Chapter 7: The Control of Microbial Growth

The Terminology of Microbial Control

Understanding the terminology of microbial control is essential for interpreting laboratory procedures and clinical practices. These terms describe various methods and outcomes in the reduction or elimination of microorganisms.

• Sepsis: Refers to bacterial contamination.

• Asepsis: The absence of significant contamination; aseptic techniques prevent microbial contamination of wounds.

• Sterilization: Removal and destruction of all microbial life.

• Commercial Sterilization: Killing Clostridium botulinum endospores in canned goods.

• Disinfection: Destruction of harmful microorganisms on inanimate surfaces.

• Antisepsis: Destruction of harmful microorganisms from living tissue.

• Degerming: Mechanical removal of microbes from a limited area.

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

• Biocide (Germicide): Treatments that kill microbes.

• Bacteriostasis: Inhibiting, not killing, microbes.

Example: Aseptic surgery techniques are used to prevent microbial contamination during operations.

The 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 Curve: 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

  • Microbial characteristics (e.g., endospores, cell wall structure)

Equation: The logarithmic death rate can be described as:

where is the number of surviving cells at time , is the initial number of cells, and is the rate constant.

Actions of Microbial Control Agents

Microbial control agents act by damaging cellular structures, leading to cell death or inhibition of growth.

• Damage to Plasma Membrane: Causes leakage of cellular contents and interferes with cell growth.

• Damage to Proteins (Enzymes): Denaturation or inactivation of essential enzymes.

• Damage to Nucleic Acids: Disruption of DNA and RNA, preventing replication and function.

Example: Phenol and phenolics disrupt plasma membranes, causing leakage and cell death.

Physical Methods of Microbial Control

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

Heat

Heat is a primary method for sterilization and disinfection, acting by denaturing proteins and enzymes.

• 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

Moist heat coagulates and denatures proteins. Methods include boiling, free-flowing steam, and autoclaving.

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

• Pasteurization: Reduces spoilage organisms and pathogens in milk and juices. High-temperature short-time (HTST): 72°C for 15 sec. Ultra-high-temperature (UHT): 140°C for 4 sec.

Dry Heat Sterilization

Dry heat kills by oxidation. Methods include flaming, incineration, and hot-air sterilization (oven at 170°C for 2 hours).

Filtration

Filtration is used for heat-sensitive materials. 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 and alters carbohydrate structure.

• Desiccation: Absence of water prevents metabolism.

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

Radiation

Radiation kills cells by damaging DNA.

• Ionizing Radiation: (X-rays, gamma rays, electron beams) ionizes water to create reactive hydroxyl radicals, causing lethal mutations. Used for sterilization of food, pharmaceuticals, and medical supplies.

• Nonionizing Radiation: (Ultraviolet, 260 nm) creates thymine dimers in DNA. UVC lamps are used for surface sterilization.

• Visible Blue Light: (470 nm) kills bacteria by forming singlet oxygen.

• Microwaves: Kill by heat, not especially antimicrobial.

Chemical Methods of Microbial Control

Chemical agents are used for disinfection, antisepsis, and sterilization. Their effectiveness depends on concentration, organic matter, pH, temperature, and time.

Principles of Effective Disinfection

• Concentration of Disinfectant

• Presence of Organic Matter

• pH

• Temperature

• Time

Use-Dilution Tests

Standard method for evaluating disinfectants. Metal cylinders are dipped in test bacteria, dried, exposed to disinfectant, and then transferred to culture media to assess survival.

Disk-Diffusion Method

Evaluates efficacy of chemical agents by placing filter paper disks soaked in chemicals on a culture plate and measuring the zone of inhibition.

Types of Chemical Disinfectants

• Phenol and Phenolics: Injure lipids of plasma membranes; remain active in presence of organic matter. Example: O-phenylphenol (ingredient in Lysol®).

• Bisphenols: Disrupt plasma membranes. Examples: Hexachlorophene (skin infections), Triclosan (antimicrobial soaps).

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

• Halogens: Iodine (impairs protein synthesis, alters membranes), Chlorine (oxidizing agent, used in water treatment).

• Alcohols: Denature proteins and dissolve lipids; not effective against endospores and nonenveloped viruses.

• Chemical Food Preservatives: Sulfur dioxide, organic acids, nitrites, and nitrates prevent spoilage and endospore germination.

• Peroxygens: Oxidizing agents (hydrogen peroxide, peracetic acid) used for sterilization and disinfection.

Microbial Characteristics and Microbial Control

The type of microbe affects the efficacy of control methods. Some microbes are more resistant due to structural or physiological features.

• Gram-negative bacteria: More resistant to biocides due to lipopolysaccharide in their outer membrane.

• Mycobacteria: Resistant to biocides; require special testing.

• Bacterial endospores: Very resistant to many biocides.

• Nonenveloped viruses: More resistant than enveloped viruses.

• Prions: Extremely resistant; require immersion in NaOH and autoclaving.

Table: Effectiveness of Chemical Antimicrobials Against Endospores and Mycobacteria

Example: Pseudomonas and Burkholderia are unusually resistant to biocides due to their outer membrane structure.

Additional info: These notes expand on brief points with academic context, definitions, and examples to ensure completeness and clarity for exam preparation.