BIOSAFETY

PROTOCOL

For the NMC Biology Department

Developed by:
K. E. Waterstripe
Science Lab Manager

Revised December 7, 2007

Table of Contents

I. PURPOSE OF THE BIOSAFETY PROTOCOL ……..………………………….. 3

II. STANDARD OPERATING PROCEDURES IN 
	MICROBIOLOIGAL LABS …………………………………………………..5

A. Procedures to be followed whenever lab work is done with
	 ANY organism, whether BSL-1 or BSL-2 …………………………...….5
B.  Special practices, safety equipment and facilities that apply 
	to organisms assigned to Biosafety Level 1 ……………………….……6
C.	Special practices, safety equipment, and facilities that apply to 
	organisms assigned to Biosafety Level 2 …………….…………………7

III. WORKING WITH GENETICALLY MODIFIED ORGANISMS (GMOs) …..8

IV. CLEANING UP SPILLS AND ACCIDENTAL EXPOSURE..............................8

IV. TRAINING …………………………………………………………………………9

V. MEDICAL WASTE MANAGEMENT…………………………………………….10

Figure 1: Biological & Medical Waste Decision Tree …………………………...…...12

APPENDIX A: BACTERIAL AND FUNGAL SPECIES 
	USED IN NMC BIOLOGY LABS ……………………………………………13

APPENDIX B:  CLASSIFICATION OF HUMAN ETIOLOGIC 
	AGENTS ON THE BASIS OF HAZARD ……………………………………14

I. PURPOSE OF THE BIOSAFETY PROTOCOL
This document established protocols and practices for minimizing exposure to biohazardous materials in the NMC Science labs.  By following these guidelines, employees and students can reduce the risk of accidents and exposure to potentially harmful organisms used in the labs.  
This Protocol specifies procedures for preventing accidental releases of genetically modified organisms into the environment.  It also works in conjunction with NMC’s Exposure Control Plan, in accordance with the OSHA Bloodborne Pathogen Standard, and the MIOSHA Bloodborne Infectious Diseases Regulations.  In addition, section III satisfies the Medical Waste Management Plan requirements of Part 138 of Michigan’s Medical Waste Regulatory Act (No. 368).

A. Definition of Biohazardous Materials

For the purposes of this policy, a biohazardous material is any biological material capable of causing harm to humans, animals or plants, including both biohazardous agents and non-replicating materials such as toxins. In addition, this term may also be used to refer to material that harbors a biohazardous agent. A biohazardous agent is a disease-causing microorganism (bacteria, chlamydia, fungi, parasites, prions, rickettsiae, viruses, etc.) capable of replication, and capable of causing diseases in humans, animals, or plants. Toxic, mutagenic, and teratogenic chemicals are not considered biohazards.

B. Classification of Biohazardous Microorganisms

Traditionally, microorganisms have been classified according to the biological containment they require. Four levels of containment have been defined and termed Biosafety Levels (BSL 1-4).  For example an organism requiring Biosafety Level 2 containment is termed a BL2 organism. Characteristics of organisms at the four biosafety levels are described in Table 1.  Biosafety levels for the microorganisms used in NMC science labs are listed in Appendix A.

Table 1:  Characteristics of infections agents at Biosafety Levels 1 - 4
BSL	Agents	
1 	Not known to consistently cause disease in healthy adults, and of minimal potential hazard to laboratory personnel and the environment.	
2	Associated with human disease, hazard is from percutaneous injury, ingestion, mucous membrane exposure	
3	Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences	
4	Dangerous/exotic agents which pose high risk of life-threatening disease, aerosol-transmitted lab infections; or related agents with unknown risk of transmission 	
	From CDC Office of Health and Safety

Biosafety Level 1(BSL-1) containment is suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. The laboratory is not necessarily separated from the general traffic patterns in the building. Work is generally conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design is neither required nor generally used. Laboratory personnel have specific training in the procedures conducted in the laboratory and are supervised by a scientist with general training in microbiology or a related science. 

Biosafety Level 2 containment is similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It differs from BSL-1 in that (1) laboratory personnel have specific training in handling pathogenic agents and are directed by competent scientists; (2) access to the laboratory is limited when work is being conducted; (3) extreme precautions are taken with contaminated sharp items; and (4) certain procedures in which infectious aerosols or splashes may be created are conducted in biological safety cabinets or other physical containment equipment.

Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents which may cause serious or potentially lethal disease as a result of exposure by the inhalation route. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents, and are supervised by competent scientists who are experienced in working with these agents.

Biosafety Level 4 is required for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections and life-threatening disease. Members of the laboratory staff have specific and thorough training in handling extremely hazardous infectious agents and they understand the primary and secondary containment functions of the standard and special practices, the containment equipment, and the laboratory design characteristics.  Access to the laboratory is strictly controlled by the laboratory director. The facility is either in a separate building or in a controlled area within a building, which is completely isolated from all other areas of the building. 

NORTHWESTERN MICHIGAN COLLEGE IS NOT EQUIPPED TO HANDLE BSL-3 AND BSL-4 INFECTIOUS AGENTS.  THESE AGENTS ARE NOT USED IN OUR LAB PROCEDURES.

More recently, the National Institutes of Health (NIH) has introduced the concept of “risk groups” in which agents are classified into four Risk Groups (RGs) based on their relative pathogenicity for healthy adult humans. Table 2 lists the four groups and the basis for classification.  A more detailed list of microorganisms in Risk Groups 1 & 2 is provided in Appendix B.

Table 2:  Risk Group system of classification of infectious agents
Risk Group 1 (RG1) 	Agents that are not associated with disease in healthy adult 
humans 	
Risk Group 2 (RG2) 	Agents that are associated with human disease which is 
rarely serious and for which preventive or therapeutic 
interventions are often available 	
Risk Group 3 (RG3) 	Agents that are associated with serious or lethal human 
disease for which preventive or therapeutic interventions 
may be available (high individual risk but low community 
risk) 	
Risk Group 4 (RG4) 	Agents that are likely to cause serious or lethal human 
disease for which preventive or therapeutic interventions 
are not usually available (high individual risk and high 
community risk) 	

II. STANDARD OPERATING PROCEDURES IN MICROBIOLOGICAL LABS

A.	These procedures must be followed whenever lab work is done with ANY organism, whether BSL-1 or BSL-2:
1.	Biosafety procedures are incorporated into standard operating procedures or in a biosafety manual (this document) adopted or prepared specifically for the laboratory by the Lab Manager and department instructors.. Instructors and students are advised of special hazards (i.e. BSL-2 organisms) and are required to read and follow instructions on practices and procedures.
2.	Persons wash their hands before working in the lab, after they handle viable materials, after removing gloves, and before leaving the laboratory.
3.	Eating, drinking, smoking, applying cosmetics or lip balm, and handling contact lenses in the laboratory.
4.	Work surfaces are decontaminated after each lab, and after any spill of viable material. A disinfectant solution, or 2% bleach (sodium hypochlorite) must be used to decontaminate equipment and work surfaces. 
5.	Minimize the use and exposure to sharps in the workplace. Never recap, bend, or shear needles. When possible, replace glassware with less damaging materials such as plastic. Keep sharps containers readily available in all locations where sharps waste may be generated. 
6.	Use a bulb or other pipetting device.  Mouth pipetting may lead to accidental ingestion of biological specimens and is strictly prohibited.
7.	Any procedures that could potentially generate aerosols or other inhalation hazards must be performed in a manner that will minimize airborne pathogen transmission. 
8.	Whenever an incubator is in use, a sign incorporating the universal biohazard label must be posted prominently on the outside of the unit.
9.	Always wear heat-resistant gloves and a laboratory coat when opening an autoclave. Be sure to allow the superheated steam to exit before attempting to remove the contents. 
10.	All infectious materials transported to and from the laboratory must be enclosed in a primary container with sealed lid or top, which must then be enclosed in a secondary leak-proof, non-breakable container (e.g., Coleman cooler) appropriately labeled with the biohazard symbol. Any specimens transported to and from off-campus satellite facilities must be handled by trained personnel. 
11.	All infectious materials to be stored must be clearly labeled with the universal biohazard symbol. The storage space (e.g., freezer, refrigerator) must also be similarly labeled. 
12.	Glass microscope slides that have been exposed to microorganisms will be placed in a disinfectant solution for at least 30 minutes, and then transferred to a container dedicated to broken glass.
13.	Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. 
14.	Spills and accidents that result in overt exposures to infectious materials are immediately reported to the Lab Manager.
15.	All cultures, stocks, and other regulated wastes are disposed of in the manner described in sec. V, Medical Waste Management.

B.  The following special practices, safety equipment and facilities apply to agents assigned to Biosafety Level 1: 
1. Safety Equipment (Primary Barriers) 
a.	Special containment devices or equipment, such as a biological safety cabinet, are generally not required for manipulations of agents assigned to Biosafety Level 1. 
b.	It is recommended that laboratory coats, gowns, or uniforms be worn to prevent contamination or soiling of street clothes.
c.	Gloves should be worn if the skin on the hands is broken or if a rash is present. Alternatives to powdered latex gloves should be available. 
d.	Protective eyewear should be worn for conduct of procedures (vacuum filtration, centrifugation) in which splashes of microorganisms or other hazardous materials are anticipated.
2. Laboratory Facilities (Secondary Barriers) 
a.	Laboratories should have doors for access control. 
b.	Each laboratory contains a sink for handwashing. 
c.	An eyewash station is readily available. 
d.	The laboratory is designed so that it can be easily cleaned. Carpets and rugs in laboratories are not appropriate. 

C.  The following standard and special practices, safety equipment, and facilities apply to agents assigned to Biosafety Level 2:
1. Special Practices
a.	Access to the laboratory is limited or restricted by the laboratory director when work with infectious agents is in progress. In general, persons who are immunocompromised or immunosuppressed may be at increased risk of acquiring infections. The Lab Manager and Science Dept. Chair have the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.
b.	A biohazard sign must be posted on the entrance to the laboratory when infectious agents are in use. 
c.	A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels.
o	Syringes which re-sheathe the needle, needle-less systems, and other safety devices are used when appropriate.
o	Containers of contaminated needles, sharp equipment, and broken glass are decontaminated before disposal, according to any local, state, or federal regulations. (See Infectious Waste Management section of this document.)
d.	Cultures, tissues, specimens of body fluids, or potentially infectious wastes are placed in a container with a cover that prevents leakage during collection, handling, processing, storage, transport, or shipping.
e.	Laboratory equipment and work surfaces should be decontaminated with an effective disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination by infectious materials. 
2. Safety Equipment (Primary Barriers) 
a.	Goggles must be used for procedures which may result in splashes or sprays of infectious or other hazardous materials to the face.
b.	Protective laboratory coats or smocks must be worn while in the laboratory. This protective clothing is removed and left in the laboratory before leaving for non-laboratory areas (e.g., cafeteria, library, administrative offices). All protective clothing is either disposed of in the laboratory or laundered by the institution; it should never be taken home by personnel.
c.	Gloves are worn when hands may contact potentially infectious materials, contaminated surfaces or equipment. Wearing two pairs of gloves may be appropriate. Gloves are disposed of when overtly contaminated, and removed when work with infectious materials is completed or when the integrity of the glove is compromised. Disposable gloves are not washed, reused, or used for touching "clean" surfaces (keyboards, telephones, etc.), and they should not be worn outside the lab. Alternatives to powdered latex gloves should be available. Hands are washed following removal of gloves.

3. Laboratory Facilities (Secondary Barriers) – same as those for BSL-1 agents.  In addition, an autoclave is readily available.

III. WORKING WITH GENETICALLY MODIFIED ORGANISMS (GMOs)

Containment must be the highest priority in procedures involving organisms that have been genetically modified, especially those modified with antibiotic resistance genes.  In most cases, the organism being modified will belong to BSL-1, and will not pose a health risk.

The following practices will be followed in any labs involving GMOs:
1.	Take special care to adhere to the Standard Operating Procedures listed above.
2.	Use disposable Petri dishes, culture tubes, and inoculating loops when possible.
3.	Wear disposable gloves, and place them in a biohazard bag after use.  Wash hands thoroughly before leaving the lab.
4.	Autoclave all materials after use.
5.	If culture plates are transported outside the lab, seal them with Parafilm.
6.	Autoclave cultures of GMOs as soon as possible after use. 

IV. CLEANING UP SPILLS AND ACCIDENTAL EXPOSURE
A.	Spills
1.	Wear protective equipment: gloves, goggles (for BSL-2), long-sleeved lab coat.
2.	Disinfect the area for 15 minutes then wipe up the spill with disposable paper towels.  Always clean spills from the periphery of the spill towards the center.  Dispose of all spill materials in an autoclaveable bag. 
3.	Broken glass should be handled only by remote means such as tongs or forceps.  
4.	Notify the Lab Manager of the spill.

B.	Accidental exposure 
All injuries and accidental autoinoculation, ingestion or inhalations of infectious agents must be reported to the instructor and to the lab director. A record of the incident must be kept by the lab manager.  Affected employees should be sent to their primary care physician for evaluation, possible treatment and/or possible referral. 

C.	Other Emergencies 
In case of natural disasters, fires, or power failure the following precautions must be taken: 
1.	In power failures, immediately discontinue all work until power is restored. If a tissue culture hood is being used, then all open containers must be closed, gas turned off and hood sash closed. 
2.	In case of fire, personnel must immediately follow standard emergency procedures (evacuate area, call 9-911). Temperatures sufficient to ignite materials will inactivate infectious agents used in the laboratory. 
3.	Emergency personnel should be notified of the presence of potentially infectious microorganisms.
4.	In natural disasters, personnel must immediately follow standard emergency procedures (take shelter, wait for further instructions). Upon return to facility, personal protective equipment (PPE) must be used when entering a lab to decontaminate any disaster-related release of infectious material. Contain released material using spill procedures. 

V.  WORKING WITH BLOOD AND BODY FLUIDS
1.	All persons shall wear gloves while working with blood or body fluids.
2.	Whenever possible, persons should only handle their own blood. 
3.	A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels.  Syringes and lancets which re-sheathe the needle, needle-less systems, and other safety devices are used when appropriate.
4.	Blood and body fluids should be replaced with synthetic materials whenever such substitutions do not compromise the educational value of the exercise.
5.	“Sharps” contaminated with blood are placed in a “sharps” container after use.
6.	All persons should wash their hands thoroughly before leaving the lab.

VI. TRAINING
Biosafety training shall be provided by the Lab Manager and will include generalized training and specialized training for specific hazards present in that laboratory. This training will be required for Biology Dept. instructors and Student Lab Assistants, and will be updated annually. 
The training agenda will include:
1.	What is a biohazard
2.	Biosafety Levels 1 & 2 – what’s included
3.	standard microbiological practices for BSL-1
4.	special procedures for BSL-2
5.	safety equipment and lab facilities for handling BSL-2 organisms
6.	“sharps” handling
7.	biohazard spill cleanup
8.	medical / bio waste disposal practices 

Names of those attending the training will be retained by the Lab Manager as record of appropriate laboratory worker training. 

Instructors in teaching laboratories should provide students with specific training for the hazards expected to be encountered in the laboratory procedures utilized. Because of the large number of students involved, it is recommended that instructors develop a training method or form emphasizing the particular elements required for their courses. Both the instructor and student should sign a safety contract.  In courses that conduct only a few labs involving biohazardous materials, the Lab Manager will provide a Hazard Information Notice to the instructor and the instructor will convey this information to the students.
V. MEDICAL WASTE MANAGEMENT 

All disposal of infectious waste, autoclave bags, pipettes, sharps and biological waste must be performed in accordance with Michigan’s medical waste regulations.  The following information and procedures comply with Northwestern Michigan College’s Medical Waste Management Plan, as required by the Medical Waste Regulatory Act of 1978, Part 138 of Act. No. 368.

A.	Types of medical waste generated by NMC Biology Department:
1.	Cultures of infectious agents and associated laboratory waste, discarded culture dishes and related devices
2.	“Sharps”: needles, syringes, scalpel blades, lancets
3.	Liquid human and animal blood, and laboratory devices contaminated with blood.  

B.	Handling procedures:
1.	Infectious organisms
a.	Cultures of infections organisms, along with associated dishes and devices are collected in an autoclave-safe bag after being used in the laboratory.  Bags are labeled with the biohazard symbol.  Such wastes are segregated from non-contaminated wastes. 
b.	Containers of contaminated materials and wastes are placed in a designated area on a lab bench near the autoclave, and sterilized at the first opportunity. 
c.	All organism cultures and blood samples are autoclaved before disposal.  After sterilization, contents of biohazard bags are emptied into trash containers.  Note: red autoclave bags are placed in the Biohazard bin, NOT in the general trash containers. Non-disposable culture tubes are washed.
d.	Non-disposable labware that is not autoclaveable is soaked in disinfectant or household bleach for at least 30 minutes.  Labware is then washed.
e.	Disposable glass slides and broken glass are soaked in disinfectant or household bleach for at least 30 minutes, then transferred to a container for broken glass.

2.	“Sharps”
a.	collected in designated labeled containers manufactured for that purpose. 
b.	Sharps containers are placed in a Biohazard bin when full.  The bin is located in HS 115A. 

3.	Blood and related devices
a.	All materials contaminated with blood are collected in an autoclave-safe bag displaying the biohazard symbol.  Such wastes are segregated from non-contaminated wastes and placed in a Biohazard bin.
b.	Amounts of blood that can be cleaned up with a paper towel may be disposed of in the regular trash container.

C.	Medical wastes are collected periodically and transported by:  Stericycle, Inc., 2695 Elmridge Drive NW, Walker, MI,  49544

D.	The Biosafety Protocol establishes Standard Operating Procedures for all lab activities involving potentially infectious organisms, genetically modified organisms, and “sharps”.  Please refer to that document for all matters pertaining to procedures, training, protection, containment, and disposal.

Figure 1: Biological & Medical Waste Decision Tree

Is it Sharp?

(needles, razor blades, scalpels)

Treat

Is it Infectious?

No

No

Yes

Don’t Know

Yes

Don’t Know

Yes

No

Dumpster

(No un-treated redbag waste may be put in the dumpster)

Can you autoclave or chemically disinfect?

Sharps container

Picked up by
Stericycle Inc.
or call:

    	Kirk Waterstripe	5-2417

APPENDIX A: BACTERIAL AND FUNGAL SPECIES USED IN NMC BIOLOGY LABS
BACTERIA	Biosafety Level	
Alcaligenes faecalis		
Bacillus cereus 	1	
B. stearothermophilus	1	
Bacillus subtilis	1	
Clostridium butyricum	1	
Clostridium sporogenes	1	
Cornybacterium xerosis	1	
Escherichia coli (no K1 antigen)	1	
Enterobacter aerogenes	1	
Enterococcus faecalis	1	
Klebsiella pneumoniae	2	
Micrococcus luteus	1	
Mycobacterium smegmatis	2	
Proteus vulgaris	1	
Pseudomonas aeruginosa	1	
Pseudomonas fluorescens	1	
Salmonella typhimurium	2	
Serratia marsescens	1	
Staphylococcus aureus	2	
Staphylococcus epidermidis	1	
Streptococcus pyrogenes	2	
FUNGI		
Aspergillus niger 	NA	
Penicillium notatum 	NA	
Rhizopus stolonifer	NA	
Saccharomyces cerevisiae 	NA	
Sordaria fimicola	NA	

APPENDIX B:  CLASSIFICATION OF HUMAN ETIOLOGIC AGENTS ON THE BASIS OF HAZARD 
This appendix includes those biological agents known to infect humans as well as selected animal agents that may pose theoretical risks if inoculated into humans. Included are lists of representative genera and species known to be pathogenic. Mutated, recombined, and non-pathogenic species and strains are not considered. Non-infectious life cycle stages of parasites are excluded. 
This appendix reflects the current state of knowledge and should be considered a resource document. The list includes the more commonly encountered agents and is not meant to be all-inclusive. Information on agent risk assessment may be found in the Agent Summary Statements of the CDC/NIH Biosafety in the Microbiological and Biomedical Laboratories. Further guidance on agents not listed in this Appendix may be obtained through: Centers for Disease Control and Prevention, Biosafety Branch, Atlanta,Georgia 30333, Phone: (404) 639-3883, Fax: (404) 639-2294; National Institutes of Health, Division of Safety, Bethesda, Maryland 20892, Phone: (301) 496-1357; National Animal Disease Center, U.S. Department of Agriculture, Ames, 
Iowa 50010, Phone: (515) 862-8258. 
Risk Group 1 (RG1) Agents 
RG1 agents are not associated with disease in healthy adult humans. Examples of RG1 agents include asporogenic Bacillus subtilis or Bacillus licheniformis. 
Those agents not listed in Risk Groups (RGs) 2, 3 and 4 are not automatically or implicitly classified in RG1; a risk assessment must be conducted based on the known and potential properties of the agents and their relationship to agents that are listed. 
Risk Group 2 (RG2) Agents 
RG2 agents are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available. 
Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia 
Acinetobacter baumannii (formerly Acinetobacter calcoaceticus) 
Actinobacillus 
Actinomyces pyogenes (formerly Corynebacterium pyogenes) 
Aeromonas hydrophila 
Amycolata autotrophica 
Archanobacterium haemolyticum (formerly Corynebacterium haemolyticum) 
Arizona hinshawii - all serotypes 
Bacillus anthracis 
Bartonella henselae, B. quintana,B. vinsonii 
Bordetella including B. pertussis 
Borrelia recurrentis, B. burgdorferi 
Burkholderia (formerly Pseudomonas species) except those listed in RG3 
Campylobacter coli, C. fetus, C. jejuni 
Chlamydia psittaci, C.trachomatis, C. pneumoniae 
Clostridium botulinum, Cl.chauvoei , Cl. haemolyticum , Cl. histolyticum, Cl. novyi. Cl. septicum, Cl. tetani 
Corynebacterium diphtheriae, C. pseudotuberculosis, C. renale 
Dermatophilus congolensis 
Edwardsiella tarda 
Erysipelothrix rhusiopathiae 
Escherichia coli - all enteropathogenic, enterotoxigenic, enteroinvasive and strains 
bearing K1 antigen, including E. coli O157:H7 
Haemophilus ducreyi, H.influenzae 
Helicobacter pylori 
Klebsiella - all species except K. oxytoca (RG1) 
Legionella including L. pneumophila 
Leptospira interrogans - all serotypes 
Listeria 
Moraxella 
Mycobacterium (except those listed in RG3) including M. avium complex, M. asiaticum, 
M. bovis BCG vaccine strain, M. chelonei, M. fortuitum, M. kansasii, M. leprae, M. malmoense, M. marinum, M. paratuberculosis, M. scrofulaceum, M. simiae, M. szulgai, M. ulcerans, M. xenopi 
Mycoplasma, except M. mycoides and M. agalactiae which are restricted animal pathogens 
Neisseria gonorrhoeae, N. meningitidis 
Nocardia asteroides, N. brasiliensis, N. otitidiscaviarum , N. transvalensis 
Rhodococcus equi 
Salmonella including S, arizonae, S. cholerasuis , S. enteritidis , S. gallinarum-pullorum, 
S. meleagridis, S. paratyphi, A, B, C, S. typhi, S. typhimurium 
Shigella including S. boydii, S. dysenteriae, type 1, S. flexneri, S. sonnei 
Sphaerophorus necrophorus 
Staphylococcus aureus 
Streptobacillus moniliformis 
Streptococcus including S. pneumoniae, S. pyogenes 
Treponema pallidum, T. carateum 
Vibrio cholerae, V. parahemolyticus , V. vulnificus 
Yersinia enterocolitica, Y. pseudotuberculosis