Laboratories:General Safety, Biosafety, Hazardous Waste and

Emergency Response

Laboratories:General Safety, Biosafety, Hazardous Waste and

Emergency Response

Laboratory Security

There are extremists who vandalize workplaces where they suspect animal research is being conducted or identify sources for chemicals/ biologicals to be used in terrorism or illegal drug production.

Protect your lab and your research:

• NEVER prop open any card or key access corridor doors.•Don’t let people ‘piggy-back’ to gain access to restricted areas.• Keep your lab doors closed and locked when unattended.•Question strangers, and report suspicious activity to campus security.

Chemical Hazards: Physical Hazards

Compressed Gas - cylinders of compressed gas are under intense pressure.

Corrosivity – strong acids and bases can cause visible destruction of living tissue at the site of contact.

Flammability - both solid and liquid chemicals can serve as fuel sources to support a fire.

Reactivity - many substances will react violently if allowed contact with air, water or other chemicals. Friction, shock, light or heat can be enough to trigger some reactions.

Oxidizability – compounds that release oxygen as they decompose. React with flammables

Irritants - cause reversible inflammation of skin, eyes or nasal passages.

Sensitizers - cause allergic reaction after repeated exposures. Individual sensitivities vary.

Carcinogens - alter DNA or cause cells with altered DNA to multiply.

Systemic Toxins - typically target a specific organ or system (liver, kidney, blood, nervous system, reproductive system, etc.).

Toxins – based on dose. Anything can be poisonous. Corrosives – cause burns to the skin, eyes, mucous

membranes and respiratory tract.

Chemical Hazards: Health Hazards

Gas Cylinder Safety

Storage and Handling Gas cylinders should not be stored in

exits or egress routes or blocking any safety equipment

Gas cylinders (excluding lecture bottles) should be stored in an upright position and with safety caps in place unless in use.

Use only the appropriate regulator for the gas

Gas cylinders must be secured with a chain or appropriate belt above the midpoint but below the shoulder of the cylinder

Electrical Safety Maintain all electrical equipment in proper and

good working order Routinely examine all wiring, plugs power strips

and extension cords for signs of exposed wire or deteriorating insulation

No tripping or hanging hazards

Safety Equipment:Chemical Fume Hood

Airflow into the hood prevents chemicals inside from migrating out into your breathing zone.

If air velocity into the hood is impeded or slowed, the hoods ability to capture chemicals is compromised.

Factors that affect airflow:

•Bulky objects inside

•Drafts

•Sash Height

Finding and Reading an MSDS

MSDS-SEARCH National Repository http://msdssearch.com/msdssearch.htm

What information does an MSDS contain- Identity of material (chemical name, synonyms)- Physical and chemical properties- Health hazards and “safe” exposure levels- Symptoms of exposure - Applicable routes of entry to the body- Carcinogen listing- Name, address, and emergency phone number of- manufacturer- Precautions for safe handling- Control measures, PPE

University of Notre Dame Chemical Hygiene Plan (CHP)

Contains information and procedures that laboratory personnel can use to protect themselves from the chemicals they work with

First place to look for answers to questions you might have regarding chemical or laboratory issues

Located on Risk Management and Safety website http://www.nd.edu/~riskman

Topics include:- Emergency Response - Personal

Protective Eq.- Chemical compatibility -Waste Management- Standard Operating Procedures (SOPs) applicable to

most labs on campus

Routes of Exposure Chemical and Biological

Percutaneous injuries Percutaneous injuries can result from needlesticks, cuts or abrasions from contaminated items. These exposures are particularly serious because of the potential for immediate entry of the agent into a normally sterile bloodstream. All sharps items should be handled and disposed of as instructed.

Inhalation of aerosols Many laboratory procedures can cause the aerosolization of infectious agents. Some of these procedures include the use of vortexes, blenders and sonicators. Proper work practices must be  implemented to minimize the aerosolization of all materials, especially those which are known to be transmitted by the aerosol route (e.g., Adenovirus, Vaccinia virus, Mycobacterium tuberculosis, etc.).

Mucous membrane Exposure of mucous membranes to infectious agents can lead to occupationally acquired infections. Mucocutaneous exposures can result from splashes to the eyes, nose or mouth, or by inadvertent inoculation via contaminated hands. Face protection should always be used if there is an likelihood of splash or splatter.

Ingestion Accidental ingestion of biohazardous materials can result from improper personal hygiene in the laboratory. Food and drink are prohibited in all areas of the laboratory in which work is conducted with potentially infectious materials. Hands must always be washed before leaving the laboratory, and immediately if visible contamination occurs

Personal Protective Equipment Gloves are required anytime you are handling

chemicals, biologicals, and radioactive materials Eye protection is required anytime you are working

with chemicals, biological and radioactive materials or involved with sanding or grinding operations

Goggles should be worn when working with corrosives or if a splash hazard is present

Lab coats are required if working with biological and radioactive materials

Open toed shoes are NOT permitted in laboratories.

General Biosafety:Who needs this training and what does it entail?

Individuals who work with or have access to biohazardous materials.

Training required on an annual basis.

Biohazardous Materials- Definition and Examples

Materials of biological origin that have the capacity to produce deleterious effects on humans or animals.

Examples Recombinant DNA molecules and/or Microorganisms containing

recombinant DNA molecules Microorganisms classified as risk group 2 (RG-2), RG-3, or RG-4

and (Biological products derived from RG-2, RG-3, or RG-4 organisms

Diagnostic specimens known or reasonably expected to contain RG-2, RG-3, or RG-4 organisms.

Clinical/ medical waste derived from the medical treatment of humans or animals or from biomedical research.

Definition

BiosafetyThe application of combinations of laboratory practice and procedure, laboratory facilities, and safety equipment when working with potentially infectious microorganisms.

We use these practices to protect the workers and those around, the product and the environment.

This involves all lab workers

Laboratory Requirements

Knowledgeable supervisor Knowledgeable personnel

Aware of potential hazards Proficient in lab practices & techniques

Availability of a lab specific biosafety manual Contact information Copies of IBC,IRB,RCC, and/or IACUC

protocols Standard Operating Procedures

For unique procedures Laboratory inspection checklist Emergency response plan

Special procedures where needed Relevant reference materials

Classification of Infectious Agents

Found in the NIH Guidelines. Classified into risk groups on

the basis of risk to the individual and to the community.

Currently, 4 risk group levels have been designated. the least risk (RG-1) to the most

risk (RG-4).

RG-1 Agents RG-2 Agents

Not associated with disease in healthy adult humans.

Generally only require a laboratory with minimal containment Biosafety Level 1

laboratory “Declaration of Dangerous

Goods” is not required for shipment.

Bacillus subtilis, Saccharomyces cerevisiae, Staphylococcus epidermidis, Escherichia coli K-12

Associated with human diseases which are rarely serious.

Preventative and therapeutic interventions are available.

Generally require a laboratory with moderate containment. Biosafety Level 2 laboratory

“Declaration of Dangerous Goods” required for shipment.

Measles virus, Salmonella spp., Toxoplasma spp., Hepatitis B virus, leshmainia, plasmodium falciparum

Combination of laboratory practices, safety equipment, and laboratory design to achieve levels of physical containment.

Currently, 4 biosafety levels have been designated. The least restrictive (BSL-1) to the most

restrictive (BSL-4).

Biosafety Levels of Containment

Containment:BSL-1 BSL-2

RG-1 Agents Not known to cause

disease in healthy adult humans

Practices Standard microbiological

practices

Safety equipment None required

Facilities Open bench top

RG-2 Agents Associated with mild to

moderate disease in humans

Practices BSL-1 plus limited access.

Safety equipment Biological Safety Cabinet

and personal protective equipment as needed.

Facilities BSL-1 plus the availability

of a mechanism for decontamination.

BSL-1 Standard Microbiological Practices

Restrict or limit access when working Prohibit eating, drinking and smoking Minimize splashes and aerosols Decontaminate work surfaces daily Decontaminate infectious waste Maintain an insect & rodent control

program Prohibit mouth pipetting/ use mechanical

pipetters Needles & sharps precautions

Use sharps containers DON’T break, bend, re-sheath or reuse

syringes or needles Use alternatives to needles when available DON’T place needles or sharps in regular trash

containers.

BSL-1 Standard Microbiological Practices

Wash hands

BSL-1 Safety Equipment (Primary and Secondary Barriers)

Personal protective equipment Eye wear Gloves Lab coat

Countertops and floors easily cleaned and decontaminated

Laboratory Requirements: Lab does not need to be separated

from other lab sections and can be of normal construction.

No specific ventilation req.

Supervisor Trained in

microbiological or related science practices.

Lab personnel Specific training in

lab procedures.

Training RequirementsBSL-1 BSL-2

Supervisor Is a competent scientist

Restricts access

Lab Personnel Aware of potential hazards Proficient in practices and

techniques

BSL-2 Standard Microbiological Practices

Follow all practices and personal equipment used for BSL-1 containment.

BSL-2 Safety Equipment (Primary and Secondary Barriers)

Class II Biological Safety Cabinet recommended to protect product,

personnel, and the environment.

Requirements: Laboratories have lockable doors and separated from

public areas Sink for hand washing Work surfaces easily cleaned Bench tops are impervious to water Sturdy furniture Adequate illumination Air flows into lab without re-circulation to non-lab areas Windows fitted with fly screens

BSL-2 Facility Design (Secondary Barrier)

Signage on the door with restricted access when work in progress or agents are present.

Autoclave available

Eyewash station available

Biological Safety Cabinets as Primary Barrier- Overview

Protection of Product Personnel Environment

Class II Biological Safety Cabinet

Equipment is laid out to not restrict airflow in the cabinet

“High efficiency particulate air” (HEPA) filter

Traps particulates 0.01 microns and larger including bacteria and viruses

chemicals, fumes, and vapors pass through

1. Load with needed supplies2. Turn on and allow to run for 10-15

minutes3. Check inward airflow with a piece of

tissue4. Enter straight into cabinet and perform

work in a slow, methodical manner5. At end of work package or

decontaminate items to be taken out of cabinet

6. Decontaminate interior of cabinet7. Allow cabinet to run for 10-15 minutes8. Shut off9. Annual certification is required.

Biological Safety Cabinet - Operation

CAUTIONS Chemicals may damage HEPA filter Volatile chemicals NOT retained by HEPA

filter Exposes personnel if not exhausted

Fans NOT spark proof Chemical use may result in fire and/or explosion Never use flammables

Biological Safety Cabinet - Safe Operation

• Check tubes for cracks/chips.• Use matched sets of tubes, buckets

etc.• Make sure the rotor is balanced

properly• Tightly seal all tubes and safety

cups.• Ensure that rotor is locked to

spindle and the bucket is seated.• Close lid during operation.• Allow to come to complete stop

before opening.• Disinfect weekly or when spills

occur.• Don’t use rotors that have been

dropped.

Centrifuge – Hazards and Operation

Mechanical failure of Mechanical failure of

machinemachine

Lab equipment failure Lab equipment failure

(tubes etc.)(tubes etc.)

Aerosol generationAerosol generation

Operator errorOperator error

Decontamination Definitions

Sterilization physical or chemical procedure used to destroy

all microbial life, including large numbers of highly resistant bacterial spores.

Disinfection physical or chemical procedure used to eliminate

all recognized pathogenic microorganisms but not all microbial forms (bacterial spores) on inanimate objects.

Antiseptic germicide that is used on skin or living tissue for

the purpose of inhibiting or destroying microorganisms.

Steam sterilization Ensure proper autoclave function Vessels should not be capped or plugged Large loads require longer contact time Excessive amounts of liquid should not be added

to a load Autoclave parameters must be appropriate for the

taskVerify for proper sterilization Chemical indicator

Per load Biological indicator

Bacillus stearothermophilis At least weekly

Log book of results must be maintained

Decontamination - Heat

Decontamination - Chemical

High level disinfection (sporocides) Kills all microorganisms

including bacterial spores

Requires 5 to10 min. exposure

Examples hydrogen peroxide glutaraldehyde

Intermediate level disinfection (tuberculocides)

kills Mycobacterium tuberculosis most vegetative bacteria viruses

Require minimum 20 min. exposure

Examples phenolics iodophores chlorine compounds (bleach 5-10% solutions)

Low level disinfection (hospital germicides) General housekeeping Kills most vegetative bacteria

not M. tuberculosis Require minimum 20 min. exposure Examples

quarternary ammonium compounds

Decontamination - Chemical

Decontamination – UV Irradiation

UV lamps may be available in the biological safety cabinet for surface decontamination

Extreme care must be taken to prevent exposure to the eyes or skin.

Periodic testing is required to determine the effectiveness of UV irradiation

Follow special procedures for lamp disposal

Decontamination: Conclusion

Disinfectants do not replace adherence to good standard microbiological practices and proper hygiene!

Waste DisposalChemical and Biological

Know and follow all disposal rules. Always keep containers closed/capped if not

adding waste Always make sure containers are properly

labeled.

Chemical Waste DisposalSatellite Accumulation

Label (3 things):

•Must be labeled as soon as first drop of waste is put into the container.

•Words “waste or “hazardous waste”•Description or Name of chemical(s)

Identify Hazardous Waste Satellite

Accumulation Area

Container•Compatible and in good condition•Has “tight-fitting” closure and container is closed unless adding waste.

Secondary Containment•Must segregate incompatibles•One container per waste stream

Generator Signs

Faculty name printed

4 Liters

Chemistry 341

Acetone 20

Methanol 40

Abbreviations and formulas NOT acceptable

Person who generates waste signs form

RM&S use ONLY

Biohazardous Waste

stock cultures materials containing or contaminated with blood blood and body fluidsSerum,

Plasma Other blood components Semen Vaginal secretions Cerebrospinal fluid Pleural fluid Peritoneal fluid Pericardial fluid Amniotic fluid Any other body fluid visibly contaminated with blood

sharps Pipette/pipette tips, Hypodermic needles, Scalpels, Breakable

containers, Glass products (i.e., slides or cover skips) animal waste other contaminated lab material

Biohazardous Waste Containers

Biohazardous waste containers shall be clearly marked with the universal biohazard symbol.

•Puncture-proof, leak-proof, sealable receptacles•avoid over-filling

Orange Biohazard Bags

Orange Biohazard Bags contain previously contaminated waste that has been decontaminated (autoclaved) by lab personnel. May be taken to the dumpster if brown bagged. Housekeepers WILL NOT handle these bags.

Biohazardous WasteInappropriate packaging

Long bore pipettes should first be boxed to prevent puncture of the plastic bag unless being autoclaved and handled by lab personnel..

Aerosol cans are not biohazardous waste and must be disposed of as hazardous waste.

Large biohazardous waste containers such as these must be sealed with a tightly fitting lid prior to transport. Don’t over pack the container. Locking rings must be fitted on the blue poly containers prior to transport.

Emergency Response

Fires/accidents911 or 631-5555 from cell Spills 631-5037

(Risk Management and Safety) After Hours Spills 911

State the type of emergency and the exact location of emergency. If a chemical is involved spell the chemical name to the dispatcher

Fire Department Direct number 631-6200

Safety Equipment:Safety Shower and Eyewash

Ask Ask yourself:yourself:

•Do I know where they are located?•Do I know how they work?•Do I know that they work?•Are they accessible?

How to Use a Fire Extinguisher

pull pin

aim at the base of the fire

squeeze handle

sweep from side to side

Biological/Chemical Exposures

Needle Sticks:Needle Sticks:

•Wash thoroughly with soap and water

•Identify source (consent?)

Ocular Exposures:Ocular Exposures: Dermal Exposures:Dermal Exposures:

•Rinse at eyewash at least 15 minutes

•Hold eye open to ensure effective wash

•Rinse under drench shower at least 15 minutes•Remove all contaminated clothing

•Seek Medical Treatment•Your supervisor must fill out an accident/exposure form

Chemical SpillsIdentify the chemical

Assess your ability to safely contain and clean up the spill:

I CANCAN safely clean it up

I CANNOTCANNOT safely clean it up

(spill size, chemical toxicity)

Notify coworkers and vacate/secure the area

Call 1-5037 (during working hours_911 after hours

Use spill kit to contain and clean up the spill (MSDS helps)

Notify coworkers and secure the area

Call Risk Management if you have any concerns

Biological Spills/Decontamination

• Cover spill with paper towels• Soak towels with disinfectant (10% Bleach is effective)

• Wait 10 minutes

• While wearing PPE, clean up the spill using paper towels

• Wipe spill area with disinfectant

• Dispose of all clean-up materials as biological waste (red bag)

Notify Risk ManagementNotify Risk Management1-5037 if you need assistance1-5037 if you need assistance

References

University of Notre Dame Biosafety Manual http://riskmgt.nd.edu/manuals/documents/

NDBiosafetyManual1.doc NIH Guidelines

http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html

Biosafety in Microbiological and Biomedical Labs

http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm

IATA Dangerous Goods Regulations http://www.iata.org