Elements of Biosafety

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 Laboratories-NIH/CDC
 UM Biosafety Manual
– On DES webpage
www.inform.umd.edu/des
 Regulations
 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
 Use 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 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
 FreeHBV 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 ?
Questions?