Mar 30, 2015
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?