Controlling Microbial Growth in the Environment: Principles and Methods

Basic Principles of Microbial Control

Terminology of Microbial Control

Understanding the terminology used in microbial control is essential for interpreting laboratory protocols and clinical procedures. The following table summarizes key terms, their definitions, examples, and comments on their use:

Term	Definition	Examples	Comments
Antisepsis	Reduction in the number of microorganisms and viruses, particularly potential pathogens, on living tissue	Iodine, alcohol	Antiseptics are frequently disinfectants whose strength has been reduced to make them safe for living tissues.
Aseptic	Refers to an environment or procedure free of pathogenic contaminants	Preparation of surgical field; hand washing; flame sterilization of laboratory equipment	Scientists, laboratory technicians, and health care workers routinely follow aseptic techniques.
-cide/-cidal	Suffixes indicating destruction of a type of microbe	Bactericide; fungicide; germicide; virucide	Germicides include ethylene oxide, propylene oxide, and aldehydes.
Degerming	Removal of microbes by mechanical means	Handwashing; alcohol swabbing at site of injection	Chemicals play a secondary role to the mechanical removal of microbes.
Disinfection	Destruction of most microorganisms and viruses on nonliving tissue	Phenolics; alcohols; aldehydes; soaps	Disinfectants are used only on inanimate objects.
Pasteurization	Use of heat to destroy pathogens and reduce the number of spoilage microorganisms in foods and beverages	Pasteurized milk and fruit juices	Heat treatment is brief to minimize alteration of taste and nutrients; microbes still remain.
Sanitization	Removal of pathogens from objects to meet public health standards	Washing tableware in scalding water in restaurants	Standards vary among governmental jurisdictions.
-stasis/-static	Suffixes indicating inhibition, but not complete destruction, of a type of microbe	Bacteriostatic; fungistatic; virustatic	Germistatic agents include some chemicals, refrigeration, and freezing.
Sterilization	Destruction of all microorganisms and viruses in or on an object	Preparation of microbiological culture media and canned food	Typically achieved by steam under pressure, incineration, or ethylene oxide gas.

Action of Antimicrobial Agents

Antimicrobial agents control microbial growth by targeting essential cellular structures and functions:

• Cell Walls and Membranes: Damage to the cell wall can cause cells to burst due to osmotic pressure. Disruption of the cytoplasmic membrane leads to leakage of cellular contents.

• Proteins and Nucleic Acids: Denaturation of proteins (by heat or chemicals) disrupts their function. Damage to nucleic acids (by chemicals, radiation, or heat) can result in fatal mutations or halt protein synthesis.

• Virus Susceptibility: Nonenveloped viruses are generally more resistant to harsh conditions than enveloped viruses.

The Selection of Microbial Control Methods

Ideal Characteristics of Antimicrobial Agents

• Inexpensive

• Fast-acting

• Stable during storage

• Harmless to humans, animals, and objects

Factors Affecting Efficacy

• Site to be Treated: The method must be appropriate for the material or tissue being treated (e.g., harsh chemicals cannot be used on living tissues).

• Relative Susceptibility of Microorganisms: Microbes vary in their resistance to antimicrobial agents. Germicides are classified as high, intermediate, or low effectiveness.

Methods for Evaluating Disinfectants and Antiseptics

• Phenol Coefficient: Compares the efficacy of an agent to phenol. A value greater than 1.0 indicates greater effectiveness than phenol.

• Use-Dilution Test: Metal cylinders are contaminated with bacteria, exposed to disinfectant, and then incubated in growth medium to assess effectiveness.

• Kelsey-Sykes Capacity Test: Bacterial suspensions are exposed to the chemical, and samples are incubated to determine the minimum effective time.

• In-Use Test: Swabs are taken before and after disinfectant application to determine real-world effectiveness.

Physical Methods of Microbial Control

Heat-Related Methods

Heat is a widely used method for controlling microbial growth, acting by denaturing proteins, disrupting membranes, and damaging nucleic acids.

• Thermal Death Point (TDP): The lowest temperature that kills all cells in a broth in 10 minutes.

• Thermal Death Time (TDT): The time required to sterilize a volume of liquid at a set temperature.

Decimal Reduction Time (D-value)

The D-value is the time required to kill 90% of the microorganisms present. It is a key parameter in sterilization protocols.

Effect of Temperature on Efficacy

Higher temperatures generally increase the rate of microbial death, reducing the time required for sterilization.

Moist Heat Methods

• Boiling: Kills most vegetative cells and viruses, but not endospores or some viruses. Boiling time is critical and varies with elevation.

• Autoclaving: Uses pressurized steam (121°C, 15 psi, 15 min) to achieve sterilization. Pressure increases the boiling point of water, allowing higher temperatures.

Sterility Indicators

Sterility indicators, such as endospore strips, are used to confirm successful autoclaving. A color change in the medium indicates whether spores survived.

• Pasteurization: Reduces pathogens in foods and beverages without sterilizing. Methods include batch, flash, and ultrahigh-temperature pasteurization.

• Dry Heat: Used for materials that cannot be sterilized with moist heat. Requires higher temperatures and longer times. Incineration is the ultimate means of sterilization.

Other Physical Methods

• Refrigeration and Freezing: Slow microbial metabolism and growth. Psychrophilic microbes can still grow at low temperatures.

• Desiccation and Lyophilization: Drying inhibits growth by removing water. Lyophilization (freeze-drying) is used for long-term preservation.

• Filtration: Physically removes microbes from liquids and air using membrane filters. HEPA filters are used in biological safety cabinets.

• Osmotic Pressure: High concentrations of salt or sugar inhibit microbial growth by causing water loss from cells. Fungi are more tolerant than bacteria.

• Radiation: Ionizing radiation (e.g., gamma rays) creates ions that damage DNA; nonionizing radiation (e.g., UV light) causes DNA mutations. Used for sterilizing food and surfaces.

Biosafety Levels

Laboratories are classified into four biosafety levels (BSL-1 to BSL-4) based on the risk associated with the pathogens handled. BSL-4 is the highest level, used for dangerous and exotic microbes.

Chemical Methods of Microbial Control

Overview

Chemical agents control microbial growth by affecting cell walls, membranes, proteins, or DNA. Their effectiveness varies with environmental conditions and the type of microbe.

Types of Chemical Agents

• Phenol and Phenolics: Intermediate- to low-level disinfectants that denature proteins and disrupt membranes. Effective in the presence of organic matter but may have unpleasant odors and side effects.

• Alcohols: Intermediate-level disinfectants that denature proteins and disrupt membranes. Commonly used for skin antisepsis (e.g., 70% ethanol).

• Halogens: Intermediate-level agents that damage. (They kill the enzymes via oxidation or denaturation.) Examples include iodine, chlorine, and bromine compounds.

Use the iodine for surgery.

• Oxidizing Agents: High-level disinfectants (e.g., hydrogen peroxide, ozone, and peracetic acid) that kill endospores by oxidizing microbial enzymes.

• Surfactants: Surface-active agents (soaps and detergents) that reduce surface tension. Quaternary ammonium compounds (quats) are low-level disinfectants.

• Heavy Metals: Ions such as silver, mercury, and copper denature proteins. Used as low-level bacteriostatic and fungistatic agents.

• Aldehydes: Compounds like glutaraldehyde and formaldehyde cross-link functional groups, denaturing proteins and inactivating nucleic acids. Used for disinfection and sterilization.

• Gaseous Agents: Sterilize items in closed chambers by denaturing proteins and DNA. Examples include ethylene oxide. These agents can be hazardous and carcinogenic.

• Enzymes: Antimicrobial enzymes (e.g., lysozyme) digest cell walls. Used in food preservation and to remove prions from medical instruments.

• Antimicrobials: Antibiotics and synthetic chemicals are used primarily for disease treatment but can also control microbes outside the body.

Development of Resistant Microbes

Overuse of antiseptics and disinfectants can promote the development of resistant microbial strains. There is little evidence that such products significantly improve human or animal health.