1. Introduction to Biohazards: Risk Management
In this lecture we will begin to discuss biosafety. We will discuss what a biohazard is and mention several types of biohazards. We will also cover some general guidelines for safely working with biohazards. Working safely while performing experiments involving biohazards requires merging the safety practices required when working with hazardous chemicals with some new practices that are tailored to the special risks associated with biological hazards. As always, safe laboratory practice will require knowledge of the risks associated with hazards and a plan to manage those risks.

2. Biohazards
Biological agents with the potential to produce harmful effects in humans
A biohazard is a biological agent with the potential to cause harm in humans. There are numerous different classes of biohazards used in the biotechnology laboratory. Such biohazards include bacteria, viruses, fungi and even smaller particles such as prions, which are biohazardous forms of naturally occurring proteins. Within each of these classes are varying degrees of risk, and it is your job to learn how to manage those risks to protect your safety and that of your laboratory co-workers. On the next few slides we will discuss a few terms that are necessary to understand in our discussion of biohazard risk management.
H1N1 flu virus

3. Pathogenicity
The relative ability of an organism to cause disease in humans or other organisms
A pathogenic agent must also be infectious
Those organisms that invade and cause a SPECIFIC host disease are called etiological agents
Pathogenicity refers to the relative ability of an organism to cause disease in humans or other organisms. Pathogens include bacteria, fungi, viruses and others biological entities. To cause harm a pathogenic agent must also be infectious. To be infectious, an entity must be able to invade a host. Those organisms that invade and cause a SPECIFIC host disease are called etiological agents. The image on this slide is of Vibrio cholerae , the etiological agent that causes cholera.
Vibrio cholerae

4. Carriers
An agent infects a host but does not cause disease in that host
If an agent infects a host but does not cause disease in that host, the host is a carrier. A carrier is an infected individual capable of spreading infectious agents to others. Carriers, because they are asymptomatic, often play important roles in the spread of disease through a population. Mary Mallon is perhaps the most famous carrier known in this country. Known as Typhoid Mary, she worked as a cook in early 1900s New York. She is believed to have infected dozens of individuals, some of whom died, with typhoid fever by spreading the bacteria through the food she prepared. She eventually died after spending decades in quarantine in a facility located on an island in the East River in New York City. An autopsy showed that she did indeed harbor live, infectious bacteria, however her lack of symptoms made it difficult for her to believe that she remained infectious throughout her life.

5. Routes of infection Inhalation Skin/eye contact Ingestion Injection
Mold spores
So how do biohazards exert their effects? We have said that pathogens are, by nature, infectious, meaning that they are able to invade a host. This invasion can occur through a number of routes. First, the infectious agents may enter the body through the respiratory system, via the process of inhalation, as is common with cold and flu viruses. Second, the agent may be introduced via the skin through contact with eyes and other mucous membranes, as is seen when pink eye, or bacterial conjuctivitis is spread. Third, the agent may enter through the mouth and the digestive system, as occurs with food-borne illnesses such as salmonella, and water-borne diseases like cholera. Finally, the agent may penetrate the body through injection. Common forms of injection include insect bites and stings. For instance, malaria is spread through the bites of mosquitoes harboring the contagion. These routes of infection are common to the illnesses that spread through the population at any point in time, such as during influenza season. However, the same routes of entry are employed by biohazardous agents used in the laboratory. In the laboratory, the most likely route of infection is through the inhalation of bioaerosols, which are sprays of small infectious particles suspended in air. In fact, 70% of all laboratory-acquired infections are the result of bioaerosols.
Many of the safety guidelines mentioned for chemical handling apply to biohazard work. But there are some special considerations when working with biohazards:

6. Special considerations when working with biohazards
Pathogenic
Associated risks
Laboratory-acquired infections
Treatments
Allergies
Imagine that you accept a position in a company that is working on a vaccine for the virus that causes Hepatitis C, one of the most common blood-borne agents in the US. You recognize that this is a biohazard, but there are a number of questions that you should ask prior to beginning work with this or any biohazard.
First, ask whether this is a know human/ primate pathogen. If you know that a particular biohazard has been shown to cause biological harm in humans or other primates, then you know that you will need to take particular care. Of course, you will then need to know what risks are associated with this hazard. For instance, Hepatitis C can cause cirrhosis of the liver. Since your liver carries out essential functions, then you will know that infection with this virus could prove fatal. Other biohazards may have considerably less serious risks associated with infection. Once you have determined what the potential outcomes of infection may be, you should consider whether this biohazard been associated with lab-acquired infections, and if so, with what health consequences. Considering that infection with the biohazard is a possibility, you should investigate whether there is either a treatment for the disease, or if, in fact, a vaccine is available that would prevent you from becoming infected in the first place. Of course, if there is a vaccine, that does not mean that the organism is not longer a serious hazard. Remember that vaccines are not effective all of the time! For instance, the virus that causes rabies can be prevented via vaccination; however, since rabies is a nearly invariably fatal illness if contracted, you would not want to handle the rabies virus in a careless way, even though you would, presumably have been vaccinated. Next, question whether any allergies are induced by this agent. For instance, various molds are known to be very allergenic.
Hepatitis C virus

7. Biohazard considerations (con.)
Infectiousness
Limiting exposure
Special safety precautions
Is the level of risk of working with this agent acceptable to the worker?
Another important consideration is the infectiousness of a biohazard. The infectiousness of biohazards varies greatly. For instance, were you to accidentally stick yourself with a needle contaminated with Hepatitis B virus, you would be ten times as likely to become infected than if the needle were contaminated with HIV. Be sure to question about the infectiousness of the biohazard of interest. Next, consider if there are ways to limit your exposure to the biohazard. Will work occur in a biological safety cabinet, can you avoid using needles, and thereby reduce the likelihood of injection of the biohazard? Then, find out if you will need to take special precautions with this particular biohazard. You may need to work in a glove box, or wear a respirator for instance. You may also be required to receive special training prior to beginning work.
Once you have gathered all of the information that you can regarding the particular biohazard that you are planning to work with, you will need to decide if the level of risk is acceptable to you, the laboratory worker. If you feel the risk level is unacceptable, you may continue to look for ways to reduce the risk, or investigate whether a less hazardous organism may be used. It is possible, also, that you will decide that you simply do not want to take this job!
Polio virus

8. Laboratory-Acquired Infections (LAI)
Infections that can be traced directly to lab organisms handled by or used in the vicinity of the infected individuals
Thousands documented
Hundreds of deaths
So what are laboratory-acquired infections? They are the event that biosafety programs are designed to prevent: infections that are the direct result of handling biohazardous agents in the laboratory. Thousands of laboratory acquired infections have been documented, resulting in hundreds of deaths. 1/4 of these cases were found in non-researchers such as dishwashers, custodians, office staff. It is easy to forget that your actions in the laboratory affect your co-workers, and even easier to forget how you may affect those workers that you rarely see. If biohazards are not managed correctly, then the biohazard can be spread throughout the laboratory and then, eventually, even outside of the laboratory. The exposure of the public to biohazardous agents must absolutely by avoided.

9. Biosafety Guidelines Centers for Disease Control and Prevention (CDC)
National Institutes of Health (NIH)
Others
As we saw when we discusses the hierarchy of responsibility for safe handling of hazardous chemicals, when we consider safe handling of biohazards we see that governmental organizations sit at the top of the hierarchy and create rules and standards for working safely with biohazards. The two most prominent governmental agencies involved are the Centers for Disease Control and Prevention (the CDC) and the National Institutes of Health (NIH). Other organizations also publish standards for workers to use to reduce risk associated with the use of biohazards in the laboratory.

10. Standard Microbiological Practices
Practices to be used when working with ALL microbiological organisms
Protect worker
Protect culture
We have discussed risk assessment when working with biohazards. Let’s now turn our discussion to the details of performing safe laboratory work with biohazards. There exists a set of guidelines that should be used whenever working with any biological organisms in the laboratory. These practices are called Standard Microbiological Practices and have two functions. The most obvious function is to protect the worker from infection by pathogens. Less obvious is the need to protect the organism from contamination with any other organisms that are harbored by the laboratory worker. In essence, Standard Microbiological Practices are designed to protect you from your laboratory culture and also to protect your laboratory culture from you!

11. Standard Rules for Working with Microbes
Only trained individuals may enter lab
Always wear lab coats and safety glasses
Wash hands after working with organisms and before leaving lab
No eating, drinking, smoking
Avoid hand to mouth or eye contact
No mouth pipetting
Minimize aerosol production
Work on clean hard benchtop and ALWAYS keep disinfectant handy
Decontaminate workspace and waste before disposing
Whenever working with biological organisms you should follow the rules shown on this slide. You can think of these rules as being designed to prevent the biological organism from gaining access to your body through any of the the four routes available: inhalation, skin/eye contact, ingestion or injection. Simple rules of good laboratory hygiene will go a long way towards preventing any contamination. And, in case any contamination has occurred, it is critical to decontaminate workspaces after each laboratory session, and to always wash hands thoroughly with soap and water prior to leaving the lab! Take a few minutes to read these rules and try to make them part of your daily laboratory practice.

12. Types of Containment
Control of biohazards by isolation and separation of organism from worker
Primary containment
Personal containment
Physical containment
Seconary containment
The harmful effects of biological hazards can be controlled using containment. Containment is the separation of the worker from the biohazard in one of several ways. This separation controls the risks associated with the particular biohazard. Primary containment includes equipment and practices that protect workers from exposure to biohazards in the lab. Secondary containment includes equipment and practices that protect the general environment outside of the laboratory from harm. Primary and secondary containment include examples of both personal containment, which includes worker practices designed to prevent spread of the biohazard, and physical containment, which includes equipment and facilities designed to contain the organism. Therefore, personal containment would include such examples as following the Standard Microbiological Practices, while physical containment would include biological safety cabinets and personal protective equipment.

13. Biosafety Levels: BSL-1
Well characterized strains not causing disease in healthy humans
Standard Microbiological Practices
As we discussed earlier, different biohazards have different associated risks. In order to characterize biohazards into groups based on the level of risk associated, the CDC created four biological safety levels. These safety levels reflect the level of risk, but also determine the types of facilities and equipment that a worker must use to work with these biohazards.
The organisms that are classified as BSL-1, or Biosafety Level 1 are all organisms that are well characterized and do not cause disease in healthy adults. Often these organisms are non-pathogenic and require no special equipment or precautions beyond the Standard Microbiological Practices that should be employed for all biological organisms. Included in BSL-1 are non-pathogenic strains of E. coli and yeasts, and most plants. BSL-1 organisms are commonly used in school settings, but are also of use in the biotechnology industry.

14. Biosafety Levels: BSL-2
Agents may cause disease
Usually treatable or preventable
Need autoclave, special disposal requirements
If aerosols produced, need biological safety cabinet (Class 1 or II)
Organisms with which more risk is associated may be classified as Biosafety Level 2. These organisms may cause disease in humans, but infection by the biological agent is usually either treatable, or preventable. Additionally, these organisms are generally not likely to spread outside of the lab, due to perhaps, lack of host, or widespread vaccination against the agent in the general population. BSL2 facilities require special disposal practices, and must have an autoclave. Additionally, any work that might generate bioaerosols (which are easily generated when working with biological organisms) must be performed in either a Class I or Class II biological safety hood.
BSL2 organisms include pathogenic strains of Salmonella and Clostridium, as well as the viruses that cause rabies and polio. While it may seem odd that the rabies and polio viruses do not rate a higher biological safety level, it is important to note that rabies and polio vaccines exist, so laboratory acquired infections can be prevented. Also, in the case of the polio virus, the general population is widely vaccinated against this agent.

15. Biosafety Levels: BSL-3
Severe or lethal human disease
Blood testing for workers
Entry to lab controlled
Physical containment for all manipulations. Extra protective gear (respirators)
No air recirculation, negative airflow
More hazardous biological organisms may receive a classification of Biosafety Level 3. BSL3 agents cause severe or lethal infection and are highly infectious. BSL3 laboratories are designed to have controlled access, negative airflow, and all manipulations must be carried out in a biological safety cabinet. Additional personal protective equipment, such as respirators, may be required. Additionally, all workers undergo blood testing to check for exposure. Generally, these organisms pose a high risk to the laboratory worker, but a relatively low risk to the community. Agents include Yersinia pestis, the causative agent of the plague, HIV, and the causative agent of tuberculosis.

16. Biosafety Levels: BSL-4
High risk of lethality, airborne or unknown agent
Clothing change, and shower upon exit
All work done in Class III BSC or Class I or II with full body, air –supplied suit
Must be done in separate building or zone with own contained air system
Those organisms that pose the highest risk level to both the laboratory worker and the general public are classified BSL4. BSL4 organisms are handled in glove boxes, or in biological safety cabinets by workers clothed in full body, air-supplied suits. BSL4 facilities are isolated from other facilities and must have their own dedicated air supply and exhaust. Workers change and decontaminate clothing upon exit. Rigorous training is required of those workers who operate in BSL4 facilities. As of 2010, only 13 operating or planned BSL4 facilities existed in the United States.
BSL4 organisms include the Ebola, Lassa and Marburg viruses.

17. Biological Safety Cabinets
Provides containment for aerosols and separates work area from operator and lab while providing clean air
Able to filter most particles (including most viruses) from air
Does not remove chemical vapors
When working with hazardous chemicals, especially those that are volatile, it is often necessary to use a fume hood. Likewise, technicians working with biohazards will often need to work in a type of hood called a biological safety cabinet. We will discuss biological safety cabinets briefly here. It is important to remember two things about biological safety cabinets. First, there are multiple types of biological safety cabinets, and you will need to know the specific type required by the biohazard with which you are working. Second, biological safety cabinets are not fume hoods and are not designed to remove chemical vapors.
In fact, biological safety cabinets are a form of physical containment designed to separate the worker from the biohazard. The organism is exposed to a flow of sterile air, which protects the organism from the worker and environment. The air flow is into the hood, and exhausted air is sterile-filtered to protect the worker and the environment from the biohazard. Biological safety cabinets can filter most particles, including most viruses, from the air.

18. Classes of BSC’s
Class I and Class II cabinets draw air from room and work something like fume hoods
Class III are glove boxes
Class I and Class II cabinets draw air from the room into the hood and look like fume hoods. Class III cabinets are glove boxes, which provide total containment. These glove boxes are fitted with air-tight gloves and have attached autoclaves, incubators and air locks. Many biological safety cabinets are also fitted with germicidal UV lamps which are turned on after work is complete and workers have left the room. These UV lamps decontaminate all work surfaces.
Regardless of the work that you are doing with biohazards, be sure that you are aware of the risks associated with the organism that you are using and that you receive appropriate training to work safely in the lab.