Sterilization Methods: Safeguarding Purity through Physical and Chemical Means

Sterilization is a critical process aimed at eliminating or destroying all forms of microbial life, including bacteria, viruses, spores, and fungi, to achieve asepsis. Various methods are employed, falling into two broad categories: physical and chemical.

Sterilization, whether through physical or chemical means, is pivotal in diverse fields, including healthcare, laboratories, and food industries. The choice of method depends on the nature of materials, desired sterility assurance levels, and practical considerations.

Understanding and implementing these sterilization methods are crucial for maintaining aseptic conditions and preventing the spread of infections.

Common Terms & Definitions

Sterilization:

Sterilization is the process by which an article, surface, or medium is made free of all microorganisms, either in vegetative or spore form, ensuring the complete elimination of viable microbes.

Disinfection:

Disinfection refers to the destruction of all pathogens or organisms that can cause infection, though not necessarily spores. While not necessarily eliminating all microorganisms, the number is reduced to a level that is no longer harmful to health.

Antiseptics:

Antiseptics are chemical disinfectants that are safe to apply on living tissues. They are used to prevent infection by arresting the growth of microorganisms. Antiseptics are commonly employed in medical settings for skin disinfection and wound care.

Asepsis:

Asepsis refers to the technique or state that helps prevent the occurrence of infection in an uninfected tissue or environment. It involves practices and procedures designed to maintain a sterile or pathogen-free condition, reducing the risk of contamination and infection. Aseptic techniques are crucial in medical and laboratory settings to ensure the safety of patients and experiments.

Methods of Sterilization

Physical Methods

Physical sterilization methods are techniques employed to eliminate or deactivate microorganisms from surfaces, equipment, or substances through physical means. Among these methods, heat sterilization, filtration, radiation, and cold sterilization are prominent approaches.

1. Heat Sterilization:
   • Autoclaving: This method employs high-pressure steam at temperatures above boiling, effectively destroying microorganisms by denaturing their proteins and disrupting cellular structures. Autoclaving is commonly used in laboratories and healthcare settings for sterilizing equipment and instruments.
   • Dry Heat Sterilization: Involves exposing items to hot air or direct flaming. This process raises temperatures, causing coagulation and oxidation of microbial proteins, ultimately disrupting their cellular integrity. Dry heat sterilization is suitable for items that may be damaged by moisture, like glassware and certain medical instruments.
2. Filtration:
   • Membrane Filtration: This technique utilizes a porous membrane to separate microorganisms and particles above a specific size threshold. By passing substances through the membrane, it effectively retains and removes contaminants. Membrane filtration is commonly used in the pharmaceutical and food industries for liquid sterilization.
3. Radiation:
   • Ultraviolet (UV) Radiation: UV radiation damages the DNA of microorganisms, specifically by forming thymine dimers that hinder replication. UV sterilization is often employed in water treatment, air purification, and surfaces that are sensitive to heat.
   • Ionizing Radiation (Gamma, X-rays): This method induces chemical and structural changes in microbial DNA, rendering the microorganisms non-viable. Gamma radiation is widely used for the sterilization of medical equipment, pharmaceuticals, and certain food products.
4. Cold Sterilization:
   • Cold Plasma: This technique employs low-temperature plasma to inactivate microorganisms. Cold plasma generates reactive species that damage microbial cell membranes and components. It is useful for sterilizing heat-sensitive materials and electronic devices.

Each physical sterilization method has its advantages and limitations, making them suitable for specific applications. The choice of method depends on factors such as the nature of the materials being sterilized, the level of sterility required, and the potential impact of the sterilization process on the integrity of the items.

Sunlight

Sunlight is a natural sterilization method with an active germicidal effect, primarily due to the presence of ultraviolet (UV) rays. Sunlight exposure reduces the number of microorganisms in various environments such as water tanks and lakes. UV radiation in sunlight damages the DNA of microorganisms, hindering their replication and rendering them non-viable.

Heat Sterilization: Heat is a widely utilized and highly effective method of sterilization, with two major approaches: dry heat and moist heat.

Dry Heat Sterilization Procedures:

1. Red Heat:
   • Objects Sterilized: Inoculation loops, wires, forceps tips, needles.
   • Method: The instruments are held in the flame of a Bunsen burner until they become red hot.
   • Purpose: To inhibit microbial contamination by achieving high temperatures that denature proteins and destroy microorganisms.
2. Flaming:
   • Objects Sterilized: Glass slides, scalpels, mouths of culture tubes, or conical flasks.
   • Method: Objects are passed through a Bunsen flame without allowing them to become red hot.
   • Purpose: Sterilization without reaching red heat, suitable for items sensitive to extreme temperatures.
3. Incineration:
   • Objects Sterilized: Soiled dressings, animal carcasses, bedding, pathological materials.
   • Method: Burning in an incinerator to reduce infective material to ashes.
   • Purpose: Complete destruction of biological materials, especially in medical waste disposal.
4. Hot Air Oven:
   • Objects Sterilized: Glass syringes, Petri dishes, flasks, pipettes, test tubes, surgical instruments, chemicals.
   • Method: Electricity maintains heat, a fan ensures even air distribution, and a thermostat controls the temperature. Sterilization is typically at 160°C for two hours, with alternative settings available.
   • Purpose: Broad sterilization application, particularly for items sensitive to moisture.
   • Uses:
     • Sterilization of glassware, surgical instruments, and chemicals.
     • Sterilization Control: Spores of Bacillus subtilis subsp. Niger (NCTC 10075 or ATCC 9372) are placed inside the oven, and their destruction indicates proper sterilization.
     • Alternative Settings: 170°C for 1 hour, 180°C for 30 minutes.
     • Sterilization Control Tools: Thermocouples and Browne’s tube with a green spot that produces a green color after proper sterilization.

Moist Heat Sterilization: Procedure

Sterilization at Temperatures below 100°C:

1. Pasteurization:
   • Types:
     • Holder Method: 63°C for 30 minutes.
     • Flash Method: 72°C for 20 seconds followed by quick cooling to 13°C.
   • Effectiveness: Targets non-sporing pathogens (e.g., mycobacteria, Salmonella) except Coxiella burnetii, which survives the Holder method due to heat-resistant characteristics.
   • Application: Commonly used for milk sterilization.
2. Inspissation:
   • Process: Media like Lowenstein-Jensen’s and Loeffler’s serum are sterilized at 80-85°C for 30 minutes daily on three consecutive days.
   • Equipment: The instrument used is called an inspissator.
   • Application: Sterilization of specific culture media.
3. Vaccine Bath:
   • Temperature and Duration: Sterilization of bacterial vaccines at 60°C for one hour. Serum or other body fluids can be sterilized in a water bath at 56°C for several successive days.
   • Application: Used for the sterilization of bacterial vaccines and body fluids.
4. Low-Temperature Steam Formaldehyde Sterilization (LTSF):
   • Temperature and Pressure: Steam at subatmospheric pressure at 75°C with formaldehyde vapor.
   • Biological Control: Bacillus stearothermophilus is used as a biological control to test the efficacy of the sterilization process.
   • Application: Suitable for materials that cannot withstand temperatures as high as 100°C.

These sterilization methods below 100°C offer alternatives for specific applications where traditional high-temperature methods may not be suitable. Each method is designed to balance the need for effective sterilization with the preservation of materials that may be sensitive to higher temperatures.

Sterilization at a Temperature of 100°C:

1. Boiling:
   • Effectiveness: Kills most vegetative cells but may not effectively eliminate spores.
   • Duration: Boiling for 10-30 minutes.
   • Application: Used when more advanced sterilization methods are not available. Suitable for sterilizing items like glass syringes, rubber stoppers, etc.
2. Tyndallisation (Intermittent Sterilization):
   • Temperature: Steam at 100°C for successive 3 days.
   • Process: Also known as intermittent sterilization, it involves multiple exposures to steam. The first exposure kills vegetative forms, while the intervals between heating allow remaining spores to germinate into vegetative forms, which are then killed in subsequent heatings.
   • Application: Used for sterilization of delicate items such as egg, serum, or sugar-containing media, where prolonged exposure to high temperatures may cause damage.
3. Steam Sterilizer (Koch’s and Arnold’s Method):
   • Temperature: Steam at 100°C.
   • Pressure: At atmospheric pressure.
   • Duration: 90 minutes.
   • Application: Used for sterilizing media that can easily decompose under the high temperature in an autoclave. The media are placed on a perforated tray, and steam at 100°C passes through for the specified duration. Effective in killing vegetative cells.

These methods, conducted at a temperature of 100°C, provide sterilization options for various items and materials, especially when the use of higher temperatures or more sophisticated equipment is not practical or available.

Sterilization at Temperature above 100°C (Under Pressure):

Autoclave

• Temperature: Steam above 121°C or saturated steam.
• Mechanism: Saturated steam has superior killing capacity compared to dry heat. Moist heat causes rapid coagulation of bacterial proteins. The ability of saturated steam to penetrate porous materials enhances its effectiveness. As steam contacts cooler surfaces, it condenses into water, releasing latent heat, creating moist conditions for microbial killing.

Autoclave Construction:

• Structure: A modified pressure cooker with a vertical or horizontal stainless steel cylinder.
• Lid: Secured tightly with screw clamps for airtight sealing.
• Components: Includes a steam discharge unit, pressure gauge, safety valve, and thermostat to monitor temperature.
• Heating: Powered by electricity.

**Sterilization Process:

1. Preheating:
   • The autoclave is filled with an adequate amount of water.
   • Preheating occurs before sterilization.
2. Loading:
   • Materials requiring sterilization are placed inside the autoclave.
3. Closure:
   • The lid is tightly closed to create an airtight seal.
4. Heating and Pressurization:
   • Temperature and pressure increase gradually.
   • Steam is generated within the autoclave.
5. Sterilization Conditions:
   • Sterilization is performed at a specific temperature and pressure.
   • Common conditions are 121°C at 15 psi (pounds per square inch).
   • Duration: Typically 15 minutes under these conditions.

Uses:

• Sterilization of:
  • Culture media.
  • Rubber materials.
  • Dressing gloves.
  • Materials unable to withstand dry heat in a hot air oven.

Advantages of Autoclaving:

• Rapid and efficient microbial destruction.
• Versatile and applicable to a wide range of materials.
• Effective penetration into porous items.
• Moist conditions enhance killing efficiency.

The autoclave is a crucial tool in microbiology, medical, and research settings, providing reliable sterilization for various instruments and materials.

Ozone Sterilizer:

Principle:

• Components: Utilizes oxygen, water, and electricity.
• Chemical Production: Produces ozone without generating toxic chemicals.

Operating Conditions:

• Temperature Range: Operates at temperatures between 25-35°C.

Sterilization Mechanism:

1. Oxygen Conversion:
   • Inside the ozone sterilizer, an intense electrical field converts oxygen into atomic oxygen.
2. Ozone Production:
   • Atomic oxygen combines with oxygen molecules to produce ozone.

Sterility Assurance:

• Sterility Level: Provides a sterility assurance level of 10^-6 (meaning a one in a million chance of a viable microorganism remaining) in approximately 4 hours.

Advantages:

1. Non-Toxic: Does not produce toxic chemicals during the sterilization process.
2. Environmental Impact: Ozone is a naturally occurring gas and, when used for sterilization, does not contribute to environmental pollution.
3. Temperature Control: Operates at a moderate temperature range (25-35°C), which may be suitable for materials sensitive to higher temperatures.
4. Efficiency: Achieves a high level of sterility assurance within a relatively short time frame (approximately 4 hours).

Application:

• Ozone sterilizers find application in various industries, including medical settings, laboratories, and food processing, where a non-toxic sterilization method is desirable, and materials may be sensitive to higher temperatures or chemical exposure.

Filtration for Sterilization:

Introduction:

• Filtration is a sterilization method suitable for materials sensitive to heat.
• Various types of filters are employed for different applications.

Types of Filters:

1. Candle Filter:
   • Application: Used for water purification.
   • Structure: Consists of hollow candles through which water passes for purification.
2. Asbestos Disc Filters:
   • Material: Made up of magnesium silicate.
3. Sintered Glass Filters:
   • Composition: Prepared by fusing finely powdered glass powders.
4. Membrane Filters:
   • Material: Made up of cellulose esters.
   • Applications:
     • Water analysis.
     • Sterility testing.
     • Solution preparation.
   • Pore Size: Available in pore sizes ranging from 0.015 to 12 microns.
     • Common Size: The 0.22-micron filter is commonly used, being smaller than bacteria.
5. Air Filters (HEPA Filters):
   • Application: Used in laminar airflow chambers to provide a bacteria-free air supply.
   • Pore Size: Can separate particles of 0.3 microns or larger.
   • Alias: Also known as High-Efficiency Particulate Air (HEPA) filters.
6. Syringe Filters:
   • Design: Syringes fitted with membranes of different diameters.

Limitation of Filtration:

• Pore Size Limitation: Filtration processes may not effectively remove viruses due to the limitation of pore sizes, as viruses can be smaller than the available filter pores.

Applications:

• Filtration is widely used in industries such as pharmaceuticals, biotechnology, water treatment, and food and beverage, providing a versatile method for achieving sterilization without exposing materials to high temperatures or chemicals.

Radiation for Sterilization:

Ionizing Radiation:

1. Types:
   • Gamma Rays: Emitted from a cobalt-60 source, commercially used for sterilization of disposable items.
   • X Rays and Cosmic Rays: Also considered ionizing radiations.
2. Mechanism:
   • Effect on Cells: High penetrating power makes ionizing radiations lethal for cells.
   • Cellular Damage: Bacterial cells are killed by damaging DNA.
   • Gamma Radiation: Utilized for cold sterilization due to its deep penetration.
3. Applications:
   • Commercial Use: Sterilization of disposable items.
   • Procedure Alias: Cold sterilization.

Non-ionizing Radiation:

1. Infrared Radiation:
   • Application: Used for mass sterilization of syringes and catheters.
   • Mechanism: Heating effect contributes to sterilization.
2. UV Radiation:
   • Wavelength: 240nm to 280nm.
   • Bactericidal Capacity: Causes protein denaturation and interferes with bacterial DNA replication.
   • Applications:
     • Sterilization of close areas.
     • Surfaces.
     • Operation theaters.
     • Laminar airflow systems.
   • UV Lamp Usage: Commonly used in various sterilization devices.

Overall Advantages:

• Lethal to Microorganisms: Both ionizing and non-ionizing radiations are effective in killing microorganisms.
• Deep Penetration: Ionizing radiation, especially gamma rays, penetrates deeply, making it suitable for sterilizing dense materials.
• Surface Sterilization: UV radiation is effective for sterilizing surfaces and confined spaces.

Considerations:

• Safety: Ionizing radiation requires safety measures due to its potential harm to living tissues.
• Material Compatibility: Compatibility of materials with radiation processes needs consideration.

Applications in Healthcare and Industry:

• Healthcare: Used for sterilizing disposable medical items.
• Industry: Applied for sterilizing various products in the manufacturing sector.

Radiation-based sterilization methods offer a range of applications, from commercial sterilization of disposable items to surface and space sterilization in controlled environments like operation theaters. Each type of radiation has unique characteristics that make it suitable for specific sterilization requirements.

Chemical Methods for Sterilization:

General Properties of Chemical Agents:

1. Broad Spectrum Activity: Effective against various microorganisms, including bacteria, viruses, protozoa, and fungi.
2. Action in the Presence of Organic Matter: Should remain effective in the presence of organic materials.
3. High Penetration Power: Ability to penetrate substances for comprehensive sterilization.
4. Chemical Stability: Stable under both acidic and basic conditions.
5. Non-Corrosive: Should not exhibit corrosive activity on metals.
6. Non-Toxic in Circulation: Non-toxic if absorbed into the circulation.
7. Availability and Cost-Effectiveness: Easily accessible and cost-effective.

Specific Chemical Agents:

1. Alcohols (Ethyl and Isopropyl Alcohol):
   • Action: Facilitate protein denaturation of bacterial proteins.
   • Standard Concentration: 70% ethyl alcohol is commonly used for disinfection.
   • Applications: Skin antiseptics and disinfection of inoculation cabinets.
2. Aldehydes (Formaldehyde, Glutaraldehyde, Orthophathalaldehyde):
   • Formaldehyde:
     • Activity: Bactericidal, sporicidal, and virucidal.
     • Usage: A 10% formalin solution is a standard disinfectant.
     • Applications: Tissue preservation, sterilization of bacterial vaccines, toxoid preparation.
   • Glutaraldehyde:
     • Activity: Effective against bacteria, fungi, viruses (including HIV, hepatitis B).
     • Form: Used as a 2% buffered solution.
     • Applications: Sterilization of endoscopes, bronchoscopes, plastic endotracheal tubes, face masks, etc.
   • Orthophathalaldehyde (OPA):
     • Activity: High-level disinfectant with stability during storage.
     • Concentration: 0.5% OPA.
     • Applications: Sterilization of medical instruments.
3. Phenols (Cresols, Chlorhexidine, Chloroxylenol, Hexachlorophene):
   • Action: Disinfection by damaging the cell membrane.
   • Toxicity: Phenols are generally toxic for the skin.
   • Applications:
     • Lysol (cresol) for sterilization of glassware and floors.
     • Chlorhexidine (Savlon) for skin disinfection.
     • Chloroxylenol (Dettol) is less toxic and irritant.
     • Hexachlorophene is bacteriostatic at high dilution.
4. Halogens (Chlorine and Iodine):
   • Chlorine:
     • Usage: Used in water supplies, swimming pools, food, and dairy industries.
     • Forms: Bleaching powder, sodium hypochlorite, and chloramines.
     • Action: Disinfection due to the release of free chlorine.
   • Iodine:
     • Usage: Skin disinfectant.
     • Forms: Alcoholic and aqueous solutions.
     • Activity: Active against M. tuberculosis, slightly active against spores.
     • Iodophors: Iodine compounds with surface-active agents.
5. Oxidizing Agents (Hydrogen Peroxide, Peracetic Acid):
   • Hydrogen Peroxide:
     • Concentration: Effective at concentrations of 3-6%.
     • Action: Kills spores at higher concentrations (10-25%).
     • Mode of Action: Liberates free hydroxyl radicals.
   • Peracetic Acid:
     • Nature: Oxidizing agent and more potent than hydrogen peroxide.
6. Salts of Heavy Metals (Copper, Silver, Mercury):
   • Toxic Effect: Exhibits toxic effects on bacteria.
   • Usage: Merthiolate (sodium ethyl mercurithiosalicylate) in a dilution of 1:10000 for serum preservation.
7. Dyes (Aniline and Acridine Dyes):
   • Groups: Aniline dyes (e.g., crystal violet, brilliant green) and acridine dyes (e.g., acriflavine, cuflavin).
   • Activity: Bacteriostatic.
   • Applications: Used as skin and wound antiseptics.
8. Vapor Phase Disinfectants (Ethylene Oxide):
   • Nature: Colorless liquid.
   • Action: Effective against microorganisms, including viruses and spores.
   • Mechanism: Alkylating amino, carboxyl, hydroxyl, and sulphydryl groups in protein molecules.

• Applications: Used for sterilizing plastic and rubber articles, respirators, heart-lung machines, dental equipment, etc.

Betapropiolactone (BPO):

Chemical Nature:

• Composition: Condensation product of ketene and formaldehyde.

Properties and Usage:

1. Rapid Action:
   • Action Time: Exhibits rapid action.
   • Efficiency: More efficient in fumigation compared to formaldehyde.
2. Concentration:
   • Usage Concentration: Used in a concentration of 0.2%.
3. Application:
   • Efficiency in Fumigation: Particularly effective in fumigation processes.
   • Vaccine Inactivation: Utilized for the inactivation of vaccines.

These chemical agents cover a wide range of applications, from skin antiseptics and wound disinfection to the sterilization of medical instruments, surfaces, and various industrial products. Each chemical has its unique properties and applications, providing diverse options for different sterilization needs.