1. Janet Peterson jpeterso@accmail.umd.edu 405-3975
Elements of Biosafety
Janet Peterson

2. BIOSAFETY: Preventing lab-acquired infections
Bacteria
Viruses
Fungi
Human blood, unfixed tissue
Human cell lines
Recombinant DNA
Biosafety is the section of Environmental Safety that addresses working safely in the laboratory with microorganisms and rDNA (organisms whose genetic material has been altered).
Its goal is to help prevent lab-acquired infections while allowing researchers to work with organisms of various levels of hazard. This is done by advising researchers of the containment practices that are appropriate for the organisms they are handling, based on the level of risk presented by the organism. 1

3. 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
Various regulations and guidelines have been developed to protect workers and the environment. They define work practices required to work safely with biological material of all levels of hazard. All Principal Investigators must register their experiments that involve the use of rDNA, infectious microorganisms, and human blood, unfixed human tissue, and primary human cell lines with DES. 2

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

5. 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
There are 4 levels of biological containment that allow laboratory researchers to work safely with infectious agents of various levels of risk. They are designed to prevent lab-associated infections.
BL1 is the lowest level of containment. At UM there are many BL1 labs, most of which are molecular biology labs that use non-pathogenic strains of E. coli for cloning. . 3

6. Levels of Containment
BL2 - microorganisms of moderate potential hazard, transmitted by contact, ingestion, puncture
Salmonella, herpesvirus, human blood
Basic laboratory
Standard Practices PLUS
BL2 is used for infectious agents that are transmitted by contact, ingestion, or puncture with contaminated sharp object. There are several BL2 labs at UM. 4

7. Levels of Containment BL2 - Standard Microbiological Practices Plus:
Training in handling pathogens
Access to lab limited
Extreme sharps precautions
Use of BSC for aerosols
BL2 labs are similar to BL1 laboratories, with the addition of these specific practices.

8. Biosafety Cabinets Courtesy of the Baker Company

9. 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

10. 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.

11. 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
BL3 containment is used for work with infectious agents that are transmitted by inhalation. We have 2 BL3 labs at UM, but neither is currently in use. 7

12. 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)
There are only a very few BL4 labs in the country: CDC in Atlanta; NIH and Ft. Detrick in MD. BL4 is used for work with the most hazardous microorganisms. Hot Zone described BL4 containment. 8

13. OSHA Bloodborne Pathogens Standard
Human blood, unfixed tissue, primary human cell culture, other potentially infectious materials
HIV,HBV, HCV
The OSHA BBP standard was published in 1991 to address concerns over occupational transmission of HIV to healthcare workers.
BBPs are microorganisms present in the blood of infected persons, which are transmitted by blood-to-blood contact. Not casual contact or by inhalation.
If you work with human material, you are covered by the BBP Standard.

14. OSHA Standard requires:
Annual training
Web-based program/DES homepage
Free HBV vaccine
Use of Universal Precautions
If you work with human material, OSHA requires:
annual training of employees. If you work with human material, please take the interactive web based training available on our web page.
provision of HBV vaccine by employer at no cost to employee;
use of Universal Precautions when handling human material
treating ALL material as if it may contain BBPs

15. Universal Precautions
Treat ALL human blood and unfixed tissue as if it contains HIV and HBV
Even if material has been tested for BBPs such as HIV and HBV, it is possible that the test was performed before a detectable level of antibodies were produced, and the virus may be present. Therefore, ALL human material should be treated as infectious. Even if it is from your lab partner, because you can’t tell if someone is infected.
If you work with human material, please take the interactive web based training available on our web page. (Copy of page in handout). 5

16. Routes of Occupational Transmission
Puncture or cut (needlestick, contaminated broken glass)
Contact with broken skin
Splash to mucous membranes of eyes, nose, mouth
Routes of occupational transmission of BBP.
Optional:
Routes of transmission in the general population:
Sexual, both homosexual and heterosexual
Sharing needles among injecting drug users.
Blood transfusions prior to screening of blood supply (1980s??).
Mother to unborn child. 6

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

18. Standard Microbiological Practices
NOT permitted in laboratories:
Eating
Drinking
Smoking
Handling contact lenses
Pipetting by mouth
Storing food and drink
These are the basic practices when working with any microorganisms, and are the foundation for BL1 containment. They are designed to prevent transmission by contact, ingestion, and puncture.
These practices are appropriate for ALL laboratories that work with radioisotopes and chemicals as well as microorganisms. 9

19. Standard Microbiological Practices
ALWAYS wash hands:
After handling microorganisms and animals
After removing gloves
Before leaving laboratory
Gloves are not a substitute for hand washing. 10

20. 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
In your handouts there is an information sheet on handling sharps safely.
Sharps containers are available from Chemistry Stores and scientific distributors such as Fisher and VWR.
Sharps containers are usually red, puncture-resistant plastic. Do not overfill sharps containers. Please call DES for pick-up of filled sharps containers. They do not need to be autoclaved before pick-up. 11

21. Standard Microbiological Practices
NEVER
recap, bend, or break needles
discard needles or sharps into biological waste bags
discard needles into regular trash
Most needle sticks occur when recapping needles. If you must recap the needle, use a one-handed technique.
If you get stuck by a needle, wash the area with soap and water, then go to the University Health Center for follow-up as soon as possible. Anti-retroviral drugs are more effective if given within 2-3 hours after needle stick. 12

22. 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
Use only autoclavable bags in autoclave. Put bags of waste in stainless steel or polypropylene tray during autoclaving to contain any leaks.
There is information on autoclave safety and Biological Waste Disposal in the handout. 13

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

24. 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.

25. 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.

26. 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
Inform your healthcare provided that you work in a laboratory, and describe the types of hazardous materials you handle. 14

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

28. Questions?