4.0 Biosafety Procedures

Biological safety is dependent on many proper practices and barriers, including:

1. Microbiological practices

a. Technical proficiency

b. Sterile technique

c. Washing hands

d. Good habits

2. Primary barriers

a. Biosafety cabinets

b. Personal protective equipment

4.1       Standard Microbiological Practices

The following standard and special practices, safety equipment, and facilities apply to agents assigned to Biosafety Level 1 (BSL-1).

1. Access to the laboratory is limited or restricted at the discretion of the PI when experiments or work with cultures and specimens are in progress.

2. Workers shall wash their hands after they handle viable materials, after removing gloves, and before leaving the laboratory.

3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, and storing food for human use are not permitted in the work areas. Persons who wear contact lenses in laboratories should also wear goggles or a face shield.

4. Food is stored outside the work area in cabinets or refrigerators designated and used for this purpose only.

5. Mouth pipetting is prohibited.

6. Policies for the safe handling and disposal of sharps are followed.

7. All procedures are performed carefully to minimize the creation of splashes or aerosols.

8. Work surfaces are decontaminated at least once each day and again after any spill of viable material.

9. All cultures, stocks, and other regulated wastes are disposed of properly. Infectious waste requires decontamination before disposal by an approved decontamination method such as autoclaving.

10. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leak proof container and closed for transport from the laboratory. Materials to be decontaminated outside of the immediate laboratory are packaged in accordance with applicable local, state, and federal regulations before removal from the facility.

11. A biohazard sign must be posted at the entrance to a laboratory designated BSL-2 or higher. The sign or additional signage near by should include the name of the agent(s) in use, the biohazard level, the name and phone number of the investigator, and emergency contact information.

12. An insect and rodent control program must be in effect.

13. A lab coat, gloves, and safety glasses must be worn when handling infectious materials.

14. The HAMM manual is available to lab staff.

15. Spills and exposures are reported immediately to the PI/LSC; large spills are reported to EH&S for assistance with clean-up.

16. Biosafety cabinets (BSCs) are used when there is potential for creating aerosols.

17. High concentrations or large volumes of infectious materials can be centrifuged outside the BSC only if the centrifuge tubes are sealed and opened inside a BSC.

18. Labeling done in accordance with section 7.3.7 Using Labels of this chapter.

4.2       Aerosols      

Aerosols are liquid droplets or solid particulates dispersed in air. Many aerosols are too small to see with the unaided eye and remain suspended for an extended period. The production of aerosols while handling infectious agents accounts for the greatest source of laboratory-acquired infections.

Generation of aerosols may be caused by using centrifuges, blenders, shakers, magnetic stirrers, sonicators, pipettes, vortex mixers, syringes and needles, freeze-dried samples, vacuum-sealed samples, mortars and pestles, culture tubes, inoculating floss, and separatory funnels.

4.2.1     Control of Aerosols     

Perform activities in a biosafety cabinet.

1. Keep tubes stoppered when vortexing or centrifuging. Consider using tubes with screw-on tops.

2. If an aerosol has been created, leave the area and allow the droplets to settle. Place cloth soaked with disinfectant over work surface to deactivate droplets of infectious agents.

3. Slowly reconstitute or dilute contents of an ampule. Soaked gauze can be wrapped around ampoules while breaking or when stoppers are removed from tubes.

4. Mix solutions by discharging the secondary fluid down the side of the container as close as possible to the surface of the primary solution.

5. Allow inoculating needle to cool before touching biological specimens.

4.3        Pipetting      

1. Mouth pipetting is forbidden.

2. No infectious mixture should be prepared by bubbling air through the liquid with the pipette.

3. No infectious materials should be forcibly discharged from pipettes.

4.4        Syringes and Needles 

Avoid the use of syringes and needles when possible. Use the needle-locking type (Luer-lock) or a disposable syringe needle unit, or other safe sharps.

Used needles must not be resheathed, bent, broken, or removed from disposable syringes.

Needles and syringes must be disposed of in approved sharps containers. These are available at no charge through the Stockroom.


4.5        Disinfection and Sterilization: General Procedures        

1. After each experiment, decontaminate work surfaces using appropriate disinfectant (See Section 4.2.6).

2. Disinfect countertops and equipment where biohazardous material is used frequently.

3. Decontaminate all infectious materials and contaminated equipment prior to their being washed, stored or discarded.

4. Use autoclavable or disposable materials whenever possible. Do not reuse disposable items.

5. When sharps containers are ¾-full, close lid and place in hall for pick-up. Housekeeping staff will collect sharps waste.

6. Housekeeping staff is not allowed entry into Biosafety Level 2 (and above) labs unless special arrangements have been made with the lab supervisor, who must ensure safe work practices are followed. BSL-2 lab staff is required to clean the lab and properly segregate all of the lab waste into the appropriate waste streams. See Chapter VI, Hazardous Waste Directory for detailed information on proper disposal of hazardous wastes.

4.5.1     Decontaminants

The ability to kill or deactivate etiologic agents is required for worker safety, to prevent contamination of experiments and proper disposal of biohazardous waste. Typically, these are physical controls (e.g., heat) and chemical controls (e.g., bleach). The ability to kill or deactivate organisms by any means is related to the construction of the organism, the method used, and the amount of time the method is applied.

Sterilization is the use of a physical or chemical procedure to destroy all microbial life, including large numbers of highly resistant bacterial spores. Disinfection is when virtually all pathogenic microorganisms but not all microbial forms (bacterial endospores) on inanimate objects are eliminated. Sanitized means that a large number of organisms have been deactivated, such that there are probably not enough viable organisms left to cause a disease in humans. Surgical instruments are sterile, most biosafety cabinets are disinfected, and food handling is sanitary.


4.5.2     Chemical Methods of Biologic Decontamination

Chemicals used for biologic decontamination are called sterilizers, disinfectants, sanitizers, antiseptics and germicides. Based on strength, length of exposure and susceptibility of the organism, these are considered high, intermediate and low level disinfectants, in that order.

Chemicals commonly used for decontamination at the Center are listed below. See also Table 4.5.2.1, Practical Disinfectants for Use in Biomedical Research, for an overview and comparison of these agents.

1. Alcohols: Contact time: immediate to 10 minutes. A 70% ethanol or isopropyl solution is made by adding three parts water to seven parts 95% ethanol. Methanol should not be substituted for ethanol or isopropyl, because it is not as effective and is a health hazard. Always keep ethanol and isopropyl solutions away from potential sources of ignition. These solutions should be labeled and dated, with an expiration date of 180 days.

2. Chlorine Dioxide: Contact time: 10 minutes. Chlorine dioxide (ClO2) is a strong and fast-acting germicide, disinfecting agent, and oxidizer, often reported to be active at concentrations lower than those needed by chlorine as bleach. However, it has a shelf-life of only one day for activated solutions, so if it is used a fresh solution must be made each day. Chlorine dioxide is unstable as a gas; however, chlorine dioxide is soluble in water and stable in an aqueous solution. Chlorine dioxide can be obtained by ordering its stabilized form, which is then activated on-site when required.

Of the oxidizing biocides, chlorine dioxide is the most selective oxidant. Ozone and chlorine are much more reactive than chlorine dioxide, and they will be consumed by most organic compounds. Chlorine dioxide, however, reacts only with reduced sulfur compounds, secondary and tertiary amines, and some other highly reduced and reactive organic compounds. A more stable residue can therefore be achieved with chlorine dioxide at much lower doses than when using either chlorine or ozone. Generated properly, chlorine dioxide can be used more effectively than ozone or chlorine in cases of higher organic loading because of its selectivity.

3. Formalin: Contact time: 10 minutes. At an aqueous concentration of 0.2-0.8%, formaldehyde (formalin) has good disinfectant properties against vegetative bacteria, spores and viruses. It has an irritating odor and is a suspected human carcinogen. Formaldehyde is not recommended for daily disinfection. Use only with proper ventilation control (e.g. chemical fume hood). Example: Sterac.

4. Glutaraldehyde: (also called a "cold disinfectant"): Contact time: 15 minutes. Two-percent solutions exhibit good activity against vegetative bacteria, spores and viruses. Glutaraldehyde is toxic, a sensitizer and is generally not used for laboratory surface disinfectant, and is capable of eye damage. Use only with proper ventilation control, such as a chemical fume hood or specially designed slot hood. Example: Cidex.

5. Hydrogen Peroxide and Peracetic Acid: Contact time: 10 minutes. Like chlorine, hydrogen peroxide (H2O2) and peracetic acid are strong oxidants and can be potent broad-spectrum germicides. They are also safer than chlorine to humans and the environment. However, they have a short shelf-life of just five days for dilute solutions, so fresh solution must be made frequently if used.

Hydrogen peroxide is supplied either as a ready-to-use 3% solution or as a 30% aqueous solution to be diluted to 5-10 times its volume with sterilized water. However, such 3-6% solutions of hydrogen peroxide alone are relatively slow and limited as germicides. Products now available have other ingredients to stabilize the hydrogen peroxide content, to accelerate its germicidal action and to make it less corrosive.

Hydrogen peroxide can be used for the decontamination of work surfaces of laboratory benches and biosafety cabinets, and stronger solutions may be suitable for disinfecting heat-sensitive medical/dental devices. The use of vaporized hydrogen peroxide or peracetic acid (CH3COOOH) for the decontamination of heat-sensitive medical/dental devices requires specialized equipment.

Hydrogen peroxide and peracetic acid can be corrosive to metals such as aluminum, copper, brass, and zinc, and can also decolorize fabrics, hair, skin, and mucous membranes. Articles treated with them must be thoroughly rinsed before contact with eyes and mucous membranes. They should always be stored away from heat and protected from light.

6. Hypochlorite (Bleach): Contact time: 10 minutes. A 10:1 bleach solution/Sodium Hypochlorite (also called 10% bleach solution) is made by adding nine parts water to one part laboratory bleach (sodium hypochlorite). Bleach solution is corrosive to stainless steel; therefore, thorough rinsing must follow its use in the biosafety cabinet. Do not autoclave bleach solutions. The present stock bleach solution is 12.5% Sodium Hypochlorite, so a 10:1 solution will result in a final concentration of 1.25%. The diluted solution should be labeled and dated, with an expiration date of 30 days. Note that household bleach is 5.25% Sodium Hypochlorite and can be used in a 10:1 solution, but has an expiration date of one day. To be an effective disinfectant for most non-HIV pathogens (HBV, HCV, etc.), the solution should be at least 0.5% but less than 2%.

Hypochlorite solutions are classified as irritant and corrosive. Appropriate precautions should be taken when using hypochlorite products: read labels carefully, adhering to cautionary warnings and following usage directions. Chlorine solutions should never be mixed or stored with cleaning products containing ammonia, ammonium chloride, or phosphoric acid. Combining these chemicals will result in the release of a chlorine gas, which can cause nausea, eye irritation, tearing, headache, and shortness of breath. These symptoms may last for several hours. If you are exposed to an unpleasantly strong odor following the mixing of a chlorine solution with a cleaning product, leave the room or area immediately until the fumes have cleared completely.

To prepare disinfectant bleach solutions:

  • Using 12.5% hypochlorite (Top-Chlor) in a 1:10 dilution (one part Top-Chlor and nine parts water) yields 12,500 ppm or a 1.25% hypochlorite solution, for use within 30 days.
  • Using 12.5% hypochlorite (Top-Chlor) in a 1:20 dilution (one part Top-Chlor and nineteen parts water) yields 6,250 ppm or a 0.625% hypochlorite solution, for use within 24 hours.
  • Using 5.25% hypochlorite (Clorox) in a 1:5 dilution (one part Clorox and four parts water) yields 10,500 ppm or a 1.05% hypochlorite solution, for use within 30 days.
  • Using 5.25% hypochlorite (Clorox) in a 1:10 dilution (one part Clorox and nine parts water) yields 5,250 ppm or a 0.53% hypochlorite solution, for use within 24 hours.

7. Iodophor Disinfectant (Wescodyne): Contact time: 10 minutes. Prepare this solution according to the instructions on the label. Final concentration should be 0.47%. These solutions should be labeled and dated, with an expiration date of 365 days (one year).

8. Phenolic Compounds: Contact time: 10 minutes. At a concentration of 1-5%, phenolic compounds are effective against vegetative bacteria, fungi and lipid-containing viruses. They have an unpleasant odor, can easily be absorbed through the skin and are irritants to the mucous membrane and respiratory tract. Examples: Amphyl, Lysol I.C.

WARNING! Phenolic compounds can cause rapid and permanent eye damage—always wear safety glasses, nitrile gloves and labcoat, and prepare in chemical fume hood, when using or preparing phenolic solutions.

9. Quaternary Ammonium Compounds (Quats): Contact time: 10 minutes. Quats are used to disinfect at concentrations of 0.1-2% for vegetative bacteria and non-lipid-containing viruses. Quaternary ammonium compounds are not effective against spores and may be neutralized by anionic detergents. Example: Lysol I.C.

Table 4.5.2.1: Practical Disinfectants for Use in Biomedical Research, Pt. 1

 

Decontaminant Category

Quaternary Ammonium Compounds

Phenolic Compounds

Chlorine Compounds   

*5*

Use Requirements

Final concentration for use

0.1%-2%

1%-5%

500 PPM-10,000 PPM

 

Lipo viruses only contact time

10 mins.

10 mins.

10 mins.

 

Broad spectrum contact time

N/E

N/E

30 mins.

Inactivates

Vegetative bacteria

•

•

•

 

Lipo viruses

•

•

•

 

Hydrophilic viruses

 

*1*

•

 

Bacterial spores

 

 

• and *6* 

 

HIV

•

•

•

 

HBV

 

•

•

Important Characteristics

Effective shelf life *2*

>1 week

>1 week

24 hrs (5k PPM); 30 days (10k PPM)

 

Corrosive

 

•

•

 

Flammable

 

 

 

 

Residue

 

•

•

 

Inactivated by organic matter

•

 

•

 

Skin irritant

•

•

•

 

Lens compatible   *3*

•

 

 

Potential Application

Work surfaces

#

#

#

 

Equipment surface decontamination

#

#

# and *4*

Proprietary Decontaminant Names

Lysol I.C. Quaternary Disinfectant

Lysol I.C. Phenolic Disinfectant, Amphyl

TopChlor, Clorox, Purex

 

N/A = Not applicable     N/E = Not effective     • = Effective disinfectant/characteristic    # = Appropriate use

*1* = Variable results dependent on virus                

*2* = Protected from light and air                        

*3* = Refers to microscope and camera lenses

*4* = Will corrode stanless steel and other metals

*5* = 10:1 dilution with stock = 12,500 ppm

*6* = >2500 ppm

Sources: Biosafety in the Laboratory: Prudent Practices for Handling and Disposal of Infectious Materials, National Research Council, National Academy Press, 1989; and "APIC Guidelines for Selection and Use of Disinfectants," W. Rutola, American Journal of Infection Control, 24:4, pp.313-342, 1996.

Table 4.5.2.1: Practical Disinfectants for Use in Biomedical Research, Pt. 2

 

Decontaminant Category

Iodophor Compounds

Alcohol Ethyl or IPA

Formalin

Glutaraldehyde

Use Requirements

Final concentration for use

0.025%-2%

70%-85%

0.2%-8%

2%

 

Lipo viruses only contact time

10 mins.

10 mins.

10 mins.

10 mins.

 

Broad spectrum contact time

30 mins.

N/E

30 mins.

30 mins.

Inactivates

Vegetative bacteria

•

•

•

•

 

Lipo viruses

•

•

•

•

 

Hydrophilic viruses

•

*1*

•

•

 

Bacterial spores

 

 

•

•

 

HIV

•

•

•

•

 

HBV

•

•

•

•

Important Characteristics

Effective shelf life *2*

>1 week

>1 week

>1 week

14 days

 

Corrosive

•

 

 

 

 

Flammable

 

•

 

 

 

Residue

•

 

•

•

 

Inactivated by organic matter

•

 

 

 

 

Skin irritant

•

 

•

•

 

Lens compatible *3*

 

 

 

•

Potential Application

Work surfaces

#

#

#

#

 

Equipment surface decontamination

#

#

#

#

Proprietary Decontaminant Names

Wescodyne

N/A

Sterac

Cidex

 

Key:   N/A = Not applicable    N/E = Not effective    • = Effective disinfectant/characteristic     # = Appropriate use

*1* = Variable results dependent on virus

*2* = Protected from light and air

*3* = Refers to microscope and camera lenses

*4* = Will corrode stainless steel and other metals

Sources: Biosafety in the Laboratory: Prudent Practices for Handling and Disposal of Infectious Materials, National Research Council, National Academy Press, 1989; and "APIC Guidelines for Selection and Use of Disinfectants," W. Rutola, American Journal of Infection Control, 24:4, pp.313-342, 1996.

4.5.3     Physical Methods of Biologic Decontamination

The physical means most frequently used in decontamination are dry heat, moist heat, and incineration. Autoclaves may provide dry or moist heat, with moist heat being the most effective. Heat is effective against most viruses and bacteria encountered, and may be effective against spores and fungi. Prions, spores, and temperature-resistant organisms may require additional time and adjunct chemical means to be completely deactivated. The amount of time the material is heated and the sustained temperature can be modified to achieve higher levels of decontamination.

The following is a comparison of the different types of sterilization and efficacy in different circumstances.

  1. Steam heat (autoclave) requires approximately 15 psi pressure with a chamber temperature of at least 250°F (121°C). The cycle time begins when the materials being sterilized reach the predetermined temperature. The duration required is dependent upon the volume of the load (usually 30-60 minutes).

    Each autoclave that uses steam sterilization must be biologically challenged using a biological indicator (
    Bacillus stearothermophilus) at least monthly. Upon failure, the unit must pass two consecutive tests prior to use.
  2. Dry heat is less effective than steam and requires more time (two to four hours) and a higher temperature (320-338°F or 160-170°C). Monitor effectiveness with a biological indicator.

    Each unit that uses dry heat must be biologically challenged using a biological indicator (
    Bacillus subtilis) at least weekly. Upon failure, the unit must pass two consecutive tests prior to use.


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