Chapter 7: Decontamination
Decontamination of cultures and objects contaminated by biological agents is 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, as well as the release of infectious organisms to the outside environment. Decontamination of media, work surfaces, and equipment is also necessary to prevent contamination of cultured organisms.
Chemical Disinfection
Decontamination of work surfaces, equipment, biological safety cabinets, and other inanimate objects using antimicrobial agents is referred to as disinfection. Several chemical agents are used as disinfectants. Laboratory workers should remember that there are hazards associated with all of these chemical disinfectants. Inhalation and skin contact should be minimized, and eye contact avoided. Appropriate gloves and safety eyewear should always be worn when handling these chemicals.
The susceptibility of microbes to chemical disinfects varies on their physical structure and physical state (e.g., more resistant spore state). For example, the waxy outer cell wall of mycobacteria provides increased resistance. Viruses that lack lipids in the outer capsid make them more hydrophilic and generally more resistant to chemical disinfectants than lipophilic viruses. The order of resistance of microbes to chemical disinfectants, from most resistant to least resistant is presented on the next page.
The following is a list of resistances of microbes to chemical disinfectants, ordered from most resistant to least resistant:
- Bacterial Spores
Bacillus anthracis
Clostridium sporogenes - Mycobacteria
Mycobacterium tuberculosis
Nontuberculosis mycobacteria - Nonlipid or Small Viruses
Coxsackievirus
Rhinovirus - Fungi
Cryptococcus spp.
Candida spp. - Vegetative Bacteria
Pseudomonas aeruginosa
Staphylococcus aureus - Lipid or medium-Size Viruses
Cytomegalovirus
Hepatitis B and C virus
* Adapted from Biological Safety: Principles and Practices, Fleming, D. O. and Hunt, D. L., eds, 4th Edition (2006), ASM Press, Washington, D. C.
Pertinent information for some of the common chemical disinfectants is summarized in table format at the end of this chapter.
Autoclaving
Autoclaving uses saturated steam under pressure (approximately 15 psi) to achieve a temperature in the autoclave of at least 121 °C (250 °F). Autoclaving can be used to destroy vegetative bacteria, bacterial spores, and viruses. When decontaminating biohazardous waste, it is recommended that the temperature in the waste reach a minimum of 115 °C for a minimum of 20 minutes. The total processing time required to meet these conditions depends on several loading factors (see below); however, a minimum autoclave cycle of one hour must be used when decontaminating biohazardous waste.
There are three factors that in combination determine the effectiveness of autoclaving:
Temperature - autoclave uses steam under a pressure of approximately 15 psi to achieve a chamber temperature of at least 121 °C. Although the autoclave chamber may reach 121 °C, this does not necessarily mean that the interior of the load will reach this temperature.
Time - a minimum autoclave cycle time of twenty minutes at a chamber temperature of 121 °C (time does not begin as soon as the autoclave cycle is initiated) is commonly recommended for sterilization of clean items. However, the total processing time required to achieve decontamination depends on several loading factors, including the load container (heat transfer properties), the amount of water contained in the load, and the weight of the load. For increased loads, a longer cycle time is required to ensure effective decontamination. For treatment of biohazardous waste, a minimum autoclave time of 60 minutes is required. Exemptions to the 60 minute minimum autoclave time will be considered on a case-by-case basis and must be approved by the Biosafety Officer or Institutional Biosafety Committee. Requests for exemption must be submitted to the Biosafety Officer with a written SOP that describes the biohazardous waste, autoclave conditions, autoclave time, and autoclave efficacy testing procedure and frequency of testing. In no case will an autoclave time of less than 30 minutes be approved.
Contact - steam saturation is essential for maximum heat transfer. Steam must contact all areas of the load. Autoclave bags and other containers should be left partially open (or otherwise permit entry of steam) to ensure adequate contact. Studies have shown that adding water to the interior of the bag improves the time-temperature profile of the autoclave cycle, increasing the sterilization efficiency of the autoclave.
There are specific requirements for decontaminating biohazardous waste prior to disposal. See Chapter 13 for autoclave procedures relating to biohazardous waste.
Autoclave Maintenance and Inspection
Autoclaves must be regularly maintained and repaired by qualified technicians. Additionally, Nevada State law requires that autoclaves with an internal volume greater than 5 cubic feet be inspected annually by a certified inspector. This service is currently performed at no cost by the University insurance carrier. Call EH&S at 775-327-5040 to schedule an autoclave for inspection. Additional autoclave use information is available in the University of Nevada, Reno Autoclave Safety Manual.
Dry Heat
Dry heat is less effective than moist heat (autoclaving); requiring higher temperature and longer contact time. Nevertheless, dry heat is preferable to moist heat for decontamination of anhydrous materials and closed containers. This is due to the fact that the moisture component of the steam used in an autoclave will not effectively penetrate anhydrous materials and closed containers. The highest dry heat equivalent temperature that these materials will reach in an autoclave is 121 °C. The highest temperature that material will reach in a dry-heat oven will be the actual temperature inside the oven. A temperature of 160-180 °C for 3-4 hours is recommended for decontamination of waste using a dry heat oven.
Summary of Chemical Disinfectants
The table is from: Laboratory Safety: Principles and Practices, second edition (1995), Diane O. Fleming, John H. Richardson, Jerry J. Tulis, and Donald Vesley, eds., American Society for Microbiology, Washington, D. C.
| Disinfectant | Use Parameters | Effective Against Vegetative cells | Effective Against Lipophilic viruses | Effective Against Tubercle bacilli | Effective Against Hydrophillic viruses | Effective Against Bacterial spores | Important Characteristics | Potential Application |
|---|---|---|---|---|---|---|---|---|
| Alcohol (ethyl, isopropyl) |
conc.: 70-85% contact time: 10-30 min. |
very positive response | very positive response | very positive response | less positive response | N/A | eye irritant, toxic, flammable, inactivated by organic matter | surfaces - work & equipment |
| Chlorine Compounds |
conc.: 0.05-0.5% (commercial bleach 5%) contact time: 10-30 min. |
very positive response | very positive response | very positive response | very positive response | less positive response | may leave residue; corrosive; skin, eye & respiratory irritant; inactivated by organic matter; makeup at least weekly | spills, equipment surfaces, instruments, glassware, water baths |
| Quaternary Ammonium Compounds |
conc.: 0.1-2% contact time: 10-30 min. |
very positive response | very positive response | N/A | N/A | N/A | toxic, inactivated by organic matter | surfaces (work & equip.), BSCs, floor maintenance, glassware, instruments |
| Phenolic Compounds |
conc.: 0.2-3% contact time: 10-30 min. |
very positive response | very positive response | very positive response | less positive response | N/A | leaves residue; corrosive, skin, eye & respiratory irritant; toxic; inactivated by organic matter | surfaces (work & equip.), BSCs, floors, spills, glassware, instruments, water baths |
| Iodophor Compounds |
conc.: 0.47% contact time: 10-30 min. |
very positive response | very positive response | very positive response | less positive response | N/A | leaves residue; corrosive, skin & eye irritant; toxic; inactivated by organic matter | surfaces (work & equip.), BSCs, glassware, water baths |
| Formaldehyde (Formalin) |
conc.: 4-8% contact time: 10-30 min. |
very positive response | very positive response | very positive response | very positive response | less positive response | leaves residue; skin, eye & respiratory irritant; toxic (carcinogen) | less effective than other disinfectants but can be used for equipment surfaces, glassware, instruments |
| Glutaraldehyde |
conc.: 2% contact time: 10-600 min. |
very positive response | very positive response | very positive response | very positive response | very positive response | leaves residue; skin, eye & respiratory irritant; toxic | equipment surfaces, glassware, instruments |
Due to its irritating characteristics and status as a carcinogen, formaldehyde should not be used without good local exhaust ventilation.
The following information pertains specifically to the inactivation of biological toxins
Inactivation of a toxin means that it is rendered non-functional and is no longer capable of exerting its toxic effects. This differs from the inactivation of a biological agent, which renders the biological material non-viable and incapable of growing, replicating, infecting, or causing diseases. Inactivation methods used for biological toxins must be specific to the toxin. Inactivation can take place using physical means, including autoclaving or by chemical means. The BMBL provides the following tables to help provide guidance on the inactivation of biological toxins.
Autoclave Inactivation of Selected Toxins
Adapted from the BMBL. The following table is from: Biosafety in Microbiological and Biomedical Laboratories, fifth edition (2009), US Department of Health and Human Services, Center for Disease Control and Prevention, National Institutes of Health.
| Toxin | Steam Autoclave | Notes |
|---|---|---|
| Botulinum neurotoxin | Yes | Steam autoclaving should be at >121°C for 1 h. For volumes larger than 1 liter, especially those containing Clostridium botulinum spores, autoclave at >121°C for 2 h to ensure that sufficient heat has penetrated to kill all spores.8,9 |
| Staphylococcal Enterotoxin | Yes | Protracted steam autoclaving, similar to that described for BoNT, followed by incineration is recommended for disposal of SE-contaminated materials. i Dry heat of >100ºC for 60 min in an ashing oven or steam autoclave treatment at >121ºC for 1 h |
| Ricin | Yes | Dry heat of >100ºC for 60 min in an ashing oven or steam autoclave treatment at >121ºC for 1 h |
| Microcystin | No | Autoclaving with 17 lb pressure (121-132º C) for 30 min failed to inactivate LMW toxins.17,19 All burnable waste from LMW toxins should be incinerated at temperatures in excess of 815°C (1,500º F) |
| Saxitoxin | No | Autoclaving with 17 lb pressure (121-132º C) for 30 min failed to inactivate LMW toxins.17,19 All burnable waste from LMW toxins should be incinerated at temperatures in excess of 815°C (1,500º F) |
| Palytoxin | No | Autoclaving with 17 lb pressure (121-132º C) for 30 min failed to inactivate LMW toxins.17,19 All burnable waste from LMW toxins should be incinerated at temperatures in excess of 815°C (1,500º F) |
| Tetrodotoxin | No | Autoclaving with 17 lb pressure (121-132º C) for 30 min failed to inactivate LMW toxins.17,19 All burnable waste from LMW toxins should be incinerated at temperatures in excess of 815°C (1,500º F) |
| T-2 mycotoxin | No | Autoclaving with 17 lb pressure (121-132º C) for 30 min failed to inactivate LMW toxins.17,19 All burnable waste from LMW toxins should be incinerated at temperatures in excess of 815°C (1,500º F) |
| Brevetoxin | No | Autoclaving with 17 lb pressure (121-132º C) for 30 min failed to inactivate LMW toxins.17,19 All burnable waste from LMW toxins should be incinerated at temperatures in excess of 815°C (1,500º F) |
Chemical Inactivation of Selected Toxins
This table is from: Biosafety in Microbiological and Biomedical Laboratories, fifth edition (2009), US Department of Health and Human Services, Center for Disease Control and Prevention, National Institutes of Health
ND indicates “not determined” from available decontamination literature.
| Toxin | NaOCl (30 minutes) | NaOH (30 minutes) | NaOCl + NaOH (30 minutes) | Ozone Treatment |
|---|---|---|---|---|
| Botulinum neurotoxin | >0.1% Solutions of NaOCl (#0.1%) or NaOH (> 0.25 N) for 30 min inactivate BoNT and are recommended for decontaminating work surfaces and spills of C. botulinum or BoNT. Chlorine at a concentration of 0.3-0.5 mg/L as a solution of hypochlorite rapidly inactivates BoNT (serotypes B or E tested) in water.20 Chlorine dioxide inactivates BoNT, but chloramine is less effective.21 |
> 0.25 N | ND | Yes Ozone (> 2 mg/L) or powdered activated charcoal treatment also completely inactivate BoNT (serotypes A, B tested) in water under defined condition.20,22 |
| Staphylococcal Enterotoxin | >0.5% SEB is inactivated with 0.5% hypochlorite for 10-15 mi.23 |
> 0.25 N | ND | ND |
| Ricin | >1.0% Ricin is inactivated by a 30 min exposure to concentrations of NaOCl ranging from 0.1-2.5%, or by a mixture of 0.25% NaOCl plus 0.25 N NaOH.17 In general, solutions of 1.0% NaOCl are effective for decontamination of ricin from laboratory surfaces, equipment, animal cages, or small spills. |
ND | > 0.1% + 0.25N The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH.17 |
ND |
| Microcystin | ≥0.1% The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH.17 |
ND | > 0.25% + 0.25N The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH.17 |
ND |
| Saxitoxin | ≥0.1% The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH.17 |
ND | > 0.25% + 0.25N The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH.17 |
ND |
| Palytoxin | ≥0.5% The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH.17 |
ND | > 0.25% + 0.25N The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH.17 |
ND |
| Tetrodotoxin | ≥0.5% The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH.17 |
ND | > 0.25% + 0.25N The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH.17 |
ND |
| T-2 mycotoxin | ≥2.5% The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH.17. For T-2 mycotoxin and brevetoxin, liquid samples, accidental spills, and nonburnable waste should be soaked in 2.5% NaOCl with 0.25% N NaOH for 4 h. Cages and bedding from animals exposed to T-2 mycotoxin or brevetoxin should be treated with 0.25% NaOCl and 0.025 N NaOH for 4 h. Exposure for 30 min to 1.0% NaOCl is an effective procedure for the laboratory (working solutions, equipment, animal cages, working area and spills) for the inactivation of saxitoxin or tetrodotoxin. Decontamination of equipment and waste contaminated with select brevetoxins has been reviewed.19 |
ND | > 0.25% + 0.25N The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH.17 |
ND |
| Brevetoxin | ≥2.5% The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH.17. For T-2 mycotoxin and brevetoxin, liquid samples, accidental spills, and nonburnable waste should be soaked in 2.5% NaOCl with 0.25% N NaOH for 4 h. Cages and bedding from animals exposed to T-2 mycotoxin or brevetoxin should be treated with 0.25% NaOCl and 0.025 N NaOH for 4 h. Exposure for 30 min to 1.0% NaOCl is an effective procedure for the laboratory (working solutions, equipment, animal cages, working area and spills) for the inactivation of saxitoxin or tetrodotoxin. Decontamination of equipment and waste contaminated with select brevetoxins has been reviewed.19 |
ND | > 0.25% + 0.25N The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 0.25% NaOCl and 0.25N NaOH.17 |
ND |