Physical Control of Microbial Growth

Essential Terminology for Microbial Control

Sterilization: The total destruction of all life forms on an object; it is considered an absolute state.
Disinfection: The destruction of vegetative pathogens, but not necessarily endospores or viruses. The goal is to reduce or inhibit microbial growth on inanimate objects.
Antisepsis: The practice of chemical disinfection applied to skin, mucous membranes, or other living tissues.
-cide: A suffix denoting a chemical agent that rapidly kills microbes, though it typically does not kill endospores.
-stasis: A suffix denoting that growth and multiplication are inhibited, but the organisms are not necessarily killed.
Asepsis: The complete absence of pathogens from an object or a specific area.
Degerming: The removal of transient microbes from the skin, typically achieved through mechanical cleansing or the use of an antiseptic.
Sanitization: The reduction of pathogens on eating utensils to levels deemed safe by public health standards, achieved by mechanical cleansing or chemical agents.
Decontamination: The treatment of an object or surface to render it safe to handle.

Kinetics of Microbial Death

Microbial death occurs at a constant rate when a population is subjected to a lethal agent such as heat.

Sample Scenario: A culture of microorganisms containing $10^6$ cells is heated at a temperature above its maximum growth temperature. Plate counts are taken from a $1.0000000000000002 \times 10^{-3} dm^3$ ( $1\,cm^3$ ) sample at $5\,min$ intervals.
Logarithmic Decline: The number of survivors decreases logarithmically over time. As the population decreases, the absolute number of cells killed in each interval also decreases, while the percentage of the remaining population killed remains constant.

Table: Survivors at intervals after heating a culture (Garbutt, 1997)

Time (min)	No. of cells killed	No. of cells surviving	$\log_{10}$ of cells surviving
$0\,min$	$0$	$1000000$	$6$
$5\,min$	$900000$	$100000$	$5$
$10\,min$	$990000$	$10000$	$4$
$15\,min$	$999000$	$1000$	$3$
$20\,min$	$999900$	$100$	$2$
$25\,min$	$999990$	$10$	$1$
$30\,min$	$999999$	$1$	$0$
$35\,min$	$999999.9$	$0.1$	$-1$
$40\,min$	$999999.99$	$0.01$	$-2$

The Microbial Growth Curve

The life cycle of a microbial population in a closed system is represented by four distinct phases:

Lag Phase: Interval where cells are adjusting to their environment and not yet dividing.
Exponential (Log) Phase: Period of maximal growth and cell division; the population increases at a constant logarithmic rate.
Stationary Phase: The growth rate slows, and the number of new cells produced equals the number of cells dying, often due to nutrient depletion or waste accumulation.
Death Phase: The number of deaths exceeds the number of new cells, leading to a decline in the population.

Conditions Influencing Microbial Control

Several factors determine the effectiveness of antimicrobial treatments:

Population Size: Larger populations require more time to achieve sterilization.
Population Composition: Includes the specific type of microorganism (e.g., endospores vs. vegetative cells) and the physiological state of the organism.
Concentration and Intensity: Higher concentrations of chemical agents or higher intensities of physical agents (like radiation) are generally more effective.
Duration of Exposure: Longer exposure times lead to higher rates of microbial death.
Temperature: Often, higher temperatures enhance the activity of antimicrobial agents.
Environmental Conditions: Factors such as pH and the presence of organic matter (e.g., blood, vomit, or feces) can inhibit the effectiveness of control methods.

Physical Methods: Heat (Moist Heat)

Moist heat kills microorganisms primarily through the degradation of nucleic acids, the denaturation of enzymes and essential proteins, and the disruption of cell membranes.

Boiling

Used for the disinfection of drinking water and various objects.
It is important to note that boiling does not achieve sterilization because it cannot reliably kill all endospores.

Steam Under Pressure (Autoclaving)

High pressure is used to increase the temperature of steam above the boiling point of water ( $100\,^{\circ}C$ ).

Relationship between Pressure and Steam Temperature (Sea Level, Tortora et al., 1995):

$0\,PSI$ : $100\,^{\circ}C$
$5\,PSI$ : $110\,^{\circ}C$
$10\,PSI$ : $116\,^{\circ}C$
$15\,PSI$ : $121\,^{\circ}C$
$20\,PSI$ : $126\,^{\circ}C$
$30\,PSI$ : $135\,^{\circ}C$

Sterilization Time and Container Size (Tortora et al., 1995):

Test tube ( $18 \times 150\,mm$ ) with $10\,cm^3$ liquid: $15\,min$
Erlenmeyer flask ( $125\,cm^3$ ) with $95\,cm^3$ liquid: $15\,min$
Erlenmeyer flask ( $2000\,cm^3$ ) with $1500\,cm^3$ liquid: $30\,min$
Fermentation bottle ( $9000\,cm^3$ ) with $6750\,cm^3$ liquid: $70\,min$

Conditions for Killing Specific Organisms with Moist Heat:

Vegetative Cells: * Yeasts: $5\,min$ at $50-60\,^{\circ}C$ * Molds: $30\,min$ at $62\,^{\circ}C$ * Bacteria (Mesophilic): $10\,min$ at $60-70\,^{\circ}C$ * Viruses: $30\,min$ at $60\,^{\circ}C$
Spores: * Yeasts: $5\,min$ at $70-80\,^{\circ}C$ * Molds: $30\,min$ at $80\,^{\circ}C$ * Bacteria: $2$ to over $800\,min$ at $100\,^{\circ}C$ ; or $0.5-12\,min$ at $121\,^{\circ}C$

Autoclave Types and Sterility Controls

Types: Common laboratory autoclaves, pressure cooker types (e.g., All American Model No. 921-25qt), vertical autoclaves, and large automatic hospital autoclaves (horizontal).
Standard Operation: Typically $15\,PSI$ for $15\,min$ . For pressure cookers, the vent pipe should let steam out for $3-5\,min$ before closing.
Sterility Indicators: * Biological indicator: Uses spores of Geobacillus stearothermophilus. A flexible vial contains a nutrient medium with a pH indicator and an endospore strip. After autoclaving, the glass ampule is crushed. If the medium turns yellow, spores are viable (not sterile); if it remains red, spores were killed (sterile). * Autoclave Tape: Chemical indicator tape (e.g., Hi-Autotape) that changes color (dark stripes appear) after exposure to steam.

Physical Methods: Pasteurization and Tyndallization

Pasteurization

This involves heating at temperatures well below boiling to kill pathogens and reduce spoilage organisms without altering the food quality.

Older Method: $63\,^{\circ}C$ for $30\,min$ .
Flash (HTST) Pasteurization: $72\,^{\circ}C$ for $15\,s$ , followed by rapid cooling.
Ultra-high-temperature (UHT) Sterilization: $140$ to $150\,^{\circ}C$ for $1$ to $3\,s$ . This allows products like milk to be stored without refrigeration (e.g., Dutch Mill UHT Yoghurt Drink).

Tyndallization (Fractional Steam Sterilization)

Involves heating at $90-100\,^{\circ}C$ for three consecutive days.
Incubation at $37\,^{\circ}C$ is performed between heating sessions to allow spores to germinate into vegetative cells, which are then killed in the subsequent heating cycle.

Physical Methods: Dry Heat

Dry heat kills by the oxidation of cell constituents and denaturation of proteins.

Hot-air Sterilization: Items are placed in an oven at $160-170\,^{\circ}C$ for $2-3\,hours$ . Dry heat is less effective than moist heat (e.g., C. botulinum spores require $5\,min$ at $121\,^{\circ}C$ in moist heat but $2\,hours$ at $160\,^{\circ}C$ in dry heat). * Advantages: It does not corrode glassware or metal instruments and can sterilize powders and oils.
Incineration: Burning materials at approximately $500\,^{\circ}C$ to physically destroy microorganisms; used for solid waste.
Direct Flaming: Used for sterilizing laboratory loops/needles.

Physical Methods: Cold

Cold temperatures inhibit growth and reproduction by slowing down microbial metabolism. They are used for short-term storage.

Effects of Low Temperature: * Flocculation of proteins. * Physical damage caused by ice crystal formation. * Alteration of membrane lipids.
Preservation of Cultures: * Deep Freezing: Rapid cooling of pure cultures in liquid suspension to $-50\,^{\circ}C$ to $-95\,^{\circ}C$ . Effective for several years. * Lyophilization (Freeze-Drying): The culture is frozen (between $-54\,^{\circ}C$ and $-72\,^{\circ}C$ ) and then dehydrated in a vacuum. Effective for several years.

Physical Methods: Filtration

Filtration removes cells rather than killing them.

Depth Filters: Thick layers of fibrous or granular materials (e.g., diatomaceous earth/Berkefeld, unglazed porcelain/Chamberlain, or asbestos) with twisting, small-diameter channels.
Membrane Filters: Porous membranes made of cellulose acetate, cellulose nitrate, polycarbonate, or polyvinylidene fluoride. Common pore diameters include $0.2\,\mu m$ and $0.4\,\mu m$ .
Air Filtration: * Surgical masks and cotton plugs. * High-efficiency particulate air (HEPA) filters: Used in clean rooms and Biosafety Cabinets. * Biosafety Cabinets (BSC): Classified as Class I, Class II, or Class III based on the level of protection provided to the personnel and the environment.

Physical Methods: Removal of Water

Desiccation prevents microbial growth by removing the water necessary for life.

Methods: Lyophilization or the addition of high concentrations of solutes (sugar or salt) to create high osmotic pressure.
Case Study: Honey: * Osmotic Pressure: Honey has such a low water concentration (fanned by bees until it reaches approximately $17\,\%$ ) that it draws water from bacteria, dehydrating them. * Chemical Properties: Bees secrete enzymes that break down sucrose into glucose and fructose. The action of bee secretions on glucose produces gluconic acid and hydrogen peroxide. * Acidity: Gluconic acid creates a low pH (between $3$ and $4$ ), making honey hostile to bacterial growth.

Physical Methods: Radiation

Radiation is categorized by wavelength and energy levels.

Ionizing Radiation: Includes X-rays and Gamma rays. They have very short wavelengths and high energy. Death or mutation results from: 1. Disruption of hydrogen bonds. 2. Oxidation of double bonds. 3. Destruction of ring structures. 4. Polymerization of molecules. 5. Destruction of DNA.
Non-ionizing Radiation (UV Rays): Specifically wavelengths between $220-300\,nm$ (with $260\,nm$ being the most absorbed by DNA). * Mechanism: Causes the formation of thymine dimers or thymine-cytosine dimers in DNA, preventing proper replication. * Usage: Disinfection of surfaces, air, and water. * Disadvantage: Low penetrating power; it cannot move through solid, opaque, or light-absorbing surfaces.

High Intensity Pulsed Electric Field Treatment

This is a non-thermal control method.
Materials are exposed to an electric field of $15-20\,kV/cm$ for a few milliseconds or less.
Electroporation: The process where pulses of electricity create temporary pores in cell membranes, often leading to cell death or enabling the introduction of foreign DNA.