Chapter 7: The Control of Microbial Growth – Study Notes

Chapter 7: The Control of Microbial Growth

Introduction

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

Key Terminology in Microbial Control

Definitions and Concepts

• Sepsis: Microbial contamination resulting from the growth and spread of bacteria in blood and tissues.

• Asepsis: The absence of significant contamination; essential for aseptic surgical techniques to prevent wound infection.

• Antisepsis: Removal of pathogens from living tissue.

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

• Commercial Sterilization: Treatment (especially of canned foods) to kill Clostridium botulinum endospores and vegetative cells.

• Disinfection: Removal of pathogens from non-living surfaces.

• Degerming: Removal of microbes from a limited area (e.g., swabbing skin with alcohol before injection).

• Sanitization: Reducing microbial counts on eating utensils and food preparation areas.

• Biocide/Germicide: Substance that kills microbes.

• Bacteriostasis: Inhibiting, but not killing, bacteria.

The Rate of Microbial Death

Microbial Death Kinetics

• Bacterial populations subjected to heat or antimicrobial chemicals usually die at a constant rate.

• When plotted logarithmically, the death curve is a straight line, indicating a constant percentage of cells die per unit time.

• The time required to kill a microbial population is proportional to the number of microbes present.

• Different microbial species and life cycle phases (e.g., endospores) have varying susceptibilities to control methods.

• Organic matter can interfere with heat treatments and chemical agents.

• Longer exposure to lower heat can have the same effect as shorter exposure to higher heat.

Table: Population Death Rate Is Constant

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

Microbial Death Curve

• Logarithmic plotting of microbial death (log of survivors vs. time) results in a straight line, indicating a constant death rate.

• Arithmetic plotting is less practical for large populations due to scale compression.

Factors Affecting Effectiveness of Antimicrobial Treatment

• Number of microbes present

• Environmental factors (organic matter, temperature, biofilms)

• Time of exposure

• Microbial characteristics (e.g., resistance mechanisms)

Actions of Microbial Control Agents

Mechanisms of Action

• Alteration of membrane permeability: Disrupts the plasma membrane, causing leakage of cellular contents.

• Damage to proteins: Denaturation by breaking hydrogen and covalent bonds, inactivating enzymes and structural proteins.

• Damage to nucleic acids: Interferes with DNA and RNA replication and protein synthesis, leading to cell death.

Physical Methods of Microbial Control

Heat

• Commonly used to eliminate microorganisms by denaturing proteins and enzymes.

• Thermal Death Point (TDP): Lowest temperature at which all cells in a culture are killed in 10 minutes. Influenced by heat resistance, cell clumping, water content, organic matter, and prior treatment.

• Thermal Death Time (TDT): Minimal time to kill all bacteria in a liquid culture at a given temperature.

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

• Boiling (100°C) kills many vegetative cells and viruses within 10 minutes.

• Autoclaving (steam under pressure) is the most effective moist heat sterilization method. Steam must contact the material directly.

Table: Pressure vs. Temperature of Steam

Pressure (psi in Excess of Atmospheric Pressure)	Temperature (°C)
0	100
5	110
10	116
15	121
20	126
30	135

Additional info: At higher altitudes, atmospheric pressure is lower, so higher psi is needed to reach the same sterilizing temperature.

Pasteurization

• Reduces spoilage organisms and pathogens in foods by killing vegetative cells.

• Equivalent treatments include:

  • 63°C for 30 min (Low-temperature long-time, LTLT)

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

  • 140°C for <1 sec (Ultra-high-temperature, UHT)

• Thermoduric organisms may survive but do not grow at storage temperatures.

Dry Heat Sterilization

• Kills by oxidation (e.g., flaming, incineration, hot-air sterilization).

Hot-air	Autoclave
180°C for 90 min	121°C, 15 min
170°C for 2 hours
160°C for 3 hours

Low Temperature

• Inhibits microbial growth (refrigeration, deep freezing, lyophilization).

• Effectiveness depends on the microorganism and application intensity.

• Most pathogens do not reproduce at 0–7°C, except Listeria.

• Many microbes survive but do not grow at subzero storage temperatures.

Filtration

• Removes microbes from liquids or gases by passing them through filters with small pores.

• Membrane filters (cellulose esters or plastic polymers) typically have pore sizes of 0.22 μm or 0.45 μm (bacteria), and 0.01 μm (viruses).

• Used for heat-sensitive materials (e.g., enzymes, vaccines, antibiotics).

• HEPA filters remove microbes from air.

Desiccation

• Absence of water prevents microbial growth, but many microbes remain viable.

• Viruses and endospores are more resistant to desiccation than vegetative cells.

Osmotic Pressure

• High concentrations of salts or sugars cause plasmolysis (cell shrinkage) due to water loss in hypertonic environments.

• Molds and yeasts tolerate low moisture/high osmotic pressure better than bacteria.

• Used in food preservation (e.g., curing meats, preserving fruits).

• High pressure can denature proteins.

Radiation

• Effectiveness depends on wavelength, intensity, and duration.

• Ionizing radiation (gamma rays, X-rays): High penetration, forms reactive hydroxyl radicals, destroys DNA. Used for sterilizing medical supplies and food products.

• Nonionizing radiation (UV): Low penetration, causes thymine dimers in DNA, most effective at 260 nm. Used for air and surface disinfection.

• Microwaves kill microbes indirectly by heating materials.

Chemical Methods of Microbial Control

Principles of Effective Disinfection

• Concentration of disinfectant

• Presence of organic matter

• pH

• Time of exposure

• Degree of contact with microorganisms

• Temperature

Conditions Influencing Microbial Control

• Type of microbe: Gram-positive bacteria are generally more susceptible than Gram-negative; Pseudomonas and Mycobacterium tuberculosis are highly resistant; endospores are very resistant.

• Environment: Organic matter (e.g., vomit, feces) can inhibit disinfectant action; higher temperatures enhance activity.

Evaluating Disinfectants

• Disk-diffusion method: Agar diffusion test (Kirby-Bauer test) to determine microbial susceptibility to chemical agents.

Types of Disinfectants

Phenol, Phenolics, and Bisphenols

• Phenol (carbolic acid): First used by Lister; rarely used now due to odor and skin irritation; bactericidal above 1% concentration.

• Phenolics: Modified phenols combined with soaps/detergents; disrupt plasma membranes, inactivate enzymes, denature proteins; effective against Mycobacteria.

• Bisphenols: Two phenol groups connected by a bridge (e.g., hexachlorophene, triclosan); effective against Gram-positive bacteria, some fungi, and Gram-negative bacteria; Pseudomonas aeruginosa is resistant to triclosan.

Biguanides

• Includes chlorhexidine; disrupts plasma membranes by blocking lipid synthesis enzymes.

• Effective against most vegetative bacteria and yeasts; less effective against mycobacteria, endospores, and protozoan cysts.

• Damages enveloped viruses; can cause eye damage.

Halogens

• Iodine and chlorine are used as disinfectants and antiseptics.

• Iodine inactivates proteins by combining with tyrosine; available as tincture or iodophor.

• Chlorine forms hypochlorous acid in water, a strong oxidizing agent; used in water treatment and as bleach.

Alcohols

• Denature proteins and dissolve lipids; enhance effectiveness of other agents in tinctures.

• Commonly used: ethanol (60–95%) and isopropanol.

Heavy Metals

• Silver, mercury, copper, and zinc exert antimicrobial action via oligodynamic action (denature proteins by binding sulfhydryl groups).

• Examples: silver nitrate (antiseptic), copper sulfate (algicide), zinc chloride (mouthwash).

Surface-Active Agents (Surfactants)

• Reduce surface tension; include soaps (mechanical removal), acid-anionic detergents (cleaning dairy equipment), and quaternary ammonium compounds (quats).

Quaternary Ammonium Compounds (Quats)

• Cationic detergents that disrupt plasma membranes, causing leakage of cell contents.

• Most effective against Gram-positive bacteria; Pseudomonas species can grow in quats.

Aldehydes

• Formaldehyde and glutaraldehyde inactivate proteins by forming covalent cross-links with functional groups.

• Formalin (37% formaldehyde) is used to preserve specimens and inactivate pathogens in vaccines.

• Among the most effective chemical disinfectants.

Gaseous Chemosterilizers

• Ozone, hydrogen peroxide, peracetic acid, and ethylene oxide are used as gaseous sterilants.

• Act by oxidizing cellular molecules, including proteins.

Chemical Food Preservatives

• Organic acids (sorbic, benzoic, calcium propionate) inhibit metabolism; used in foods and cosmetics.

• Nitrate/nitrite salts prevent endospore germination of Clostridium botulinum in meats.

• Antibiotics (nisin, natamycin) prevent spoilage of cheese.

Microbial Characteristics and Microbial Control

Relative Resistance of Microbes

• Gram-negative bacteria are generally more resistant than Gram-positive bacteria to disinfectants and antiseptics.

• Endospores and mycobacteria are highly resistant.

• Nonenveloped viruses are more resistant than enveloped viruses.

Factors to Consider When Selecting a Disinfectant

• Acts rapidly

• Destroys a wide range of microbes

• Penetrates surfaces

• Mixes easily with water

• Effective in presence of organic matter

• Stable, non-staining, non-corrosive, non-toxic, odorless, safe to transport, and economical