Microbiology: Control of Microbial Growth

COMMON PROTOCOLS FOR THE CONTROL OF MICROBIAL GROWTH

Microbial control protocols are categorized based on whether they are applied to inanimate objects (fomites) or living tissue.

Protocols for Use on Fomites

Disinfection
- Definition: The process of reducing or destroying the microbial load of an inanimate item through the application of heat or antimicrobial chemicals.
- Common Application: Cleaning surfaces such as laboratory benches, clinical surfaces, and bathrooms.
- Common Agents: Chlorine bleach, phenols (e.g., Lysol), and glutaraldehyde.
Sanitization
- Definition: The reduction of microbial load on an inanimate item to safe public health levels through the application of heat or antimicrobial chemicals.
- Common Application: Commercial dishwashing of eating utensils and cleaning public restrooms.
- Common Agents: Detergents containing phosphates (e.g., Finish) and industrial-strength cleaners containing quaternary ammonium compounds.
Sterilization
- Definition: The complete elimination of all vegetative cells, endospores, and viruses from an inanimate item.
- Common Application: Preparation of surgical equipment and needles used for injection.
- Common Agents: Pressurized steam (autoclave), specific chemicals, and radiation.

Protocols for Use on Living Tissue

Antisepsis
- Definition: The reduction of microbial load on skin or tissue through the application of an antimicrobial chemical.
- Common Application: Cleaning skin broken due to injury or cleaning skin before surgery.
- Common Agents: Boric acid, isopropyl alcohol, hydrogen peroxide, and iodine (betadine).
Degerming
- Definition: The reduction of microbial load on skin or tissue through gentle to firm scrubbing and the use of mild chemicals.
- Common Application: Routine handwashing.
- Common Agents: Soap and alcohol swabs.

MEASURING MICROBIAL CONTROL AND EFFICIENCY

Microbial Death Kinetics: Microbial death is logarithmic. It is more effectively observed and analyzed using a semilog plot rather than an arithmetic one.
Decimal Reduction Time (D-value): The D-value is defined as the time required to kill $90\%$ of the microbial population (representing a 1-log decrease in the total population) when exposed to a specific control protocol.
Environmental Microbial Load: Microbes are ubiquitous. In a study of cars, the center console was found to harbor the most microbes ( $506\,$ \text{CFUs}), likely due to spilled drinks. Frequently touched sites also show high concentrations of colony-forming units (CFUs).

BIOSAFETY LEVELS (BSL)

The CDC classifies infectious agents into four biosafety levels based on the potential risk to laboratory personnel and the community. Each level requires progressively more stringent precautions:

BSL-1: Low risk; basic precautions.
BSL-2: Moderate risk; specific training and restricted access.
BSL-3: Potential for serious disease; specialized ventilation.
BSL-4: High risk of life-threatening disease; full-body pressure suits and maximum containment.

PHYSICAL METHODS OF MICROBIAL CONTROL: HEAT

Heat is one of the most common physical methods for controlling growth by denaturing proteins and altering membranes.

Boiling: Exposure to $100\,^\circ\text{C}$ at sea level. Typically used for cooking, personal use, and preparing certain laboratory media. It denatures proteins and alters membranes.
Dry-heat Oven: Exposure to $170\,^\circ\text{C}$ for $2\,$ \text{hours}. Used for the sterilization of heat-stable medical and laboratory equipment and glassware. Mode of action includes protein denaturation, membrane alteration, dehydration, and desiccation.
Incineration: Direct exposure to flame (e.g., flaming a loop or using a microincinerator/bactericinerator). This destroys organisms by burning them to ash.
Autoclave: Uses pressurized steam. Typical settings are $121\,^\circ\text{C}$ for $15\,$ \text{minutes} at $15\,$ \text{pounds per square inch (psi)}. Used for sterilizing microbiological media and heat-stable medical/laboratory items. Autoclave tape features white strips that turn dark to indicate a successful run.
Pasteurization: Prevents spoilage in liquids like milk, apple juice, and honey. It denatures proteins and alters membranes.
- High-Temperature Short-Time (HTST): $72\,^\circ\text{C}$ for $15\,$ \text{seconds}.
- Ultra-High-Temperature (UHT): Used for long-term storage of milk without refrigeration.

PHYSICAL METHODS: COLD, PRESSURE, AND DESICCATION

Refrigeration: Temperatures between $0\,^\circ\text{C}$ to $7\,^\circ\text{C}$ . Inhibits metabolism to slow or arrest cell division. Used for preserving food and laboratory materials.
Freezing: Temperatures below $-2\,^\circ\text{C}$ . Stops metabolism and may kill microbes. Ultra-low freezers maintain temperatures at or below $-70\,^\circ\text{C}$ for long-term storage of cultures or medical specimens (e.g., liquid nitrogen storage).
High-pressure Processing: Uses $100-800\,$ \text{MPa}. Denatures proteins and can cause cell lysis. Used in food preservation.
Hyperbaric Oxygen Therapy: Air pressure three times higher than normal. Inhibits metabolism and growth of anaerobic microbes; used for treating infections like gas gangrene.
Simple Desiccation: Drying (reducing water activity) to inhibit metabolism. Used for dried fruits and jerky.
Osmotic Pressure: Addition of salt or sugar to inhibit metabolism and cause lysis. Used for salted meats, fish, honey, jams, and jellies.
Lyophilization: Rapid freezing under a vacuum. Inhibits metabolism; used for preserving food, reagents, and laboratory cultures.

PHYSICAL METHODS: RADIATION, SONICATION, AND FILTRATION

Ionizing Radiation: Exposure to X-rays or gamma rays. It alters molecular structures and introduces double-strand breaks into DNA. Used for sterilizing spices and heat-sensitive medical items.
Nonionizing Radiation: Exposure to Ultraviolet (UV) light. It causes the formation of thymine dimers in DNA, leading to lethal mutations. Used for surface sterilization and water purification via germicidal lamps.
Sonication: Exposure to ultrasonic waves. Creates cavitation (empty spaces) that disrupts and lyses cells. Used in laboratory research for cell lysis and cleaning jewelry or lenses.
HEPA Filtration: High-efficiency particulate air filters with a $0.3\,\mu\text{m}$ pore size. Physically removes microbes from air in biological safety cabinets, operating rooms, and HVAC systems.
Membrane Filtration: Use of filters with $0.2\,\mu\text{m}$ or smaller pore sizes. Removes bacteria from heat-sensitive solutions such as vitamins, antibiotics, and specialized media.

CHEMICAL METHODS OF MICROBIAL CONTROL

Triclosan: A common ingredient in many antibacterial soaps. Despite its use, the FDA notes no significant health benefit over conventional soap and evidence suggests it poses environmental and health risks.
Iodophors: Examples include Betadine (a povidone-iodine solution). These are used as topical antiseptics on skin before surgical incisions.
Alcohols: Includes Ethyl alcohol (found in drinks) and Isopropyl alcohol (rubbing alcohol). Used commonly as disinfectants and antiseptics.
Soaps: Salts of fatty acids. They function by emulsifying lipids, fats, and oils. They have hydrophilic heads and hydrophobic tails that interact with water and lipids respectively to lift away microbes.
Peroxygens: Hydrogen peroxide ( $H_2O_2$ ) is used for cleaning wounds and sterilizing contact lenses. The enzyme catalase converts $H_2O_2$ into water and oxygen ( $2H_2O_2 \rightarrow 2H_2O + O_2$ ).

PRACTICAL APPLICATIONS AND HYGIENE

Clostridium botulinum: The causative agent of botulism. Home canning requires a pressure canner because the endospores of this bacterium can survive temperatures above the boiling point of water ( $100\,^\circ\text{C}$ ).
Hand Hygiene for the Public: The CDC recommends a standard five-step process for general handwashing.
Surgical Scrubbing: A more extensive procedure than routine handwashing. It requires scrubbing from the fingertips, extending through the hands and forearms, and moving beyond the elbows.