Overview
Robert M. Rioux and Zhifeng Chen, Pennsylvania State University, University Park, PA
Decontamination is essential for laboratory biosafety, as the accumulation of microbial contamination in the laboratory can lead to the transmission of disease. The degree of decontamination can be classified as either disinfection or sterilization. Disinfection aims to eliminate all pathogenic microorganisms, with the exception of bacterial spores on lab surfaces or equipment. Sterilization, on the other hand, aims to eliminate all microbial life. Different methods are available which include chemicals, heat, and radiation, and once again depend on the degree of decontamination, as well as the concentration of the contaminating microorganisms, presence of organic matter, and type of equipment or surface to be cleaned. Each method has its advantages and cautionary measures that need to be taken to avoid hazards.
Principles
Be clear about the degree of decontamination that needs to be conducted in the laboratory and then inspect the type, concentration, and location of microorganisms present in the lab. With this information, choose suitable methods depending on the features of each method and determine the most appropriate plan to resolve contamination issues. For example, if a chemical decontamination method is used, a decision must be made regarding the appropriate temperature and contact time applied. Precautions are needed for each method to avoid subjecting individuals to chemical and physical hazards and radiation during decontamination.
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Procedure
1. Chemicals
- Liquid Chemicals
Liquid disinfectants are widely used for lab decontamination. The effectiveness of liquid disinfectants depends on a number of factors, such as the chemical nature of the disinfectant, concentration and quantity of disinfectant, contact time, and temperature. Remember, no liquid disinfectants are applicable in all situations. Make sure to select suitable disinfectants according to the detected microorganisms, using the following criteria:
a. Type of contaminating microorganism: Different microorganisms have different resistance towards disinfectants. For example, bacterial spores are much more chemically resistant than lipophilic viruses.
b. Amount of proteinaceous material present: For example, high protein materials absorb and neutralize some chemical disinfectants, such as formaldehyde and quaternary ammonium compounds.
c. Amount of organic material present: For example, quaternary ammonium compounds are less effective in the presence of soap and detergents.
d. Other important factors include the chemical nature, concentration, quantity, pH, application temperature, and toxicity of disinfectants utilized.
NOTE: Make sure suitable PPE is worn when working with chemical disinfectants.- Low-Level Disinfectants
A. Quaternary Ammonium (QA) Compounds: (such as benzalkonium chloride, ammonium chloride)
• Effective against Gram+ bacteria, Gram- bacteria, and enveloped viruses.
• NOT effective against non-enveloped viruses, fungi, and bacterial spores.
• Contain NH4+ and provide good contact with negatively charged surfaces, making them good cleaning agents.
• Low in toxicity but can be irritating when exposed for long durations.
• Commonly used in noncritical surfaces such as floors, furniture, and walls.
B. Phenolics: (O-phenophenoate-based compounds)
• Effective against bacteria, especially Gram+ bacteria and enveloped viruses.
• NOT effective against non-enveloped viruses and spores.
• Compatible with organic materials.
• Low in toxicity but can be irritating when exposed for long durations.
• Commonly used in hospital environments and laboratory surfaces. - Intermediate-Level Disinfectants
A. Alcohols (such as ethyl alcohol and isopropyl alcohol)
• Effective against Gram+, Gram- bacteria, and enveloped viruses.
• NOT effective against spores and limited effective against non-enveloped viruses.
• Optimum concentration is in the range of 60-90%. Activity drops quickly when diluted below 50%.
• Commonly used in healthcare settings.
• Alcohols are flammable and quickly evaporate.
B. Halogen-Based Biocides: (Chlorine-based compounds and Iodophores)
Chlorine Compounds.
• Hypochlorites are the most widely used chlorine disinfectants.
• Effective against both enveloped and non-enveloped viruses, fungi, bacteria, and algae.
• NOT effective against spores.
• Quickly inactivated by organic matter.
• Degraded quickly due to the high oxidizing power.
C. Iodophores: An iodophor is a combination of iodine and a solubilizing agent or carrier; the resulting complex provides a sustained-release reservoir of iodine and releases small amounts of free iodine in aqueous solution.
• Effective against bacteria, spores, and fungi.
• Needs prolonged contact time.
• NOT effective in the presence of organic matter.
• Commonly used as antiseptics, for blood culture bottles, and medical equipment. - High-Level Disinfectants
A. Oxidizers and Acids: (Hydrogen peroxide, Peracetic Acid)
The effect is not dependent on pH alone. For example, weak organic acids are more potent than inorganic acids despite the low dissociation constant.
Hydrogen peroxide:
• Effective against enveloped and non-enveloped viruses, vegetative bacteria, fungi, and bacterial spores.
• Often used as antiseptics to clean wounds and disinfect environmental surfaces.
• High concentration is harmful for tissues.
Peracetic Acid:
• Effective against all microorganisms with fast action.
• Effective in the presence of organic matter and low temperatures.
• Safe with no harmful decomposition products.
• NOT suitable for metals due to corrosion.
• Commonly used in automated machines to sterilize medical, surgical, and dental instruments.
B. Aldehydes (Formaldehyde, Glutaraldehyde)
Formaldehyde:
• Used as a disinfectant and sterilant both in gases and liquid states.
• Often used in a 37% percent in water solution, known as formalin.
• Effective against bacteria, fungi, viruses, and spores.
• Hazardous with an 8 hour time weighted exposure limit of 0.75 ppm.
• Polymerized solid form-Paraformaldehyde-is also a strong disinfectant.
Glutaraldehyde:
• 10 times more effective than formaldehyde.
• Effective against vegetative bacteria, spores, and viruses.
• Used to sterilize equipment.
• Effective in present of organic material.
• Hazardous with ceiling threshold limit 0.2 ppm and avoid skin contact.
- Low-Level Disinfectants
- Gases or Vapor
Vapors and gases of disinfectants include chlorine dioxide, ethylene oxide, hydrogen peroxide, peracetic acid, etc. These vapors and gases show excellent disinfection properties in closed systems such as biosafety cabinets and animal room facilities. However, well-controlled conditions of temperature, humidity, and inert gas-if flammable-must be maintained for safety. These gases or vapors are used in hospitals and commercial facilities with a need for a closed system with tight control of the temperature, humidity, and concertation.
2. Heat
- Dry Heat
Dry heat is used under conditions of 160-170 °C for periods of 2-4 h in an appropriate oven. This method is often used for glassware or other non-porous heat conductive materials. However, it's ineffective for insulation materials or heat-labile materials. - Wet Heat
Wet heat, also known as autoclaving, is usually under the conditions of at least 120 °C for periods of 30-60 min. Autoclaving is the most convenient and dependable method to achieve effective and rapid sterilization of most forms of microbial life. Wet heat is more efficient than dry heat due to the shorter time and lower temperature required.
3. Radiation
- Ionizing Radiation
Ionizing radiation is not used in general laboratory sterilization due to potential issues associated with radiation safety. - Non-ionizing Radiation (Ultraviolet, UV)
Ultraviolet radiation is typically used for decontamination in air, water, and surfaces due to its strong ability to destroy microorganisms. UV is also widely used in biological safety cabinets. The wavelength of ultraviolet radiation ranges from 250 nm to 270 nm with 265 nm as the optimum. However, UV lamp intensity drops with time, and maintenance needs to be performed after a certain time to maintain the power. Additionally, precautions need to be taken for UV light, as it can cause burns to the eyes or skin.
Decontamination of laboratory space is essential to prevent accumulation and spreading of microbes that can lead to the transmission of diseases.
Decontamination falls into two categories: disinfection and sterilization. Disinfection involves eliminating nearly all pathogenic microorganisms, with the exception of microbial spores on laboratory surfaces and equipment. Sterilization, on the other hand, is a more lethal process, eliminating all microbial life.
Decontamination is carried out using a variety of methods, such as chemicals, heat, or radiation. The choice of method depends on the degree of contamination as well as the type and concentration of the contaminant.
This video will illustrate the types of decontamination and the procedures for disinfection and sterilization of machines, surfaces, and equipment.
Prior to establishing a decontamination procedure, the type, concentration, and location of the microorganism must be determined. Types of microorganisms include Gram-positive or -negative bacteria; viruses; fungi; bacterial spores; and algae. Once the type of microorganism is established, a suitable disinfectant should be chosen.
When selecting a decontamination method the effectiveness of a disinfectant has to be considered, which is dependent on factors such as its chemical composition; the amount, concentration, contact time; and temperature.
Now that we have discussed how to choose a method for decontamination, let's explore the various types used for an actual procedure.
Liquid chemicals are categorized in three levels, as low-, intermediate-, and high-degree disinfectants. Regardless of which you choose, always wear appropriate personal protective equipment when working with hazardous materials.
Most non-critical microorganisms require only low-level disinfectants, which are low in toxicity, but cause irritation upon long exposure times. Common low-level disinfectants are quaternary ammonium compounds, such as benzalkonium chloride and ammonium chloride, and phenolic compounds, such as o-phenylphenol and chloroxylenol.
For the decontamination of more resistant microorganisms, alcohol-based chemicals are used in areas ranging from healthcare to laboratories.
Additionally, halogen-based compounds, such as hypochlorites and iodophors are often applied as antiseptics and disinfectants of medical equipment. However these agents have prolonged contact times and their effectiveness is decreased in the presence of organic matter.
High level disinfectants, which can be classified as oxidizers, acids, and aldehydes are used if decontamination of all microorganisms is required.
Oxidizers such as hydrogen peroxide are fast-acting and often used as antiseptics for wound cleaning and to disinfect environmental surfaces like benchtops. But be careful, as exposure to high concentrations of hydrogen peroxide can be harmful to tissue and airways.
Peracetic acid is generally used to disinfect automated machines and to sterilize medical, surgical, and dental instruments. The advantage of peracetic acid and other oxidizers is a short contact time; however, the use of material to be disinfected can be limited, due to corrosion of metals in acids, for example.
Aldehydes on the other hand, such as formaldehyde or gluteraldehyde, are non-corrosive, but are still hazardous. These chemicals are used to sterilize various types of equipment, but suffer from prolonged contact time.
In addition to liquid chemicals, gaseous chemicals may also be used for decontamination purposes. Gases such as chlorine dioxide and ethylene oxide, as well as vaporized hydrogen peroxide and peracetic acid are frequently used to rid closed equipment, such as biosafety cabinets, of bacteria, viruses, and spores.
In addition to chemicals, heat is a common physical agent for the decontamination of pathogens.
There are two forms of heat. "Dry" heat is used under conditions of 160 to 170 degrees Celsius for 2 to 4 hours to disinfect glassware, but it is not suitable for heat-labile materials. On the other hand, "Wet" heat, also known as autoclaving, is used by heating samples and equipment to only 120 degrees Celsius for 30 to 60 minutes under high pressure.
Besides heat, ultraviolet radiation in the wavelength range of 250 to 270 nanometers is often used for decontamination. This method is effective against bacteria and viruses, but not against spores, and is used to decontaminate air, water, and surfaces such as in biological safety cabinets. Furthermore UV light in this range can cause burns of skin and eyes, thus proper PPE should be worn.
You've just watched JoVE's introduction to Decontamination for Laboratory Safety. You should now understand the various types of microbial contaminants, how to choose a suitable method, and the types of disinfection and sterilization available. Thanks for watching!
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Applications and Summary
To avoid infection transmission and maintain biosafety in the lab, periodic decontamination in the lab is important. Three methods are available including chemical, heat, and radiation. Each method has its own strength and suitable applications. Awareness of the type of microorganism in the laboratory environment is useful for selection of a suitable decontamination method. Appropriate safety protocols should be in place during the decontamination procedure.
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References
- Center for Disease Control. A Guide to Selection and Use of Disinfectants. (2003)
- Biosafety: Decontamination Methods for Laboratory Use, 2016, Blink, University of California, San Diego. at http://blink.ucsd.edu/safety/research-lab/biosafety/decontamination/#Vapors-and-gases
- Disinfectants and Sterilization Methods, 2008, Environmental Health & Safety, University of Colorado Boulder. at https://ehs.colorado.edu/resources/disinfectants-and-sterilization-methods/
