elements of biosafety janet peterson jpeterso@accmail.umd.edu 405-3975

Elements of Biosafety Elements of Biosafety

Janet Peterson

jpeterso@accmail.umd.edu

405-3975

BIOSAFETY: Preventing BIOSAFETY: Preventing lab-acquired infectionslab-acquired infections

Bacteria Viruses Fungi Human blood, unfixed tissue Human cell lines Recombinant DNA

GuidelinesGuidelines NIH Guidelines for Experiments

Involving Recombinant DNA Molecules– Large Scale > 10 liters

Biosafety in Microbiological and Biomedical Laboratories-NIH/CDC

UM Biosafety Manual– On DES webpage

www.inform.umd.edu/des

RegulationsRegulations

OSHA Bloodborne Pathogens Standard

Maryland Waste Regulations Shipping and packaging infectious

substances– DOT, UN, CDC, IATA

Levels of Containment Levels of Containment

BL1 - microorganisms that don’t consistently cause disease in healthy adults– E. coli K12, S. cerevisiae,

polyomavirus

– Basic laboratory

– Standard Microbiological Practices

Levels of Containment Levels of Containment

BL2 - microorganisms of moderate potential hazard, transmitted by contact, ingestion, puncture– Salmonella, herpesvirus, human

blood

– Basic laboratory

– Standard Practices PLUS

Levels of ContainmentLevels of Containment

BL2 - Standard Microbiological Practices Plus:– Training in handling pathogens

– Access to lab limited

– Extreme sharps precautions

– Use of BSC for aerosols

Biosafety CabinetsBiosafety CabinetsCourtesy of the Baker CompanyCourtesy of the Baker Company

Use of Biosafety CabinetUse of Biosafety Cabinet

Turn on fan 15 min before starting Don’t block grille Disinfect work surface w/ 70% etoh Discard pipets inside cabinet Minimize movement of hands Avoid use of flame unless necessary Have cabinet certified annually

Clean BenchClean Bench

This is not a BSC Air flows from back

of cabinet, across work surface, and onto user.

This does not provide worker protection.

Levels of Containment Levels of Containment

BL3 - microorganisms that cause serious disease, transmitted by inhalation– M. tuberculosis, yellow fever virus,

hantavirus, Y. pestis (plague) – Containment lab: double door entry;

directional airflow; all work in biosafety cabinet

Levels of Containment Levels of Containment

BL4 - microorganisms that cause lethal disease, with no known treatment or vaccineEbola virus, Marburg virus Maximum containment lab; positive

pressure ventilated suits (moon suits)

OSHA Bloodborne OSHA Bloodborne Pathogens StandardPathogens Standard

Human blood, unfixed tissue, primary human cell culture, other potentially infectious materials

HIV,HBV, HCV

OSHA Standard requires:OSHA Standard requires:

Annual training– Web-based program/DES homepage

Free HBV vaccine Use of Universal Precautions

Universal Precautions Universal Precautions

Treat ALL human blood and unfixed tissue as if it contains HIV and HBV

Routes of Occupational Routes of Occupational TransmissionTransmission

Puncture or cut (needlestick, contaminated broken glass)

Contact with broken skin Splash to mucous membranes of

eyes, nose, mouth

Precautions for First AidPrecautions for First Aid

Wear gloves If conscious, have patient put

pressure on wound Use one-way valve for CPR

Standard Microbiological Standard Microbiological PracticesPractices

NOT permitted in laboratories: Eating DrinkingSmokingHandling contact lensesPipetting by mouthStoring food and drink

Standard Microbiological Standard Microbiological PracticesPractices

ALWAYS wash hands: After handling microorganisms and

animalsAfter removing glovesBefore leaving laboratory

Standard Microbiological Standard Microbiological PracticesPractices

Discard needles, razor blades, and scalpel blades into red, puncture resistant sharps containers

Dispose of broken glass into “broken glass” containers, never regular trash

Standard Microbiological Standard Microbiological PracticesPractices

NEVER – recap, bend, or break needles

– discard needles or sharps into biological waste bags

– discard needles into regular trash

Standard Microbiological Standard Microbiological PracticesPractices

Decontaminate all biological waste (including BL1) before disposal– Solid waste (Petri dishes, cultures):

autoclave and put in dumpster

– Liquid waste: add disinfectant (bleach to 10%) and pour down drain

AutoclavesAutoclaves

Autoclaves use pressurized steam to sterilize materials.

There is usually steam remaining in the chamber at the end of a liquid cycle.

Autoclave SafetyAutoclave Safety

Opening door at end of liquid cycle:– Wear eye and face protection.

– Stand behind door when opening it.

– Slowly open door only a crack to allow residual steam to escape.

– Keep face away from door as it opens.

Autoclave SafetyAutoclave Safety

Removing liquids at end of cycle:– Wait 5 min. before removing liquids.

– Liquids removed too soon may be super-heated and boil up and out of container.

– Aim mouth of flask away from face.

– Don’t knock flask against bench.

Standard Microbiological Standard Microbiological PracticesPractices

Decontaminate work surfaces daily and after any spill of viable material

Report accidents to the PI Tell Health Care Provider that you

work with infectious agents or chemicals

Think AgainThink Again

You've carefully thought out all the angles.

You've done it a thousand times.

Nothing could possibly go wrong, right ?

Questions?Questions?