Sterilization and Disinfection Methods

Sterilization is a process of killing or removal of all microorganisms, including highly resistant bacterial endospores. Depending on the material, sterilization can be achieved by moist heat (autoclave), dry heat (hot-air oven), ethylene oxide gas, or filtration. 

However, sterilization is not attainable or practical in certain circumstances, such as fresh produce processing. In those cases, we limit the rapid microbial growth by decontamination and disinfection. 

Methods of sterilization and disinfection
Methods of sterilization and disinfection

Disinfection is the killing of pathogenic organisms, but not all, microorganisms. Pathogenic bacteria are destroyed during disinfection, but some microorganisms, including bacterial endospores, may remain viable. The use of disinfectant varies depending on the purpose of use, i.e., inanimate objects or skin surfaces. Some common disinfectants are phenol-containing compounds, quaternary ammonium compounds, alcohol (ethanol), chlorine, iodine, etc.

Decontamination is the removal of pathogenic microorganisms from inanimate objects or surfaces, so items are safe to handle or dispose of. For example, simply wiping a table after a meal removes contaminating microorganisms and their potential nutrients. 

Factors affecting Sterilization and Disinfection

Sterilization and disinfection procedures aim to prevent the transmission of microbes to patients. The numbers of factors that affect the success of sterilization and disinfection methods are;

  1. Organic load (organisms and other contaminating materials such as blood or body fluids). The time necessary for killing microorganisms increases directly to the microbial load (the number of viable microorganisms in a sample). Because of this, organic materials are mechanically removed before chemical sterilization to decrease the microbial load. 
  2. The type of organisms present; For example, spores of Bacillus spp are the most resistant, and enveloped viruses (e.g., herpes simplex virus) are the most susceptible to the action of disinfectants. 
  3. The concentration and exposure time to the germicide: For example, chemicals that have demonstrated bactericidal, viricidal, sporicidal, and fungicidal activities, when used for shorter periods, act as disinfectants.
  4. The physical and chemical nature of the surface (hinges, cracks, rough or smooth surfaces): For example, UV rays do not penetrate well. Hence, it kills organisms that are exposed directly on the surface of biological safety cabinets (BSCs) only.
  5. Temperature: Steam under pressure, when applied for a specific duration, can sterilize materials but only boiling it in water acts as a disinfectant. 
  6. Other factors affecting sterilization are pH, humidity, and the presence of a biofilm. 

Sterilization Methods 

Physical Agents of Sterilization 

Applications of heat, radiation, and filtration are commonly used physical sterilization methods. Among them, heat is the most widely used method. Temperature, duration, and nature (moist or dry heat) affect a microorganism’s susceptibility to heat. 

Incineration 

Incineration is a method of treating infectious wastes by burning them to ashes at temperatures of 870°C to 980°C. Incineration is the safest method to destroy pathogenic microorganisms, bacterial endospores, and even prions and infective proteins. 

Toxic air emissions and heavy metals in ash have limited the use of incineration.

Moist Heat Sterilization 

Using autoclaves, moist heat (saturated steam under pressure) sterilizes bio-hazardous trash and heat-stable objects. It is the fastest and simplest physical method of sterilization. Moist heat has better penetrating power than dry heat and, at a given temperature, produces a faster reduction in microbial loads. The two common sterilization temperatures are 121°C and 132°C. 

Medical autoclave
Medical Autoclave Sterilizer

Read more about the moist heat sterilization method.

An autoclave is a large pressurized chamber that uses moist heat for sterilization. An autoclave sterilizes infectious medical waste containing body fluids or blood, biological waste including culture media, and broth. The gravity displacement autoclave is the most commonly used steam sterilizer in the microbiology laboratory. 

Read more about the working mechanism of autoclaves and the types of autoclaves.  

Dry Heat Sterilization 

Dry heat requires longer exposure times (1.5 to 3 hours) and higher temperatures than moist heat (160°C to 180°C). Dry heat ovens sterilize items such as glassware (Petri dishes, flasks, pipettes, and test tubes), oil, petrolatum, or powders. 

Read more about dry heat sterilization method.

hot air oven
Hot air oven Source: Acumenlabware

Hot air oven is the most widely used method of dry heat sterilization. This equipment is electrically heated and fitted with a fan to ensure adequate and even air distribution in the chamber.

Read more about hot air oven: parts, types, and uses

Radiation

Ionizing radiation produces ions such as electrons, hydroxy radicals, hydride radicals, and other reactive molecular species from molecules with which the radiation particles collide. Each of these highly reactive molecules is capable of altering and disrupting macromolecules such as DNA, lipids, and protein. The ionization and subsequent degradation of these biomolecules lead to the death of irradiated cells.

UV light Radiation Sterilization
Use of UV light for sterilization

Radiation is used for sterilization and decontamination in the medical supplies and food industries. FDA has approved the use of radiation for sterilization of surgical supplies, disposable labware, drugs, and even tissue grafts. WHO has approved radiation for decontaminating foods susceptible to microbial contamination, such as fresh produce, meat products, chicken, and spices. 

Read more about radiation sterilization: types, mechanism, and uses 

Filtration

Filtration of an aqueous liquid through the tiny pores of a membrane filter traps any microorganisms present in the liquid and renders it sterile. Filtration of liquids is accomplished by using cellulose acetate or cellulose nitrate membrane with a vacuum, whereas high-efficiency particulate air (HEPA) filters are used for air filtration. 

Concept of filtration
Filtration sterilization by removing organisms

Filtration is the method of choice for sterilizing gases and heat-sensitive materials such as antibiotic solutions, toxic chemicals, radioisotopes, vaccines, and carbohydrates.

Read more about filtration sterilization

Chemical Agents of Sterilization

Chemicals used to destroy all life are called chemical sterilants or biocides. Ethylene oxide (Eto) and hydrogen peroxide are common chemical sterilants. Commonly used chemicals in a laboratory include alcohols, phenols, detergents, chlorine, iodine, heavy metals, formaldehyde, glutaraldehyde, acids, and alkalis. Sterilants, when used in low concentrations, serve as disinfectants. 

Ethylene oxide (EtO) is the most common chemical sterilant for sterilizing heat-sensitive objects. Vapor-phase hydrogen peroxide sterilizes HEPA filters in biological safety cabinets (BSCs), metals, and nonmetal instruments such as medical instruments.

Disinfection Methods 

Physical Methods of Disinfection

Boiling 

Boiling instruments at 100°C for 15 minutes may kill the most vegetative bacteria but not the bacterial endospores. Heating in boiling water at 100°C for 20 minutes at altitudes below 600 meters is sufficient to kill all non-sporing bacteria, some bacterial spores, fungi, protozoa, and viruses, including hepatitis viruses and HIV.

Though boiling is a simple, easily available option to most people, it is not a suitable method for sterilizing surgical instruments.

Pasteurization 

Pasteurization uses controlled heat (71°C for 15 seconds or 63-66°C for 30 minutes) to kill pathogens without damaging the nutritional value or flavor of milk or other food products. Pasteurization does not kill all organisms but reduces the microbial load. 

Pathogenic bacteria are killed, especially those causing tuberculosis, brucellosis, Q fever, and typhoid fever. Pasteurization also controls commonly encountered pathogens such as Listeria monocytogenes, Campylobacter species, Escherichia coli O157:H7, and Salmonella

Non-ionizing Radiation 

Nonionizing radiation, such as ultraviolet (UV) rays, are long wavelength and low energy. UV rays of 220 to 300 nm can cause modifications or actual breaks in the DNA of organisms, causing death.

UV rays do not penetrate solid, opaque, or light-absorbing surfaces, so UV light is useful for disinfecting surfaces, air, and materials such as water that do not absorb UV waves. For example, laboratory laminar flow hoods are equipped with a germicidal UV light to decontaminate the work surface after use. 

Chemical Methods of Disinfection

Chemicals act as sterilants (biocides) and disinfectants. Chemicals in low concentration are generally used as disinfectants on inanimate surfaces.  For example, stabilized hydrogen peroxide is a sterilant, and commercially available 3% hydrogen peroxide is a disinfectant. The working solution of biocides, antiseptics, or disinfectants has to prepare according to the manufacturer’s package insert to match water to the active ingredient, which is critical for efficacy. 

Chemical disinfectants
Chemicals disinfectants

Chemical disinfectants include alcohols, phenolics, aldehydes, halogens (chlorine and chlorine compounds), hydrogen peroxide, peracetic acid, and quaternary ammonium compounds. Bleach (sodium hypochlorite) solution is a disinfectant used to clean and disinfect food preparation areas.

Find more about disinfection methods and their uses.

Antiseptics

Antiseptics are disinfectants used on living tissue, such as the skin’s surface or mucous membrane.  Antiseptics are required for personal laboratory safety and the preparation of patients for invasive procedures such as blood drawing, operations, etc. 

Iodine compounds (tincture-iodine or povidone-iodine) are widely used antiseptics. Iodine is prepared either as a tincture with alcohol (tincture-iodine) or as an iodophor coupled to a neutral polymer (povidone-iodine). 70% ethyl alcohol, followed by an iodophor, is the most common compound used for skin disinfection before drawing blood specimens for culture or surgery. 

Clinical Use

Sterilization and disinfection are the basic components of hospital infection control activities. According to CDC, in the United States alone, more than 99,000 death occurs each year due to hospital-acquired infections.  Proper sterilization and disinfection techniques are pivotal in managing hospital-acquired infection. 

Sterilization of medical equipment that comes in contact with sterile body parts, disinfection of hospital floors using proper chemicals, surgical site preparation, handwashing, etc., can reduce the incidence of hospital-acquired infections. For example, medical devices intended to enter normally sterile sites, such as surgical instruments, cardiac catheters, implants, needles, etc., should be sterilized by steam, plasma, or ethylene oxide. Devices like blood pressure cuffs, and stethoscopes, which come in contact with intact skin only, are decontaminated using an intermediate or low-level disinfectant.

Commonly used disinfection or sterilization methods and their clinical use is mentioned in the table below. 

Clinical UseCommonly used Disinfection or Sterilization Method
Surgical hand disinfection (Disinfect surgeon’s hands before surgery)Chlorohexidine
Surgical site skin preparation Iodophor
Skin preparation for venipuncture or immunization 70% ethanol
Disinfect skin before blood culture or inserting a vascular catheterTincture of iodine followed by 70% ethanol, or iodophor, or chlorhexidine
Cleanse woundsThimerosal, chlorhexidine, hydrogen peroxide
Cleanse burn woundsSilver sulfadiazine
Cleanup of blood spill from a patient with hepatitis B or C (disinfect area)Hypochlorite (bleach)
Sterilize surgical instruments and heat-sensitive materials (e.g., endoscopes, respiratory therapy equipment)Ethylene oxide or glutaraldehyde
Sterilize heat-resistant materials (e.g., surgical gowns, drapes)Autoclave
Sterilize intravenous solutionsFiltration 
Disinfect air in operation theatre (when not in use)Ultraviolet light 
Disinfect the floor of an operating roomBenzalkonium chloride (Lysol)
Disinfect stethoscope70% ethanol
Preservation of vaccines Thimerosal

References and further readings 

  1. Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
  2. Madigan, M., Martinko, J., Stahl, D., & Clark, D. (2012). Brock Biology of Microorganisms (13th ed). Pearson Education
  3. Review of Medical Microbiology and Immunology, Lange Medical Books, 13th edition
  4. Mohapatra S. Sterilization and Disinfection. Essentials of Neuroanesthesia. 2017:929–44. doi: 10.1016/B978-0-12-805299-0.00059-2. Epub 2017 Mar 31. PMCID: PMC7158362.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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