Janet Peterson jpeterso@accmail.umd.edu
405-3975

BIOSAFETY: Preventing lab-acquired infections
 Bacteria
 Viruses
 Fungi
 Human blood, unfixed tissue
 Human cell lines
 Recombinant DNA

Guidelines
 NIH Guidelines for Experiments
Involving Recombinant DNA Molecules
– Large Scale > 10 liters
 Biosafety in Microbiological and
Biomedical LaboratoriesNIH/CDC
 UM Biosafety Manual
– On DES webpage www.inform.umd.edu/des

 OSHA Bloodborne Pathogens
Standard
 Maryland Waste Regulations
 Shipping and packaging infectious substances
– DOT, UN, CDC, IATA

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
 BL2 - microorganisms of moderate potential hazard, transmitted by contact, ingestion, puncture
– Salmonella , herpesvirus, human blood
– Basic laboratory
– Standard Practices PLUS

Levels of Containment
 BL2 - Standard Microbiological
Practices Plus:
– Training in handling pathogens
– Access to lab limited
– Extreme sharps precautions
– Use of BSC for aerosols

Biosafety Cabinets
Courtesy of the Baker Company

 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 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
 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
 BL4 - microorganisms that cause lethal disease, with no known treatment or vaccine
 Ebola virus, Marburg virus
 Maximum containment lab; positive pressure ventilated suits (moon suits)

OSHA Bloodborne
Pathogens Standard
 Human blood, unfixed tissue, primary human cell culture, other potentially infectious materials
 HIV,HBV, HCV

OSHA Standard requires:
 Annual training
– Web-based program/DES homepage
 Free HBV vaccine
 Use of Universal Precautions

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

Routes of Occupational
Transmission
 Puncture or cut (needlestick, contaminated broken glass)
 Contact with broken skin
 Splash to mucous membranes of eyes, nose, mouth

Precautions for First Aid
 Wear gloves
 If conscious, have patient put pressure on wound
 Use one-way valve for CPR

Standard Microbiological

Practices
NOT permitted in laboratories:
 Eating
 Drinking
 Smoking
 Handling contact lenses
 Pipetting by mouth
 Storing food and drink

Standard Microbiological
Practices
 ALWAYS wash hands:
 After handling microorganisms and animals
 After removing gloves
 Before leaving laboratory

Standard Microbiological
Practices
 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
Practices
 NEVER
– recap, bend, or break needles
– discard needles or sharps into biological waste bags
– discard needles into regular trash

Standard Microbiological
Practices
 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

Autoclaves
 Autoclaves use pressurized steam to sterilize materials.
 There is usually steam remaining in the chamber at the end of a liquid cycle.

Autoclave 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 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
Practices
 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 Again
 You've carefully thought out all the angles.
 You've done it a thousand times.
 Nothing could possibly go wrong, right ?