Control of Microbial Growth: Principles, Methods, and Applications

Control of Microbial Growth

Terminology of Controlling Microbial Growth

Understanding the terminology is essential for discussing methods to control microbial growth in clinical and laboratory settings.

• Sepsis: Microbial contamination of body tissues or blood.

• Asepsis: The absence of significant contamination; aseptic techniques prevent contamination during procedures.

• Antisepsis: Destruction of harmful microorganisms from living tissue.

• Sterilization: Removal and destruction of all microbial life.

• Degerming: Mechanical removal of microbes from a limited area.

• Disinfection: Destruction of harmful microorganisms on surfaces.

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

• Biocide (germicide): Treatments that kill microbes.

• Bacteriostasis: Inhibition of microbial growth without killing.

The Rate of Microbial Death

The effectiveness of microbial control treatments depends on several factors, including the number of microbes, environmental conditions, exposure time, and microbial characteristics. Microbial death often follows an exponential pattern.

• Microbial Death Curve: When plotted logarithmically, the death curve is linear, indicating a constant percentage of cells killed per unit time.

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

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

Actions of Microbial Growth Control Agents

Agents that control microbial growth act by disrupting vital cellular components.

• Alteration of membrane permeability: Leads to leakage of cellular contents.

• Damage to proteins (enzymes): Denaturation impairs metabolic functions.

• Damage to nucleic acids: Prevents replication and transcription.

Physical Methods of Controlling Microbial Growth

Moist Heat Sterilization

Moist heat is highly effective for sterilization, especially in the form of steam under pressure (autoclaving).

• Autoclave: Uses steam at 121°C and 15 psi for 15 minutes to kill all organisms and endospores. Steam must contact the item's surface.

• Boiling and Free-flowing Steam: Less effective than autoclaving; may not kill endospores.

• Test strips: Used to indicate sterility.

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

Pasteurization

Pasteurization reduces spoilage organisms and pathogens using high temperature for a short time (HTST: 72°C for 15 sec). Thermoduric organisms may survive.

Dry Heat Sterilization

• Kills by oxidation: Methods include flaming, incineration, and hot-air sterilization.

Filtration

Filtration is used for heat-sensitive materials, allowing passage through a screenlike material.

• HEPA filters: Remove microbes > 0.3 μm.

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

Other Physical Methods

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

• High pressure: Denatures proteins.

• Desiccation: Absence of water prevents metabolism.

• Osmotic pressure: High salt/sugar concentrations cause plasmolysis.

Radiation

• Ionizing radiation: (X-rays, gamma rays, electron beams) creates reactive hydroxyl radicals, damaging DNA.

• Nonionizing radiation: (UV, 260 nm) creates thymine dimers in DNA.

• Microwaves: Kill by heat, not especially antimicrobial.

Principles of Effective Disinfection

Disinfection efficacy depends on concentration, organic matter, pH, and time. Standardized tests evaluate disinfectant effectiveness.

• Use-Dilution Test: Metal cylinders dipped in test bacteria, dried, exposed to disinfectant, then cultured to assess survival.

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

Chemical Methods of Controlling Microbial Growth

Phenol and Phenolics

Disrupt plasma membranes, causing leakage. Bisphenols contain two phenol groups connected by a bridge (e.g., hexachlorophene, triclosan).

Essential Oils

Mixtures of hydrocarbons from plants (e.g., peppermint, pine, orange oils). Microbial action is due to phenolics and terpenes; more effective against gram-positive bacteria.

Halogens

• Iodine: Impairs protein synthesis and alters membranes; available as tincture or iodophor.

• Chlorine derivatives: Oxidizing agents (e.g., bleach, chloramine) shut down metabolic pathways.

Alcohols

• Denature proteins and dissolve lipids: Ineffective against endospores and nonenveloped viruses.

• Ethanol and isopropanol: Require water for effectiveness.

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
40	G	G	G	G	G

Heavy Metals

• Oligodynamic action: Small amounts exert antimicrobial activity by denaturing proteins (e.g., Ag, Hg, Cu, Zn).

• Applications: Silver nitrate prevents ophthalmia neonatorum; copper sulfate is an algicide; zinc chloride in mouthwash.

Surface-Active Agents

• Soap: Degerming and emulsification.

• Acid-anionic sanitizers: Anions react with plasma membrane.

• Quaternary ammonium compounds (Quats): Cations are bactericidal, denature proteins, disrupt plasma membrane.

Effectiveness of Antiseptics

Other Chemical Methods

• Sulfur dioxide: Prevents wine spoilage.

• Organic acids: Inhibit metabolism; sorbic acid, benzoic acid, calcium propionate prevent molds in acidic foods.

• Nitrites and nitrates: Prevent endospore germination.

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

Aldehydes

• Inactivate proteins: Cross-linking with functional groups; used for preserving specimens and medical equipment.

• Glutaraldehyde: One of the few liquid chemical sterilizing agents.

Oxidizing Agents

• Used for contaminated surfaces and food packaging: O3, H2O2, peracetic acid.

Gaseous Agents and Plasma

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

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

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

Additional info: Decimal reduction time (DRT) is mathematically expressed as: $D = \frac{t}{\log_{10}(N_0/N) }$ where D is the DRT, t is time, N_0 is initial population, and N is final population.