1. Tim Wetzel Chemical Engineering December 10, 2009
Biological Safety
Tim Wetzel
Chemical Engineering
December 10, 2009

2. Biohazard according to CDC
An agent of biological origin that has the capacity to produce deleterious effects on humans, i.e. microorganisms, toxins and allergens derived from those organisms; and allergens and toxins derived from higher plants and animals.
The main government bodies that set standards for biological hazards are the Center for Disease Control and the National Institute of Health. This is the CDC definition of what constitutes a biohazard.

3. Biosafety Hazards
Between 1978 and 1998 were 1,267 overt infections found in the literature
663 more cases were subclinical
22 deaths
5 fetuses were aborted due to infection
80% of cases no specific accident was identified as being the cause
Breathing of aerosols is most often attributed
There have been several large studies on the hazards of working with biological agents. One of the largest studies was a literature search over 20 years of accidents from 1978 to There were approximately 1900 accidents attributed to biological laboratory work, but only 20% of the cases were associated with a specific incident. Most of the cases were attributed to breathing of biohazards in the form of aerosols, enforcing the need to be vigilant about inhalation hazards in the laboratory and preventing aerosol formation in microbiological work.

4. Goals of Biosafety Prevent biohazard from harming individuals
You (and fetus)
Correct PPE and microbiological procedures
Other lab workers
Promptly lean up spills
Minimize aerosols
Custodians
Proper waste removal
Researchers in other labs
No wearing protective equipment outside of laboratory
Researchers need to be concerned with how their actions affect other people in the laboratory and in the university. Your first priority is to do no harm to yourself. Likewise if you are pregnant you have an obligation to protect the unborn child from being affected from the biological agents.
Likewise you have a responsibility to prevent any possible injury to other people in the laboratory. Clean up all spills immediately. Spills should be decontaminated with freshly made, 10% bleach. Adequate contact time (20 minutes) should be allowed to kill the biological agents. Pouring the bleach solution can cause aerosols to be formed, so it is best to evacuate the laboratory after applying bleach. In addition to spill clean up, follow correct biological procedures to reduce the possibility of contamination of outside material.
Researchers should properly dispose of waste. This includes using the red biowaste bags and following correct autoclave procedures for decontaminating waste material.
The researchers should be conscientious about sharp disposal. Directly disposing of sharps into the biowaste bags can lead to an accidental injection of custodial staff that are handling the boxes.
Researchers should make sure that the biological work stays within the laboratory.

5. Biohazards in the Laboratory
Infectious agents and pathogens:
Bacteria, virus, parasites, fungi
Human-derived tissues, cells, body fluids
Non-human primate tissues, cells, body fluids
Animals – wild trapped or lab stock
Biological toxins:
Botulinum, tetrodotoxin, ricin, etc.
Recombinant DNA, RNAi:
Plasmids, linear naked DNA, synthesized oligos, etc.
Viral vectors:
Adenovirus, MuLV, lentivirus, etc.
All are designed to express transgenes. Many insert in the genome. Modifications like VSV G can increase your risk.
This is a list of the major categories of bioagents ranging for viruses, to bacteria, to fungi, and other cellular agents.

6. Biohazards in Real World
We face biological hazards every day of our lives. Anytime that we come into close contact with people we run the risk of inhaling or touching objects that have bacteria or viral agents. Crowded situations like buses, malls, and public restrooms are places to cognizant of the agents left by other people.
Animals are often carriers of blood born pathogens, and many epidemics were passed by animals like mosquitoes and rats.

7. Resources CDC/NIH guide on Biohazards
Free PDF download at
The authoritative manual on Biosafety is put out by the CDC entitled “Biosafety in Microbiological and Biomedical Laboratories”. The book is currently on the 5th addition available for download, and 4th edition in print.
The book details standard safety practices for microbiological work and establishes guidelines for the risk level for most agents found in the laboratory. The risk level is classified from I to IV, and the safety level of the laboratory corresponds to the agent with highest risk.

8. Biosafety Resources
NIH Guidelines for Research Involving Recombinant DNA Molecules
Approval process for rDNA goes through Johns Hopkins Institutional Biosafety Committee
For work done with recombinant DNA molecules, the NIH Guidelines are the accepted practices. All guidelines listed in the NIH are accepted by Johns Hopkins as mandatory. All experiments involving rDNA must have approval of the Hopkins Institutional Biosafety Committee.

9. Resources Biological Safety at Johns Hopkins 2024 E. Monument Street
rDNA Registration
Infectious Agent Registration
Biological Toxin Registration
Human Tissue Registration
Select Agent Registration
Biological Safety Cabinet Certification and Maintenance
Laboratory Training
Emergency Response
The Biosafety office is located at the medical school on East Monument Street. The safety office handles registration of toxic biological substances, maintenance and certification of biological safety cabinets, safety training and emergency response.

10. Blood Born Pathogens
Work with human or primate blood and tissue have risk of transmitting disease
HIV, Hepatitis B,C
Free immunization available for Hep B
Free online and instructor training available to researchers
Most diseases are prevented by preventing direct access to mucous membrane or into the blood stream
Laboratory work with blood requires extra precautions, with additional risk of being infected with blood born pathogens like HIV Hep B and Hep C. There are free immunizations provided for students at risk for blood born diseases.
The Johns Hopkins Health and Safety Office offers courses on blood born pathogens online and instructor led.

11. Accident Prevention Analyze agents for potential harm
Analyze routes of exposure and determine appropriate personal protection
Follow standard microbiological safety procedures
Use engineering controls if necessary
Decontaminate and disinfect waste

12. Risk Assessment Biological agents are classified by risk group
Laboratories are classified into Biological Safety Levels on ability to contain biohazards
BSL 1 – No known human hazards
E. Coli and S. cerevisiae
BSL 2 – Moderate risk to people
Blood or body tissue
Bacteria
Human cells
BSL 3 – Severe risk to people
Tuberculosis
Anthrax
BSL 4 – Severe risk to people and greater society
Ebola
Biological agents are specified according to their hazard to people and their ability to spread. Laboratories are classified according to the agents that are specified in the laboratory, with the Biological Safety Level corresponding to the agent with highest risk. The lowest safety level of 1 corresponds to a laboratory that only has E. Coli or S. Cerevisiae present. Most microbiological laboratories at Johns Hopkins are BSL 1 or 2. There are no BSL 3 labs on homewood campus but some in the school of public health. There are no BSL 4 laboratories on campus, and most are military institutions

13. BSL 1 Laboratories Follow general microbiological laboratory practices
Personal Protective Equipment
Wear gloves when handling material
Eye glasses with splash hazard
Recommend wearing lab coat
Decontaminate surfaces
Decontaminate cultures
Wash hands after removing gloves
BSL 1 laboratories have the minimum safety standards for microbiological work. Even though the agents are not considered harmful, correct procedures should be followed. This serves the purpose of preparing the researcher for more dangerous agents, but also puts the researcher in the right mindset to get accurate results.
The minimum protective equipment for BSL 1 laboratories are gloves and protective glasses. Lab coats are not required but are recommended.
All surfaces should be decontaminated with appropriate disinfectant, and cultures should be decontaminated before disposing of them in the biomedical waste container.
Every time a researcher removes their gloves they should be in the habit of washing their hands. Even if they were not working with anything hazardous, it will be make this safety step routine.

14. BSL 2 Laboratories Everything in BSL 1 plus Limited lab access
Specific training provided by PI
Immunizations for laboratory risks
Report all exposures to hazardous materials
All splash/aerosol formation processes performed in biological safety cabinet
Gloves, eyewear, lab coat required
Lab manual outlining standard operating procedures
BSL 2 laboratories have the same requirements as BSL 1 laboratories plus additional requirements. Since there are microbiological agents that are capable of causing human harm, the access to the laboratory should be restricted to qualified personnel. Kids and other unrelated personnel should not be in the laboratory during experiments.
Researchers should be trained to work with agents and procedures that have the potential to cause harm to the researcher and other people in the laboratory. This training can be provided by reading literature, tutelage from older graduate students and post docs, or the PI themselves, but the ultimate responsibility is on the principle investigator. If the person is not suitably trained and accident occurs from negligence, the PI is held responsible.
Certain agents have identifiable risks that pre-research immunizations can help prevent. Researchers should be aware of the risks and the preventive measures before commencing research. If in the course of research, a student is exposed to an agent, they should report that to the occupational health office immediately to receive treatment. Researchers should error on the side of caution on treating possible infections.
The biological safety cabinet should be used to contain microbiological work. This is especially important if there the possibility of an inhalation risk with the formation of aerosols or if there is a procedure that has a significant splash hazard.
The personal protective equipment requirements are the same as BSL 1 except that a lab coat is required instead of being recommended.

15. BSL 3 and 4 Laboratories Respiration hazards
Requires significant training
BSL 3 and 4 are laboratories that have significant hazards and usually extreme respiratory hazards. These laboratories usually have contained breathing apparatus that prevent any contact with the air in the laboratory. The requirements for these laboratories are much more extensive, and are beyond the scope of an elementary safety discussion.

16. Routes of Entry Adsorption through the skin Splash to the eyes
Wear gloves and lab coat, closed toed shoes, no application of make up
Splash to the eyes
Wear safety glasses
Ingestion into Digestive Tract
No food or drinks, no chewing on gum or pens
Injection to the blood stream
Proper use of sharps
Inhalation
Prevent aerosols
Use Biological Safety Cabinet
Researchers should be concerned about how the microbiological agents can enter their body, and recognize how to prevent these dangers from actualizing. Direct adsorption through the skin should be prevented by wearing correct safety attire. Always wear gloves and lab coat and cover the legs and toes. Avoid touching skin by rubbing the face and wash hands whenever removing gloves.
Prevent adsorption through the mucous membrane by wearing safety glasses. Make sure the glasses have side shields that protect from splashes.
Preventing ingestion means being careful about possible entrance through the mouth. Besides the obvious no food and drink, less obvious is chewing on gum, pens, shirt collars or touching the face with contaminated gloves.
Prevention of injection is mostly concerned with proper use of sharps. When using sharps be vigilant against possible cutting skin and dropping sharps which have a tendency to land on toes. Dispose of sharps using approved sharps containers and do not overflow the container.
Prevent the formation of aerosol particles. Anything that has the risk of causing an inhalation hazard and is a dangerous agent should be handled in a biological safety cabinet.

17. Biological Safety Practices
Leave the bio in the lab
Wash hands after removing gloves and before leaving
Don’t wear lab coat and gloves outside of the lab
Researchers should contain the dangers of the laboratories to prevent them from spreading outside of the lab. After you remove your gloves make sure you wash your hands to prevent the spread of biological agents.
Do not wear your gloves outside of the laboratory. If you touched something in the laboratory, that will adhere to your gloves and every object that you touch will become infected – doorknobs, keyboards, handrails, etc… Anyone who touches these objects without gloves then is at a risk of adsorption through the skin or ingestion hazard.

18. Biological Safety Practices
Use disposable sharps
Use plastic instead of glass when possible
A major risk of biological agents is direct application to the blood is a major danger. The most common method is handling broken glass, and the easiest method is to use plastic material instead of glass whenever possible.

19. Biological Safety Practices
Reduce Aerosols
Gently expel fluids against the walls of tubes or flasks
Use contaminated container in the BSC to reduce drips to the biohazard bag.
Inhalation of biological agents is blamed for a majority of microbiological infections. The main risk is the formation of aerosol particle that are breathed in by the researcher and then get passed into the blood stream in the lungs. Laboratory exercises that produce aerosols should be conducted in biological safety hazard, and even then should be kept to a minimum.

20. Decontamination of Work Spaces
Liquid Disinfectants
70% Isopropyl alcohol or ethyl alcohol
Volatile and flammable
Acts quickly with no residue
10% bleach solution
Must be prepared daily
Effective against wide range of agents
Requires contact time to deactivate agent
Leaves residue
Disinfect work surfaces daily and after spill
Surfaces should be disinfected daily to kill all bacterial cultures. Two common disinfectants are alcohol or 10% bleach solution. Alcohol is a quick disinfectant that leaves no residue. The downside is that it is volatile and can easily ignite if near an ignition source.
10% bleach is very effective disinfectant but needs to be made fresh daily. The bleach requires adequate contact time and leaves a residue that must be cleaned with water afterwards.

21. Decontamination of Waste
Autoclave liquid waste
Solid waste can go directly into the biohazard box
Autoclave safety
Follow SOP for sterilization
Wear heat resistant gloves, lab coat, goggles
Do not autoclave chemicals
Do not overload autoclave bags
Liquid waste must be autoclaved before disposing into the red biomedical waste bags. Solid waste can be disposed of directly into the box without being autoclaved. Follow autoclave safety and cleanup to prevent accidents. Follow the correct operating instructions for the autoclave to ensure sterilization of the cultures.
The autoclave uses high pressure steam to decrease the necessary contact time. As a result the bags are hot and should be handled with care to prevent minor burns. Since the autoclave applies heat, chemicals should not be autoclaved for the possibility of causing a fire due to the high heat.

22. Aerosol formation Common operations that produce aerosols
Pipetting
Centrifuging
Grinding
Blending
Shaking
Mixing
Sonicating
Opening containers of hazardous materials
Perform aerosol forming experiments in Biological Safety Cabinet
These are examples of laboratory exercises that cause aerosols to form in microbiological laboratories. These exercises should be performed in a Biological Safety Cabinet to reduce the risk of inhalation of biological agents by the researcher and coworkers.

23. Biological Safety Cabinet
Requires yearly certification from Health Safety Environment office
$185

24. Biological Safety Cabinet
Biological Safety Cabinets operate similarly to a fume hood with some important differences. Fume hoods draw air from the room at a constant flow, and dispose the air directly into the buildings exhaust system. Fume hoods prevent the flow of material from inside of the hood both by flushing air out and not recycling the stream.
The Biological Safety Cabinet pulls air from the room but protects the sample by passing the air through a HEPA filter before cycling back into the cabinet. Part of the air that is recycle is sent back into the laboratory. The HEPA does not filter out small molecules, so there is a possibility of a build up of chemical in the laboratory with a BSC that isn’t present with a fume hood. The HEPA filter is designed to remove particle 99.97% of molecules 0.3 microns in size, and is more efficient on either side of 0.3 microns.

25. Biological Safety Cabinet
Prevent turbulent air flow within the cabinet
Keep sash pulled down
Do not block grill
Keep materials towards the back of the cabinet
Move arms slowly
Using a biological safety cabinet is similar to using a fume hood. The goal is to prevent turbulent air flow, which is bad for two reasons. In a biological safety cabinet turbulent air can bring contaminated air over a sample spoiling the sample. Also, turbulent air causes backflow into the laboratory causing potential harm to the researcher and other people in the laboratory.
It is important to keep air flow laminar into the cabinet. Do not have materials close to the sash and do not block the grills. Keep the sash pulled down as far as possible to prevent backflow of air into the room.

26. Biological Safety Cabinet Operation
Prevent Cross Contamination
The stream lines in a biological safety cabinet are not perfectly linear, but instead can be turbulent due to arm movements inside the cabinet and body movements outside. It is important to separate the areas of the cabinet so that there is no cross contamination. Keep a working area in the middle that can easily be disposed in a contaminated area. Make sure there is no cross over from the clean area to the contaminated area.
Clean Area
Working Area
Contaminated Area

27. Biological Safety Cabinet
BSC are not fume hoods
Biological Safety Cabinets should not be used as fume hoods. The air from inside the cabinet is recycled to the cabinet and the room and thus a volatile chemicals can build up inside the cabinet. If there is an ignition source this could cause an explosion in the laboratory.
This picture was taken after a flame was left on inside the cabinet. There was a build up of flammable chemical which exploded when a spark was produced.

28. Protection of Vacuum Lines
Protect the vacuum lines from aspiration flask by using filter
The researcher must protect vacuum lines. If a microbiological agent got into the vacuum line, this would infect the entire system.
Protect the vacuum line by separating the aspiration flask from the line by using a filter.

29. Work with hazardous chemicals
Biological Safety Cabinet is not suitable for work with highly flammable or toxic material
Material is recycled into cabinet and not removed by HEPA filter
Material is recycled into the room
Use chemical fume hood or specially designed BSC instead
Standard biological safety cabinets are not designed to handle toxic material. The hazardous material is recycled back into the room posing an inhalation hazard to the laboratory. The HEPA filter is designed for large molecules and does not significantly reduce the amount of small chemicals passing through.
Work should be performed in a fume hood or in a specialized Biological Safety Cabinet instead.

30. Biological Waste Solids go into biohazard box
Sharps containers go into box with lid fastened
Autoclave or chemically deactivate liquids
Researchers should correctly dispose of waste in the laboratory. Solid waste can be disposed directly into the biohazard box – material like gloves, dishes, and pipettes. Sharps need to be secured to prevent accidental injections. The sharps container needs to have a secure base and should not be overfilled. The sharps container can be placed directly into the biomedical waste box with the lid fastened.
Cell cultures must be autoclaved or chemically deactivated before disposing into the biological waste containers.

31. Cleaning up Spills Small Spills
Replace contaminated clothing and put on appropriate protection
Cover spill in adsorbent material soaked 10% freshly made bleach solution
Wait 20 minutes and then clean up
Dispose of materials into biohazard bag and wash hands
Researches should be prepared to clean up small spills without much assistance. The first step is to remove the biological agent from contaminated clothing. This reduces the contact time and minimizes the chances of adsorption. Sufficiently wash any skin area that was exposed and don correct protective attire to clean up the spill.
Contain the spill using an adsorbent material so that it doesn’t spread. 10% bleach is very effective against many microbiological materials but requires 20 minutes of contact time to effectually kill cultures. The process of applying bleach might cause the production of aerosols so it might be best to evacuate the room if the spill is large.
Dispose of the materials into the biohazard bag and wash hands to remove any agents that might have gotten onto the hands.

32. Cleaning up Spills Big Spills Alert coworkers and contain spill
If hazardous aerosols are possible evacuate room for 30 minutes
Replace contaminated PPE
Cover with adsorbent saturated with fresh 10% bleach
Wait minutes then dispose of materials in biohazard box
Change PPE and wash hands
Call Biosafety for assistance in cleaning up spill
Notify PI of spill
These are the instructions for larger spills. Large spills are defined by the researchers ability to contain and clea nup. For large spills contact the Biosafety office to have assistance on how and if the spill should be cleaned up by the researcher or by a biosafety officer. All large spills should be reported to the PI and biosafety.

33. Exposure to Biohazards
Remove contaminated clothing
Wash area with soap and water
Call (410) and tell them nature of exposure
Route of Entry
Concentration
Amount
Notify lab supervisor
Go to Occupational Health or ER after hours
If you become exposed to a biohazard immediately remove contaminated clothing. Sufficiently wash the area to minimize the effect of the exposure and have someone call biosafety immediately to inform them of the situation. Time is of the essence in these cases and medical assistance needs to be gotten immediately. Report all incidents so that sufficient follow up can be maintained.

34. Lab Coat Cleaning Decontaminate lab coat before using cleaner
Use autoclave or chemical disinfectant
Use appropriate commercial cleaning agency or contractor service
Do not clean laboratory coats in commercial cleaners or at home. Instead use a contracting service or a specialized cleaning agency.

35. Sharps Put all used sharps into approved sharps container
Do not overfill sharps container
When filled secure lid and place in biohazard box
Correct disposal of sharps is one of the most important aspects of biological safety. Accidental injections are an easy way to introduce agents into the blood stream. Use only approved sharps containers that have a metal base that prevent tipping. Containers should not overfilled past the fill line to prevent accident injections from handling the container. Once the container is filled secure the lid and place directly into the biohazard box.

36. Biosafety Conclusions
Know hazards of agents
Correctly use biosafety cabinet to reduce laboratory aerosols
Correctly use sharps
Correctly dispose of material
Correctly clean up spills
Treat and report exposures
The basics of biosafety are be prepared. Before performing any experiments know the agents that you are working with. Know the hazards associated with each agent, and the best methods for preventing those agents from getting into the blood stream.
Proper handling of sharps is the best method for preventing direct injection into the blood stream. Never handle broken glass directly, always sweep up pieces without touching.
Inhalation is the most common risk of exposure, and the production of aerosols should be minimized. If aerosols are inevitable, use a biological safety cabinet to reduce the researchers exposure to inhalation hazards.
Plan ahead for possible accidents. Know how to handle common accidents and spills. Do a risk assessment and prepare for accidental exposures. Know how to handle waste and the proper methods to dispose of it.