Ch 9 - Controlling Microbial Growth in the Environment

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Controlling Microbial Growth in the Environment

Introduction

Microbial control in the environment is essential for preventing infection, ensuring food safety, and maintaining sterile conditions in medical and laboratory settings. This chapter covers the principles, methods, and applications of physical and chemical agents used to control microbial growth.

Basic Principles of Microbial Control

Terminology of Microbial Control

Understanding the terminology is crucial for distinguishing between different microbial control strategies. The following table summarizes key terms, their definitions, examples, and comments:

Term	Definition	Examples	Comments
Antisepsis	Reduction in the number of microorganisms and viruses, particularly potential pathogens, on living tissue	Use of iodine or alcohol to prepare skin for injection	Antiseptics are frequently disinfectants whose strength has been reduced for use on living tissue
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	Use of phenolics, alcohols, aldehydes, or soaps on equipment or surfaces	Term is used primarily in relation to pathogens
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 avoid altering taste and nutrients; microbes still remain and eventually cause spoilage
Sanitization	Removal of pathogens from objects to meet public health standards	Washing tableware in scalding water in restaurants	Standards of sanitization vary among governmental jurisdictions
-stasis/-static	Suffixes indicating inhibition, but not complete destruction, of a type of microbe	Bacteriostatic; fungistatic; virustatic	Germistats 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 by ethylene oxide gas

Table of terminology of microbial control

Microbial Death Rates

Microbial death is defined as the permanent loss of reproductive ability under ideal environmental conditions. The microbial death rate is a measurement of the efficacy of an antimicrobial agent, indicating the constant percentage of the extant population killed per unit time.

Significance: Understanding death rates helps in designing effective sterilization and disinfection protocols.
Mathematical Representation: Microbial death often follows a logarithmic pattern, where a constant proportion of cells die per unit time.

Plot of microbial death rate

Action of Antimicrobial Agents

Antimicrobial agents act by targeting essential cellular structures and functions:

Cell Walls: Damage leads to osmotic lysis.
Cytoplasmic Membranes: Disruption causes leakage of cellular contents.
Proteins: Denaturation by heat or chemicals disrupts function.
Nucleic Acids: Damage by chemicals, radiation, or heat can produce fatal mutations or halt protein synthesis.

Relative susceptibilities of microbes to antimicrobial agents

The Selection of Microbial Control Methods

Factors Affecting the Efficacy of Antimicrobial Methods

Several factors influence the choice and effectiveness of microbial control methods:

Site to be Treated: The nature of the surface or tissue determines the method (e.g., harsh chemicals cannot be used on living tissue).
Relative Susceptibility of Microorganisms: Microbes vary in resistance; prions, endospores, and protozoan cysts are among the most resistant.
Environmental Conditions: Temperature, pH, and presence of organic materials can enhance or inhibit antimicrobial efficacy.

Levels of Germicides

High-level germicides: Kill all pathogens, including endospores.
Intermediate-level germicides: Kill fungal spores, protozoan cysts, viruses, and pathogenic bacteria.
Low-level germicides: Kill vegetative bacteria, fungi, protozoa, and some viruses.

Biosafety Levels

Laboratories are classified into four biosafety levels (BSL) based on the risk associated with the pathogens handled:

BSL-1: Non-pathogenic microbes.
BSL-2: Moderately hazardous agents.
BSL-3: Pathogens handled in safety cabinets.
BSL-4: Dangerous and exotic microbes causing severe or fatal diseases.

BSL-4 laboratory workers

Physical Methods of Microbial Control

Overview of Physical Methods

Physical methods include heat, cold, desiccation, filtration, osmotic pressure, and radiation. Each method has specific applications and limitations.

Heat-Related Methods

Moist Heat: Denatures proteins and destroys membranes. Methods include boiling, autoclaving, pasteurization, and ultra-high-temperature sterilization.
Dry Heat: Used for materials that cannot be sterilized with moist heat; requires higher temperatures and longer times.

Key Definitions

Thermal Death Point: Lowest temperature that kills all cells in 10 minutes.
Thermal Death Time: Time to sterilize a volume of liquid at a set temperature.
Decimal Reduction Time (D): Time required to destroy 90% of microbes in a sample.

Decimal reduction time (D) as a measure of microbial death rate

Autoclaving and Sterility Indicators

Autoclaving uses pressurized steam (121°C, 15 psi, 15 min) to achieve sterilization. Sterility indicators, such as endospore strips, confirm successful sterilization.

Sterility indicators using endospore strips Autoclave and its schematic diagram

Pasteurization Methods

Pasteurization reduces microbial load in foods and beverages without sterilizing. Methods include batch, flash, and ultra-high-temperature pasteurization.

Process	Treatment
Historical (batch) pasteurization	63°C for 30 minutes
Flash pasteurization	72°C for 15 seconds
Ultra-high-temperature pasteurization	135°C for 1 second
Ultra-high-temperature sterilization	140°C for 1–3 seconds

Table of moist heat treatments of milk

Refrigeration and Freezing

Slows microbial metabolism and growth.
Some microbes can survive or multiply at low temperatures.
Slow freezing is more effective than quick freezing.

Desiccation and Lyophilization

Desiccation: Inhibits growth by removing water.
Lyophilization: Freeze-drying for long-term preservation; prevents ice crystal formation.

Desiccation as a means of preserving apricots

Filtration

Filtration physically removes microbes from air or liquids using membrane filters of defined pore sizes. HEPA filters are used in safety cabinets and hospital air systems.

Filtration equipment and filtered bacteria

Pore Size (µm)	Smallest Microbes That Are Trapped
5	Multicellular algae, animals, and fungi
3	Yeasts and larger unicellular algae
1.2	Protozoa and small unicellular algae
0.45	Largest bacteria
0.22	Largest viruses and most bacteria
0.025	Larger viruses and pliable bacteria
0.01	Smallest viruses

HEPA filters in biological safety cabinets Diagram of HEPA filter in a safety cabinet

Osmotic Pressure

High concentrations of salt or sugar create hypertonic environments, causing cells to lose water and inhibiting growth.
Fungi are more tolerant of hypertonic conditions than bacteria.

Radiation

Ionizing Radiation: (e.g., gamma rays, X-rays) Ejects electrons, creating ions that damage DNA and proteins. Used for sterilizing medical equipment and food.
Nonionizing Radiation: (e.g., UV light) Causes DNA damage (pyrimidine dimers); used for disinfecting air, surfaces, and transparent fluids.

Increased shelf life of food by ionizing radiation

Chemical Methods of Microbial Control

Overview of Chemical Methods

Chemical agents target cell walls, membranes, proteins, or DNA. Their effectiveness depends on environmental conditions and the type of microbe.

Method	Action(s)	Level of Activity	Some Uses
Phenol	Denatures proteins and disrupts cell membranes	Intermediate to low	Original surgical antiseptic, now replaced by less odorous and injurious phenolics
Phenolics	Denature proteins and disrupt cell membranes	Intermediate to low	Disinfectants and antiseptics
Alcohols	Denature proteins and disrupt cell membranes	Intermediate	Disinfectants, antiseptics, and as a solvent in tinctures
Halogens	Denature proteins	Intermediate	Disinfectants, antiseptics, and water purification
Oxidizing Agents	Denature proteins by oxidation	High	Disinfectants, antiseptics for deep wounds, water purification, and sterilization of food-processing and medical equipment
Surfactants	Decrease surface tension of water and disrupt cell membranes	Low	Soaps, detergents, and antiseptics
Heavy Metals	Denature proteins	Low	Fungistats in paints, silver nitrate cream, surgical dressings, burn creams, and catheters
Aldehydes	Denature proteins	High	Disinfectant and embalming fluid
Gaseous Agents	Denature proteins and DNA by cross-linking functional groups	High	Sterilization of heat- and water-sensitive objects
Enzymes	Denature proteins	High against target substrate	Removal of prions on medical instruments
Antimicrobials	Act against cell walls, cell membranes, protein synthesis, and DNA transcription and replication	Intermediate to low	Disinfectants and treatment of infectious diseases

Table of chemical methods of microbial control

Phenol and Phenolics

Denature proteins and disrupt cell membranes.
Effective in the presence of organic matter and remain active for prolonged periods.
Commonly used in healthcare settings, but have a strong odor and potential side effects.

Structures of phenol, a phenolic, and a bisphenolic

Alcohols

Intermediate-level disinfectants that denature proteins and disrupt membranes.
Most effective at concentrations of 70–90%.
Used for skin antisepsis and instrument disinfection.

Halogens

Intermediate-level agents that damage enzymes by denaturation.
Include iodine, chlorine, bromine, and fluorine compounds.
Used for water disinfection, antiseptics, and surface cleaning.

Degerming in preparation for surgery using Betadine

Oxidizing Agents

High-level disinfectants and antiseptics (e.g., hydrogen peroxide, ozone, peracetic acid).
Kill by oxidation of microbial enzymes.
Used for sterilizing equipment and treating water.

Surfactants

Reduce surface tension of solvents, aiding in mechanical removal of microbes.
Soaps are good degerming agents; detergents (quats) disrupt membranes and are low-level disinfectants.

Heavy Metals

Denature proteins; low-level bacteriostatic and fungistatic agents.
Examples: silver nitrate, thimerosal, copper sulfate.

Effect of heavy-metal ions on bacterial growth

Aldehydes

Compounds with terminal –CHO groups; cross-link functional groups to denature proteins and inactivate nucleic acids.
Glutaraldehyde and formaldehyde are common examples.

Gaseous Agents

Used in closed chambers to sterilize items; denature proteins and DNA.
Hazardous, potentially explosive, and carcinogenic.

Enzymes

Antimicrobial enzymes (e.g., lysozyme, prionzyme) target specific microbial structures.
Used in food processing and medical instrument sterilization.

Antimicrobial Drugs

Include antibiotics, semisynthetics, and synthetics.
Primarily used for disease treatment, but some are used for environmental control.

Methods for Evaluating Disinfectants and Antiseptics

Evaluation Methods

Phenol Coefficient: Compares efficacy to phenol; values >1 indicate greater effectiveness.
Use-Dilution Test: Measures effectiveness against specific microbes on metal cylinders.
Kelsey-Sykes Capacity Test: Assesses minimum time required for a disinfectant to be effective.
In-Use Test: Monitors effectiveness in real-world conditions by sampling surfaces before and after disinfection.

Petri dish showing zone of inhibition for disinfectant effectiveness

Development of Resistant Microbes

Overuse of antiseptics and disinfectants can promote the development of resistant microbial strains, reducing the effectiveness of these agents over time.