Chapter 7: The Control of Microbial Growth – Study Notes

The Control of Microbial Growth

Introduction

Controlling microbial growth is essential in medical, industrial, and food settings to prevent infection, spoilage, and contamination. This chapter covers terminology, mechanisms, and methods used to control microorganisms, including both physical and chemical approaches.

Terminology of Microbial Control

Definitions and Concepts

• Sepsis: Refers to bacterial contamination, often leading to infection.

• Asepsis: The absence of significant contamination; aseptic techniques are crucial in surgery to prevent infection.

• Sterilization: Removal and destruction of all microbial life, including endospores.

• Commercial Sterilization: Specifically targets Clostridium botulinum endospores in canned goods.

• Disinfection: Destruction of harmful microorganisms on surfaces.

• Antisepsis: Destruction of harmful microorganisms on living tissue.

• Degerming: Mechanical removal of microbes from a limited area (e.g., hand washing).

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

• Biocide (germicide): Treatments that kill microbes.

• Bacteriostasis: Inhibition, not killing, of microbes.

The Rate of Microbial Death

Microbial Death Curve and Factors Affecting Death Rate

The rate at which microbes die during treatment is exponential and can be represented mathematically and graphically. Several factors influence the effectiveness of microbial control methods:

• 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: Some microbes are more resistant than others.

Decimal Reduction Time (DRT): The time required to kill 90% of a microbial population at a given temperature.

Mathematical representation:

$N_t = N_0 \times 10^{-t/D}$

where $N_t$ is the number of survivors at time $t$, $N_0$ is the initial number, and $D$ is the decimal reduction time.

Actions of Microbial Control Agents

Mechanisms of Action

• Alteration of membrane permeability: Disrupts cell membranes, causing leakage of cellular contents.

• Damage to proteins (enzymes): Denaturation or inactivation of enzymes impairs metabolism.

• Damage to nucleic acids: Prevents replication and transcription, leading to cell death.

Physical Methods of Microbial Control

Heat

Heat is a widely used method for sterilization and disinfection. It denatures proteins and 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 required to kill all bacteria at a specific temperature.

• Pasteurization: Reduces spoilage organisms and pathogens by heating materials for a short time.

  • High-temperature short-time (HTST): 72°C for 15 sec

  • Ultra-high-temperature (UHT): 140°C for 4 sec

  Thermoduric organisms may survive these treatments.

Moist Heat Sterilization

• Boiling and Free-flowing Steam: Coagulates and denatures proteins.

• Autoclave: Uses steam under pressure (121°C at 15 psi for 15 min) to kill all organisms and endospores. Steam must contact the item's surface for effectiveness.

Large containers require longer sterilization times. Test strips are used to indicate sterility.

Dry Heat Sterilization

• Kills by oxidation.

• Methods include flaming, incineration, and hot-air sterilization.

Filtration

Filtration is used for heat-sensitive materials. It involves passing a substance through a screenlike material to remove microbes.

• HEPA filters: Remove microbes > 0.3 μm in diameter.

• Membrane filters: Remove microbes > 0.22 μm; pore sizes as small as 0.01 μm can filter out viruses and large proteins.

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) Ionizes water to create reactive hydroxyl radicals, damaging DNA.

• 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

Testing Disinfectant Efficacy

• Use-Dilution Tests: Metal cylinders dipped in test bacteria, dried, exposed to disinfectant, then transferred to culture media to check survival.

• Disk-Diffusion Method: Filter paper disks soaked in chemical placed on culture; zone of inhibition indicates efficacy.

Types of Chemical Agents

• Phenol and Phenolics: Injure lipids of plasma membranes, causing leakage.

• Bisphenols: Two phenol groups connected by a bridge; disrupt plasma membranes (e.g., hexachlorophene, triclosan).

• Biguanides: Chlorhexidine; used in surgical hand scrubs, disrupts plasma membranes.

• Essential Oils: Mixtures of hydrocarbons from plants; microbial action due to phenolics and terpenes, stronger against gram-positive bacteria.

• Halogens:

  • Iodine: Impairs protein synthesis and alters membranes; tincture and iodophor forms.

  • Chlorine: Oxidizing agent; shuts down cellular enzyme systems (bleach, chloramine).

• Alcohols: Denature proteins and dissolve lipids; ineffective against endospores and nonenveloped viruses. Ethanol and isopropanol require water for activity.

• Heavy Metals: Oligodynamic action; denature proteins. Examples: Ag, Hg, Cu, Zn. Silver nitrate prevents ophthalmia neonatorum; copper sulfate is an algicide; zinc chloride in mouthwash.

• Surface-Active Agents: Lower surface tension, aiding removal of microbes.

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

• Antibiotics: Bacteriocins inhibit other bacteria; nisin and natamycin prevent cheese spoilage.

• Aldehydes: Inactivate proteins by cross-linking; used for specimen preservation and medical equipment sterilization (formaldehyde, glutaraldehyde).

• Chemical Sterilization: Gaseous sterilants (e.g., ethylene oxide) cause alkylation, cross-linking nucleic acids and proteins; used for heat-sensitive materials.

• Plasma: Electrically excited gas; free radicals destroy microbes, used for tubular instruments.

• Supercritical Fluids: Used for sterilization, especially in medical and food industries.

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

Microbial Characteristics and Microbial Control

Resistance of Microbes to Chemical Agents

Microbial resistance varies by species and type. Endospores and mycobacteria are particularly resistant to many chemical agents.

Chemical Agent	Effect against Endospores	Effect against Mycobacteria
Glutaraldehyde	Fair	Good
Chlorines	Fair	Fair
Alcohols	Poor	Good
Iodine	Poor	Good
Phenolics	None	Good
Chlorhexidine	None	Fair
Bisphenols	None	None
Quats	None	None
Silver	None	None

Summary Table: Microbial Exponential Death Rate

Time (min)	Deaths per Minute	Number of Survivors
0	0	1,000,000
1	900,000	100,000
2	90,000	10,000
3	9,000	1,000
4	900	100
5	90	10
6	9	1

Conclusion

Effective microbial control requires understanding the terminology, mechanisms, and methods available. Both physical and chemical approaches are used, and the choice depends on the type of microorganism, environment, and intended application. Resistance varies among microbes, making it essential to select appropriate methods for each situation.