Chapter 7: The Control of Microbial Growth – Study Notes

The Control of Microbial Growth

Introduction

The control of microbial growth is essential in healthcare, food safety, and laboratory settings. This chapter explores the terminology, mechanisms, and methods used to inhibit or eliminate 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 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 (e.g., skin before injection).

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

• Biocide (Germicide): Treatments that kill microbes.

• Bacteriostasis: Inhibiting, not killing, microbes.

The Rate of Microbial Death

Factors Affecting Microbial Death

• Number of Microbes: Larger populations take longer to eliminate.

• Environment: Presence of organic matter, temperature, and biofilms can affect effectiveness.

• Time of Exposure: Longer exposure increases effectiveness.

• Microbial Characteristics: Endospores, cell wall structure, and other traits influence resistance.

Microbial Death Curve

Microbial death typically follows a logarithmic pattern, where a constant percentage of cells die per unit time. This is important for understanding sterilization and disinfection kinetics.

Table: Microbial Exponential Death Rate Example

Time (min)	Deaths per Minute	No. 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

Actions of Microbial Control Agents

Mechanisms of Action

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

• Damage to Proteins (Enzymes): Denaturation leads to loss of function.

• Damage to Nucleic Acids: Prevents replication and function.

Physical Methods of Microbial Control

Heat

• Denatures enzymes and proteins.

• 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 given temperature.

• Decimal Reduction Time (DRT): Minutes to kill 90% of a specific population at a given temperature.

Moist Heat Sterilization

• Coagulates/denatures proteins.

• Boiling and Free-flowing Steam: Effective but may not kill all endospores.

• 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.

• Large containers require longer sterilization times.

• Test strips are used to indicate sterility.

Table: Effect of Container Size on Autoclave Sterilization Times

Container Size	Liquid Volume	Sterilization Time (min)
Test tube: 18 × 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

Pasteurization and UHT

• 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 seconds, sterilizes milk and juices for storage without refrigeration.

Dry Heat Sterilization

• Kills by oxidation: Flaming, incineration, hot-air sterilization (170°C for 2 hours).

Filtration

• Used for heat-sensitive materials.

• HEPA filters: Remove microbes >0.3 μm.

• Membrane filters: Remove microbes >0.22 μm; pore sizes as small as 0.05 μm can filter out viruses and large proteins.

Other Physical Methods

• Low Temperature: Bacteriostatic effect (refrigeration, deep-freezing, lyophilization).

• High Pressure: Denatures proteins, alters carbohydrate structure.

• 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, damages DNA, used for sterilizing pharmaceuticals, medical supplies, and food.

• Nonionizing Radiation: (UV, 260 nm) Damages DNA by creating thymine dimers, used for surface sterilization.

• Visible Blue Light (470 nm): Kills bacteria via singlet oxygen formation.

• Microwaves: Kill by heat, not especially antimicrobial.

Chemical Methods of Microbial Control

Principles of Effective Disinfection

• Concentration of disinfectant

• Presence of organic matter

• pH

• Temperature

• Time of exposure

Testing Disinfectant Efficacy

• Use-Dilution Test: Metal cylinders with dried bacteria are 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.

Major Types of Chemical Agents

• Phenol and Phenolics: Disrupt plasma membranes; remain active in organic matter. Example: O-phenylphenol (Lysol®).

• Bisphenols: Disrupt plasma membranes. Examples: Hexachlorophene, Triclosan.

• Biguanides: Disrupt plasma membranes. Example: Chlorhexidine (surgical scrubs).

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

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

• Alcohols: Denature proteins, dissolve lipids; ineffective against endospores and nonenveloped viruses. Examples: Ethanol, isopropanol.

Table: Biocidal Action of Various Concentrations of Ethanol Against Streptococcus pyogenes

Concentration of Ethanol (%)	10 sec	20 sec	30 sec	40 sec	50 sec
100	G	G	G	G	G
95	NG	NG	NG	NG	NG
90	NG	NG	NG	NG	NG
80	NG	NG	NG	NG	NG
70	NG	NG	NG	NG	NG
60	NG	NG	NG	NG	NG
50	G	G	NG	NG	NG
40	G	G	G	G	G

G = growth; NG = no growth

• Heavy Metals: Oligodynamic action, denature proteins. Examples: Silver nitrate (prevents ophthalmia neonatorum), copper sulfate (algicide), zinc chloride (mouthwash).

• Surface-Active Agents: Soaps (degerming), acid-anionic sanitizers (cleaning food facilities), quaternary ammonium compounds (quats; broad spectrum, not effective against endospores or mycobacteria).

• Chemical Food Preservatives: Sulfur dioxide (wine), organic acids (sorbic, benzoic, calcium propionate), nitrites/nitrates (meat products).

• Antibiotics for Food Preservation: Bacteriocins (nisin, natamycin) prevent cheese spoilage.

• Aldehydes: Inactivate proteins by cross-linking. Examples: Formalin (preserving specimens), glutaraldehyde (liquid sterilant for medical equipment).

• Gaseous Chemosterilants: Ethylene oxide (sterilizes heat-sensitive materials), chlorine dioxide (building and water disinfection).

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

• Supercritical Fluids: CO2 in a supercritical state, used for food and medical implants.

• Peroxygens: Oxidizing agents (hydrogen peroxide, peracetic acid, ozone) used for disinfecting surfaces, packaging, and water.

Microbial Characteristics and Microbial Control

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

• Mycobacteria: Highly resistant; require special testing for tuberculocidal activity.

• Bacterial Endospores: Very resistant to many biocides.

• Nonenveloped Viruses: More resistant than enveloped viruses.

• Prions: Extremely resistant; require immersion in NaOH and autoclaving at 121°C for 1 hour.

Table: Effectiveness of Chemical Antimicrobials Against Endospores and Mycobacteria

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

Summary

Understanding the principles and methods of microbial control is essential for preventing infection, ensuring food safety, and maintaining sterile environments in healthcare and research. Both physical and chemical methods have specific applications, advantages, and limitations depending on the type of microorganism and the context of use.