Chapter 7 - The Control of Microbial Growth – Study Notes

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The Control of Microbial Growth

Introduction

The control of microbial growth is essential in medical, industrial, and everyday settings to prevent infection, spoilage, and contamination. This chapter explores the terminology, mechanisms, and methods used to control the growth of microorganisms, including both physical and chemical approaches.

Terminology of Microbial Control

Key Definitions

Sepsis: Refers to bacterial contamination.
Asepsis: The absence of significant contamination; aseptic techniques are crucial in surgery to prevent microbial contamination of wounds.
Antisepsis: The destruction of harmful microorganisms from living tissue.
Sterilization: The removal and destruction of all microbial life, including endospores.
Disinfection: The destruction of harmful microorganisms, typically on inanimate objects.
Degerming: Mechanical removal of microbes from a limited area (e.g., skin before injection).
Sanitization: Lowering microbial counts on eating utensils to safe levels.
Biocide (germicide): Treatments that kill microbes.
Bacteriostasis: Inhibition of microbial growth, without killing the organisms.

The Rate of Microbial Death

Patterns and Principles

When microbial populations are exposed to control agents (such as heat or chemicals), they typically die at a constant rate. For each minute of treatment, 90% of the population is killed, demonstrating an exponential death rate.

Decimal Reduction Time (D value): The time (in minutes) required to kill 90% of a specific microbial population at a given temperature.

Table showing microbial exponential death rate Microbial death curve plotted arithmetically and logarithmically Microbial death curve with different population loads

Factors Affecting Effectiveness of Antimicrobial Treatments

Number of microbes: Larger populations require longer treatment times.
Environment: Presence of organic matter, temperature, and biofilms can affect efficacy.
Time of exposure: Longer exposure increases effectiveness.
Microbial characteristics: Different microbes vary in susceptibility to control methods.

Actions of Microbial Control Agents

Mechanisms of Action

Alteration of membrane permeability: Damages the plasma membrane, causing leakage of cellular contents.
Damage to proteins (enzymes): Denaturation or inactivation of essential enzymes.
Damage to nucleic acids: Prevents replication and cellular function.

Physical Methods of Microbial Control

Heat

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

Thermal Death Point (TDP): The lowest temperature at which all cells in a liquid culture are killed in 10 minutes.
Thermal Death Time (TDT): The minimal time required to kill all bacteria in a liquid culture at a given temperature.
Decimal Reduction Time (D value): Minutes to kill 90% of a population at a given temperature.

Moist Heat Sterilization

Mechanism: Coagulates and denatures proteins.
Boiling: Kills most vegetative cells and viruses within 10 minutes but does not reliably kill endospores.
Autoclaving: Uses steam under pressure (121°C at 15 psi for 15 minutes) to kill all organisms and endospores. Steam must contact all surfaces for effective sterilization.

Diagram of an autoclave

Effect of Container Size on Autoclave Sterilization

Container Size	Liquid Volume	Sterilization Time (min)
Test tube (18 x 150 mm)	10 ml	15
Erlenmeyer flask (125 ml)	95 ml	15
Erlenmeyer flask (2000 ml)	1500 ml	30
Fermentation bottle (9000 ml)	6750 ml	70

Table showing effect of container size on autoclave sterilization times

Sterilization Indicators

Test strips are used to confirm sterility after autoclaving.

Sterilization indicators

Pasteurization

Purpose: Reduces spoilage organisms and pathogens in food and beverages.
High-Temperature Short-Time (HTST): 72°C for 15 seconds.

Dry Heat Sterilization

Mechanism: Kills by oxidation.
Methods: Flaming, incineration, and hot-air sterilization.

Filtration

Filtration is used for sterilizing heat-sensitive materials by passing them through filters that remove microbes.

HEPA filters: Remove microbes >0.3 μm in diameter.
Membrane filters: Remove microbes >0.22 μm; some can filter out viruses and large proteins (pore sizes as small as 0.01 μm).

Filter sterilization with a disposable unit

Other Physical Methods

Low Temperature: Bacteriostatic effect (refrigeration, deep-freezing, lyophilization).
High Pressure: Denatures proteins.
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

Principles of Effective Disinfection

Concentration of disinfectant
Presence of organic matter
pH
Time of exposure

Evaluation of Disinfectants

Use-Dilution Test: Metal cylinders are dipped in test bacteria, dried, exposed to disinfectant, then transferred to culture media to check for survival.
Disk-Diffusion Method: Filter paper disks soaked in chemical agents are placed on a culture; zones of inhibition indicate effectiveness.

Disk-diffusion method for evaluating disinfectants

Types of Disinfectants

Phenol and Phenolics: Injure plasma membrane lipids, causing leakage.
Bisphenols: Two phenol groups connected by a bridge (e.g., hexachlorophene, triclosan); disrupt plasma membranes.
Biguanides: (e.g., chlorhexidine) used in surgical hand scrubs; disrupt plasma membranes.
Essential Oils: Plant-derived, effective mainly against gram-positive bacteria.
Halogens: Iodine (impairs protein synthesis, alters membranes), chlorine (oxidizing agent, inactivates enzymes).
Alcohols: Denature proteins, dissolve lipids; ineffective against endospores and nonenveloped viruses. Ethanol and isopropanol require water for activity.

Table showing biocidal action of ethanol concentrations

Heavy Metals: Oligodynamic action; denature proteins (e.g., copper sulfate, zinc chloride).
Chemical Food Preservatives: Sulfur dioxide, organic acids, nitrites/nitrates inhibit microbial growth in foods.
Surface-Active Agents (Surfactants): Soaps (mechanical removal), acid-anionic sanitizers, quaternary ammonium compounds (quats).
Antibiotics: Natamycin (antifungal, used in cheese).
Plasmas: Ionized gases used for sterilizing instruments.
Aldehydes: Inactivate proteins by cross-linking; used for preserving specimens and sterilizing medical equipment (e.g., glutaraldehyde, formaldehyde).
Chemical Sterilization: Ethylene oxide and other gases cross-link nucleic acids and proteins; used for heat-sensitive materials.
Peroxygens: Oxidizing agents (e.g., ozone, hydrogen peroxide) used for surfaces and packaging.

Effectiveness of Chemical Antimicrobials

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

Table showing effectiveness of chemical antimicrobials

Summary

Microbial control is achieved through physical and chemical methods, each with specific mechanisms and effectiveness.
Understanding the terminology, mechanisms, and factors influencing microbial death is essential for effective application in healthcare, industry, and daily life.