-threatening systemic response to infection.

• 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. Commercial sterilization targets Clostridium botulinum endospores in canned goods but may not achieve complete sterilization.

• Disinfection: The destruction of only harmful microorganisms. Disinfectants are used on surfaces and objects, not on living tissue.

• Antisepsis: The destruction of harmful microorganisms from 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, not killing, microbes. The suffix -stat means to inhibit.

• Aseptic techniques: Procedures that prevent contamination by unwanted microorganisms.

Example: Disinfecting a surface with Clorox spray removes most pathogens but may not kill endospores, whereas sterilization in an autoclave destroys all forms of microbial life.

The Rate of Microbial Death

Microbial populations typically die at a constant rate when exposed to antimicrobial agents. The effectiveness of treatment depends on several factors, including the number of microbes, environmental conditions, time of exposure, and microbial characteristics.

• Microbial death curve: When plotted logarithmically, the death curve is a straight line, indicating a constant rate of death.

• Key factors affecting microbial death:

  • Number of microbes

  • Environment (organic matter, temperature, biofilms)

  • Time of exposure

  • Microbial characteristics

• Actions of microbial control agents:

  • Alteration of membrane permeability

  • Damage to proteins (enzymes)

  • Damage to nucleic acids

Example: Heating food to a specific temperature for a set time ensures a logarithmic reduction in microbial population, aiding in food preservation.

Physical Methods of Microbial Control

Physical methods are widely used to control microbial growth, especially in clinical and food industry settings. These include heat, filtration, low temperature, high pressure, desiccation, osmotic pressure, and radiation.

Heat

• Heat denatures enzymes, leading to microbial death.

• 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 specific population of bacteria at a given temperature.

Moist Heat Sterilization

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

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

• Pasteurization: High-temperature short-time (HTST) pasteurization (72°C for 15 sec) reduces spoilage organisms and pathogens. Ultra-high-temperature (UHT) treatments (140°C for 4 sec) sterilize and allow storage at room temperature.

Example: Autoclaving surgical instruments ensures complete sterilization, while pasteurization of milk reduces pathogens without altering flavor.

Dry Heat Sterilization

• Kills by oxidation: Methods include flaming, incineration, and hot-air sterilization.

Filtration

• Filtration: Passage of substances through a screen-like material, used for heat-sensitive materials. HEPA filters remove microbes >0.3 μm; membrane filters can remove viruses and large proteins.

Other Physical Methods

• Low temperature: Bacteriostatic effect (prevents growth). Methods include 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

• Ionizing radiation: (X-rays, gamma rays, electron beams) damages DNA by causing lethal mutations.

• Nonionizing radiation: (ultraviolet, 260 nm) damages DNA by creating thymine dimers.

• Microwaves: Kill by heat; not especially antimicrobial.

Chemical Methods of Microbial Control

Chemical agents are used to disinfect, sanitize, and sterilize surfaces, instruments, and living tissues. Their effectiveness depends on concentration, presence of organic matter, pH, and time of exposure.

Principles of Effective Disinfection

• Concentration of disinfectant

• Organic matter

• pH

• Time

Use-Dilution Tests and Disk-Diffusion Method

• Use-dilution test: Determines effectiveness of disinfectants by exposing microbes to different concentrations.

• Disk-diffusion method: Evaluates efficacy of chemical agents by measuring the zone of inhibition around disks on a culture.

Types of Chemical Disinfectants

• Phenol and Phenolics: Injure lipids of plasma membranes, causing leakage. Remain active in presence of organic compounds.

• Bisphenols: Derivatives of phenol with two phenol groups. Examples: hexachlorophene, triclosan. Disrupt plasma membranes.

• Biguanides: Broad spectrum activity, especially against gram-positive bacteria. Example: chlorhexidine, used in surgical hand scrubs.

• Essential Oils: Mixtures of hydrocarbons from plants. Microbial action due to phenolics and terpenes. Stronger activity against gram-positive bacteria.

• Halogens: Iodine (tincture, iodophor) impairs protein synthesis and alters membranes. Chlorine (bleach) is an oxidizing agent that shuts down cellular enzyme systems.

• Alcohols: Denature proteins and dissolve lipids. No effect on endospores and nonenveloped viruses. Ethanol and isopropanol require water for effectiveness.

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

• Surface-Active Agents: Soap (degerming, emulsification), acid-anionic sanitizers, quaternary ammonium compounds (quats) are bactericidal, denature proteins, and disrupt plasma membranes.

Comparison of Antiseptic Effectiveness

Different antiseptics vary in their effectiveness, as shown by survival rates of bacteria after exposure.

Example: Tincture of iodine is highly effective, reducing bacterial survival to 10% in 90 seconds.

Chemical Food Preservatives

• Sulfur dioxide prevents wine spoilage.

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

• Nitrites and nitrates prevent endospore germination.

Antibiotics and Aldehydes

• Bacteriocins: Proteins produced by one bacterium that inhibit another. Nisin and natamycin prevent spoilage of cheese.

• Aldehydes: Inactivate proteins by cross-linking with functional groups. Used for preserving specimens and medical equipment. Glutaraldehyde is a liquid chemical sterilizer.

Chemical Sterilization

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

• Plasma sterilizers: Operate at low temperatures using H2O2 gas plasma. Free radicals destroy microbes.

• Supercritical fluids: Used for medical implants; CO2 above critical temperature and pressure.

• Peroxygens: Oxidizing agents (O3, H2O2, peracetic acid) used for contaminated surfaces and food packaging.

Microbial Characteristics and Microbial Control

The type of microbe affects the effectiveness of chemical antimicrobials. Endospores and mycobacteria are more resistant than other forms.

Example: Glutaraldehyde is effective against both endospores and mycobacteria, making it suitable for sterilizing medical equipment.

Summary Table: Key Terms in Microbial Control

Additional info: Gram-negative bacteria are more resistant to chemical biocides due to their outer membrane, which restricts the entry of many agents. Endospores are highly resistant because of their tough spore coat and metabolic inactivity.