Decontamination of cultures and objects contaminated by biological agents should be routinely performed in microbiological laboratories. Decontamination is a vital component of microbiological safety practice and serves to protect laboratory personnel (as well as others) from infection and the release of infectious organisms to the outside environment (through person-to-person transmission, or contact with fomites). Decontamination of media, work surfaces, and equipment is also necessary to prevent contamination of cultured organisms.

Disinfection refers to the elimination of virtually all pathogenic organisms on inanimate objects and surfaces thereby reducing the level of microbial contamination to an acceptably safe level, with the use of chemical disinfectants.  In contrast, sterilization refers to the destruction of all microbial life. At UVM, sterilization is accomplished with heat and steam by autoclaving. Cleaning refers to the general removal of organic matter from a surface, and should always precede disinfection.

Choosing a Disinfectant

Choosing an appropriate disinfectant will depend on a number of factors. First, you will have to determine what disinfectant will inactivate the organism you are using. Your application will also be important. Do you need a full decontamination in a spill condition, or are you looking for a product for surface disinfection for general cleanup? Finally, practical factors such as cost, shelf life, contact time, and Personal Protective Equipment (PPE) requirements can affect your choice. Please consult the flow chart below for information on selecting an appropriate disinfectant.

 

#1 - What disinfectants are effective on my organism?

Classify your organism

  • Is it a bacteria, virus, fungus, protozoa or prion?
  • Does it form spores?
  • If it's a virus, is it enveloped or non-enveloped?
  • Pure cultures are easy to identify, but for animal, human or environmental samples, consider what organisms are likely to be present.

Determine organism susceptibility or resistance to disinfection:

  • Bacteria and enveloped viruses are generally more susceptible
  • Spore former are more resistant
  • Other organisms can very
  • Consult the general effectiveness chart below
  • Bleach is effective against most organisms at the appropriate concentration and contact time

 

#2 - What is my application?

Liquid waste decontamination:

  • Add concentrated household bleach (5% or greater concentration of sodium hypochlorite) until a 10% final concentration is reached
  • Clorox regular or Clorox germicidal bleach is recommended
  • Do not use laundry bleach
  • Pleas contact Biosafety for other options if bleach cannot be used

Spill cleanup:

  • Make a fresh dilution of 10% final concentration of household bleach (5% or greater concentration of sodium hypochlorite)
  • Bleach must be fresh as dilute bleach loses effectiveness after 1-2 weeks
  • Clorox regular of Clorox germicidal bleach is recommended

Surface decontamination or tool/equipment decontamination:

  • Consider contact time, A shorter contact time would be recommended
  • Consider the type of material you're decontamination.
  • some products like bleach are corrosive and may require a rinse or flush step when used on certain surfaces (such as the stainless steel surfaces of biosafety cabinet)

Work with human materials:

  • OSHA requires a disinfectant that is effective against HIV and Hepatitis B.
  • 70% ethanol is not appropriate for surface decontamination
  • Most products recommended on this website are appropriate
  • EPA registration numbers will confirm whether the product is appropriate. See Human Material below.

 

#3 - What else should I consider?

Contact Time:

  • Contact time will vary depending on the product and the organism targeted
  • Some products require 10 minutes contact (20 minutes for spill/splash or equipment decontamination)
  • Newer Quaternary Ammonium products and some Activate Hydrogen Peroxide products are effective in 5 minutes or less
  • Alcohols can evaporate too quickly to achieve sufficient contact time

Product safety/toxicity:

  • Some aldehydes, phenolics, and activated hydrogen peroxide/acid products (Peridox) require special Personal Protection Equipment
  • always consult the product label and SDS prior to using a disinfectant
  • If possible, select a less hazardous product
  • Most of these products are used to inactivate bacterial spores

Product Costs:

  • Concentrated products generally cost less than ready to use products but require more preparation time
  • Ready to use products are more expensive but do not require valuable research time in measuring and diluting
  • Buying in bulk can reduce costs (but is not always the best option)

Residue or odor:

  • Bleach and some quaternary ammonium products can leave residue
  • Iodophours can stain
  • Phenolics have a strong, unpleasant odor
  • Activated hydrogen peroxide and alcohols leave no reside

Product expiration/shelf life

  • Shelf life can vary greatly by products
  • Concentrated products that require dilution generally lose effectiveness quicker once dilutes as compared to ready to use products
  • Ready to use products include stabilizers and generally remain effective longer
  • Always check product labels and be aware of expiration date of your disinfectant

Concentrated or Ready to use products

  • Concentrated products can be more cost effective up front but require more preparation time
  • Disinfectants diluted from a concentrate must be replaced more frequently and require more monitoring to avid using expired dilutions
  • Ready to use products can be more expensive up front, but require not preparation time and tend to last longer
  • Consider your individual needs

Choosing a Disinfectant Q+A Chart (PDF)
Visual chart from text above.

Liquid Chemical Disinfectants

There are a wide variety of chemical disinfectants available for laboratory use, both conventional and proprietary. Concentration is an important factor when using these chemicals. A concentration that is too low may not completely kill the biological agent. A concentration that is too high may put you or others at an increased (and unnecessary) risk for adverse health effects.

To avoid inhalation during use, pour the disinfectant or use disinfectant wipes instead of sprays. If purchasing a proprietary disinfectant (such as Lysol, Cavicide, Virkon, etc.), choose one that has an EPA registration number on the label. This indicates that the EPA has evaluated and accepted the efficacy data for that disinfectant.

Never mix liquid disinfectants with other chemicals (i.e. bleach and ammonia), as this could cause a potentially dangerous chemical reaction. Before disposing of a liquid disinfectant, make sure you are using the appropriate method. For instance, diluted bleach is OK for drain disposal, but some other chemicals may need to be tagged as hazardous waste. Please consult the Chemical Waste Management for more information.

Important: Removal of organic matter must always precede the use of any disinfectant. Otherwise, the efficacy of the disinfectant may be greatly reduced.

Disinfectant Type and Practical Characteristics

Disinfectant effectiveness will vary depending on the active ingredient in the disinfectant and the nature of the organism. There are seven broad categories of disinfectants. Each has benefits and limitations:

Disinfectant Type and Practical Characteristics
Type

Properties

Limitations
Activated Hydrogen Peroxide
  • Broad effectiveness
  • Non-corrosive
  • Lower contact time
  • No scent or residue
  • Non-toxic and non-staining
  • Longer shelf life
  • Generally not active against bacterial spores
  • Combined products including acid to inactivate bacterial spores require special PPE
Alcohols
  • Inexpensive
  • Non residue
  • No odor
  • Varied activity
  • Sufficient contact time can be challenging
  • Flammable
  • NOT appropriate for use with human material
Aldehydes
  • Non-corrosive
  • Broad effectiveness
  • Bacterial sporicidal at extended contact time
  • Limited shelf life once diluted
  • May require specially PPE
Chlorine (bleach)
  • Broad effectiveness
  • Effective against bacterial spores at higher contact time
  • Cheap
  • Limited residue
  • Readily available
  • Corrosive
  • May be inactivated by organic matter
  • Longer contact time
  • Limited shelf life once diluted unless ready to use product with stabilizers
Iodophors
  • Broad effectiveness
  • Long shelf life
  • Inactivated by organic matter
  • Poor residual activity
  • Concentration must be 1-3% for efficacy
  • May stain surfaces
Quaternary ammonium compounds (quats)
  • Non-corrosive
  • Slight residue
  • Some scent
  • Lower contact time
  • Longer shelf life
  • Varied activity
  • Not active against bacterial spores
  • Can be inactivated by organic matter
Phenolics
  • Non-corrosive
  • Not easily inactivated by organic matter
  • May require PPE due to toxicity
  • Unpleasant smell
  • Residue

 

Disinfectant Property Table - Chart (PDF)
Visual chart from text above.

Source: MIT Environmental Health & Safety

Disinfectants Commonly Used at UVM

Commonly used disinfectants (PDF) shows many disinfectants that are commonly used at UVM and the contact time necessary.  This list is not exhaustive, and other disinfectants are available.  Please contact your biosafety officer if you have questions.

Source: MIT Environmental Health & Safety

Disinfectants appropriate for use with human material

The OSHA Bloodborne Pathogen Standard is designed to protect workers from exposure to Bloodborne Pathogens, including but not limited to HIV, Hepatitis B, and Hepatitis C.  It requires that work surfaces that are contaminated with blood or other potentially infectious material (anything from the human body, including human cell lines) or that have the potential to have become contaminated during the course of work be decontaminated with an appropriate disinfectant.

Any lab that works with human material must use an appropriate disinfectant to disinfect work surfaces or to decontaminate surfaces and equipment after a spill.  Currently, OSHA recognizes the following as appropriate disinfectants:

Please note that 70% ethanol is NOT recognized as an appropriate surface disinfectant by OSHA. Evaporation of the alcohol prevents sufficient contact time to inactivate Hepatitis B.

To check if your product meets the OSHA requirements:

  • Locate the EPA registration number found on the label of your product.
  • Go to the appropriate EPA list using the links above.
  • Search the list using the first two sets of numbers in the EPA registration number.  For example, if your product label lists the EPA registration number as 777-99-675, search the list for 777-99 (omit 675 from your search).
  • The last number is the distributor identification number. These will not be included on the EPA lists.  Only the initial formulation submitted to the EPA is listed.

For disinfectants commonly used at UVM that meet these requirements, please see Commonly Used Disinfectants at UVM section, above.

For more information about using human material at UVM, please see the Bloodborne Pathogens page.

Bleach Dilutions

When using household bleach as a disinfectant, it is important to check the concentration of sodium hypochlorite (NaOCl) as this may vary between manufacturers. Household bleach usually contains between 4 - 6 % sodium hyphochlorite. Sodium hypochlorite breaks down relatively quickly when mixed with water, so it is also important to make bleach solutions fresh daily to ensure effectiveness.

NaOCl % (w/v) Dilution of Liquid Bleach (~4-6% NaOCl) in water Bleach Solution
0.1% 1 to 50 2%
0.25% 1 to 20 5%
0.5% 1 to 10 10%
1.0% 1 to 5 20%
2.5% 1 to 2 50%

 

Gases and Vapors

Sterilizing gas (e.g., ethylene oxide) or vapors from a chemical (e.g., formaldehyde) may also be used for decontamination in some instances. Because of the hazardous nature of the gases and vapors used, this requires specially designed equipment and facilities.

Physical Inactivation

Autoclaving or exposure to dry heat are common physical methods of inactivating biological agents. Generally, autoclaving (moist heat) is more effective than dry heat. Dry heat requires a much longer contact time to be effective, but may be more appropriate for anhydrous materials that the steam from an autoclave cannot penetrate. Pressure or radiation may also be used, but check to make sure that these methods are effective and safe to use.

UVM Health and Safety has adopted the position of the NSF, NIH, CDC and the American Biological Safety Association (ABSA) in regard to the use of UV lamps within biosafety cabinets; UV lamps are neither recommended nor required in biological safety cabinets.  UVM Health and Safety believes they are not appropriate for disinfection within a BSC and are potentially dangerous when used improperly. Many factors can negatively alter the effectiveness of these lights, and they should not be relied on for decontamination. Even if all conditions are optimal, UV light has very poor penetrating ability.

UV light is not an acceptable method of disinfection, unless approved by both the Biosafety and Radiation Safety offices.

Contact Time

No matter which disinfection agent or method is used, sufficient contact time is extremely important in order to completely disinfect. Insufficient contact time may not kill all of the biological agent in question, and instead gives the user a false sense of security. Always check the manufacturer's recommendations or ask safety staff if you are unsure.

Disinfectant Effectiveness & Microbial Resistance

Disinfectant Effectiveness

Chemical disinfectants will vary in their effectiveness. Choosing an appropriate disinfectant will depend on a number of factors such as: the organism you are handling, the item to be disinfected, and the nature of the disinfectant (cost, ease of use, contact time, residue, odor, etc).

Individual products may vary. For example, many of the quaternary ammonium products we recommend have been developed to be effective against Mycobacterium tuberculosis. Please check the product label for specific efficacy.

General Effectiveness by Disinfectant Type (PDF)
Visual supporting text below. Source: MIT Environmental Health & Safety

General Effectiveness by Disinfectant Type
  Quatrnary Ammonium Alcohols Phenolics Chlorine Bleach Peroxides Aldehydes
Bacterial spores Resistant Resistant Resistant Moderate Moderate Moderate
Protozoal o ocysts (cryptosporidium) Resistant Resistant Effective Effective Effective Effective
Helmith eggs (Ascaris) Risistant Resistant Effective Effective Effective Effective
Mycobacteriun tuberculosis Product Specific Moderate Effective Effective Effective Effective
Small nonenveloped viruses (parvovirus, Hepatitis B) Product Specific Moderate Effective Effective Effective  
Protozoal cycsts (Giardia) Effective Moderate Effective Effective Effective Effective
Fungal spores Effective Moderate Effective Effective Effective Effective
Gram negative bacteria Effective Effective Effective Effective Effective Effective
Vegetative fungi & algae Effective Effective Effective Effective Effective Effective
Large nonenveloped viruses (Adenovirus) Effective Effective Effective Effective Effective Effective
Gram positive bacteria Effective Effective Effective Effective Effective Effective
Lipid-containing (envelope) viruses (HIV) Effective Effective Effective Effective Effective Effective

 

 

Microbial Resistance

Some classes of biological agents tend to be generally more resistant to decontamination methods than others. Make sure to use a decontamination method that is appropriate and effective for the biological agents used in your lab.

( More Resistant )

more resistant↓ Prions (Creutzfeld-Jakob Disease)

less resistant↓ Bacterial spores (Bacillus)

less resistant↓ Coccidia (Cryptosporidium)

less resistant↓ Helminth eggs (Ascaris)

less resistant↓ Mycobacteria (M. tuberculosis)

less resistant↓ Non-enveloped viruses (Polio, Coxsackie)

less resistant↓ Protozoal cysts (Giardia)

less resistant↓ Fungi (Aspergillus, Candida)

less resistant↓ Vegetative bacteria (Staph, Pseudomonas)

less resistant↓ Enveloped viruses (HIV, Hepatitis B)

( More Susceptible )