The Biosafety/Bloodborne Pathogens Course is required for all Wayne State University investigators, staff, and students who work in a lab with materials that are potentially infectious, including human blood, body fluids, tissue, cell lines, animals infected with human pathogens, mammalian viruses, or any agents that are handled at Biosafety Level 2 (BSL2).

1. Biosafety/Bloodborne Pathogens:
Working Safely With
Biological Materials in Laboratories
http://research.wayne.edu/oehs

2. What is a Biohazard?
A biohazard is an agent of biological origin that
has the capacity to produce deleterious effects
on humans, e.g.; microorganisms, toxins and
allergens derived from those organisms; and
allergens and toxins derived from higher plants
and animals.
1

3. Laboratories working with biohazards have a set of guidelines to
follow from the Centers for Disease Control (CDC) and the National
Institutes of Health (NIH):
 The guidelines are in place to protect
human health and prevent the release
of pathogens into the environment.
 The guidelines include:
 laboratory practices and procedures
 facility design
 safety equipment requirements
Link to the CDC/NIH BMBL 5th Edition
2

4. Research may require review and approval by
the WSU Institutional Biosafety Committee (IBC)
 An IBC is mandated by the NIH at every institution.
 IBC members include WSU faculty, staff and PhDs
from neighboring institutions.
 The IBC may increase the Biosafety Level
requirements if the vector is more infectious than
wild-type virus.
3

5. Research requiring IBC review and approval:
 Native or recombinant mammalian viruses
 Pathogenic/infectious agents (non-rDNA)
 Biological toxins or rDNA encoding a toxin
 Work with, or creation of, transgenic animals.
 Exempt rDNA research does not need approval - researchers
must sign off on exemption form.
All research requiring IBC review must be submitted through
eProtocol: https://research.wayne.edu/eprotocol
4

6. Risk Groups (RG) & Biosafety Levels (BSL)
 The appropriate Risk Group and Biosafety Level is
determined by the CDC/NIH, based on characteristics
of the infectious agent, including;
 Pathogenicity of material - disease incidence/severity
 Documented route of transmission (bloodborne, airborne,
ingestion)
 Availability of protective immunization (HBV Vaccine) or
effective therapy
 Risk of exposure created by manipulation in handling the
agent & caring for infected animals
5

7. Risk Group (RG) Classifications
6

8. Biosafety Levels 1- 4 provide:
 Increasing levels of personnel & environmental protection,
and appropriate guidelines for:
 Laboratory Practices and Techniques
 Standard Practices and Special Practices
 Knowledge of supervisor and personnel
 Lab specific SOPs/Biosafety manual
 Safety Equipment (Primary Barriers)
 Laboratory Facilities (Secondary Barriers)
 Buildings (Tertiary Barriers)
7

9. 8

10. This presentation will focus on
the CDC/NIH guidelines for
biosafety levels 1 – 2 research
laboratories.
9

11. Proper handwashing is
important at all
biosafety levels!
 Wash with warm, running water, mild, preferably liquid
soap, (doesn’t have to be antibacterial).
 Rub hands together vigorously for at least 15 seconds:
scrub between fingers, under nails, tops & palms of hands.
 Rinse with warm, running water.
 Dry with disposable paper towel.
 Use lotion to prevent chapping of hands.
10

12. 11

13.  LAB COAT
 protects clothing/skin
 GLOVES:
 disposable latex/non-latex exam gloves
 change when torn or contaminated
Basic Personal Protective
Equipment (PPE) is required at
all Biosafety Levels
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Remove PPE before leaving
the work area!

14.  If risk of splashes or aerosols,
protect the eyes & face.
 safety glasses/goggles
 face mask
 If necessary, other PPE should
be worn, including:
 gown, face shield, booties, etc.
Basic Personal Protective Equipment (PPE) is
required at all Biosafety Levels
13

15.  Use of a N95 respirator
requires enrollment in the
employer’s respiratory
protection program.
 Users must receive a
medical evaluation, training,
and fit-testing.
 Contact OEHS for more info.
Personal Protective Equipment

16. Biosafety Level 1 (BSL1)
 BSL 1 is suitable for work involving well-characterized
agents not known to cause disease in healthy adults and
of minimal potential hazard to lab personnel and the
environment. Some examples include:
 Bacillus subtilis
 Naegleria gruberi
 Infectious canine hepatitis virus
 Non-entero hemorrhagic E. coli
 Exempt recombinant DNA experiments
15

17. Biosafety Level 2 (BSL 2)
 BSL 2 is suitable for work involving agents of moderate
potential hazard to personnel and the environment.
 Immunization or antibiotic treatment is available
 Examples include:
 Measles virus
 Salmonellae
 Toxoplasma spp.
 Hepatitis B virus
 Adenoviruses
16

18. All Biosafety Level 2 (or higher) labs
are inspected to assure they meet the
CDC/NIH guidelines.
17

19. Biosafety Levels 1 – 2
Facility Design Guidelines
 Lab doors are lockable.
 Sinks available for hand washing.
 Work surfaces easily cleaned,
impervious to water.
18
 BSL2 labs should be under negative pressure. Air flows
into lab and doesn’t re-circulate to non-lab areas.

20. Biosafety Level 2 Additional Requirements
 Biosafety Cabinet
 Autoclave
 Eye Wash 19

21. Biosafety Levels 1 – 2
Standard Microbiological Practices
 Restrict/limit access when working
 No eating, drinking, storing food, etc.
 No mouth pipetting
 Minimize splashes and aerosols
 Decontaminate wastes
 Decontaminate work surfaces daily
 Maintain insect & rodent control program
20

22.  Extreme precaution with
SHARPS
 Gloves and additional PPE
 Use of mechanical pipetting
devices
21
Biosafety Levels 1 – 2
Standard Microbiological Practices +

23.  Extra care should be taken when using any contaminated
sharp item, including needles and syringes, slides, pipettes,
capillary tubes, razor blades, lancets, and scalpels.
 Plastic should be
substituted for glass
whenever possible.
22
Biosafety Level 2
Precautions with SHARPS

24.  Used disposable needles must not be bent, sheared,
broken, recapped, removed from disposable syringes, or
otherwise manipulated by hand before disposal.
 Always dispose of whole
unit in SHARPS containers!
23
Biosafety Level 2
Precautions with SHARPS

25.  Inspection by the Biosafety Officer
 WSU Biosafety Manual available.
 Biosafety SOPs are read & signed by all staff.
 Labs using materials from human source must review
and sign off on WSU Bloodborne Diseases Exposure
Control Plan.
 Principal Investigator must train staff annually.
24
Biosafety Level 2
Additional Requirements

26. Hazard warning signs
Include the following:
• Entry requirements
• Agent name/Human cells
• Equipment
• BSC
• Refrigerators/freezers
• Incubators
• Regulated waste

27. Hazard warning signs
 Risk Group 2 Agents
26
 Universal Precautions
 Labs using human/non-human
primate cells, cell lines, organ
cultures and body fluids –
doesn’t require IBC approval.
 Require IBC Approval

28.  Cultures, tissue, specimens should be placed in a container
with a cover that prevents leakage during collection,
handling, processing, transport, storage or shipping.
27
Biosafety Level 2
Additional precautions

29. MIOSHA Occupational Exposure to
Bloodborne Diseases Standard
R325.0001-325.0018
 Standard applies to all jobs where there is a potential for
exposure to human bloodborne diseases, including
healthcare workers, laboratory workers, firefighters, EMTs,
custodians and maintenance workers, etc.
28
 Retraining is required annually!
 Based on the concept of UNIVERSAL
PRECAUTIONS: treating all human blood,
tissue, cells, etc. as if they ARE infectious.

30. WSU Exposure Control Plan
MIOSHA Bloodborne Infectious
Disease Standard
29

31. Use of human materials in research
 BSL-2 is appropriate for activities with all primate cell lines,
even well established ones, all cells derived from primate
lymphoid or tumor tissues; all primate tissue; all human
clinical material*; cultured cells new to the laboratory until
proven contaminant-free; and, cells exposed to or
transformed by a primate oncogenic virus.
* These activities and the use of any cells purposely infected
with or suspected of harboring agents defined as bloodborne
pathogens are covered by the Bloodborne Pathogens Standard)
and WSU’s Exposure Control Plan.

32. Use of human materials in research
 Laboratories using human cell strains (non-transformed
cells) propagated from primary explants must also comply
with the Bloodborne Pathogens Standard) and WSU’s
Exposure Control Plan because they are considered
“unfixed human tissue” and may carry bloodborne diseases
such as Hepatitis B, Hepatitis C, HIV.

33. Bloodborne Diseases
 HIV: Human Immunodeficiency Virus
causes AIDS - no cure or vaccination
32
 HBV: Hepatitis B virus causes liver disease
vaccination available
 HCV: Hepatitis C virus causes liver disease
no vaccination available

34. Bloodborne diseases are only spread when blood and
certain other body fluids from an infected source get into
the bloodstream of an uninfected person.
An occupational exposure is contact with infected
material through:
 needlestick or cut with
contaminated sharp object
 cuts/breaks in skin
 mucous membranes
(eyes, nose, mouth)
33

35. Bloodborne Diseases are NOT
transmitted through:
 Kissing or hugging
 Sneezing or coughing
 Food or water
 Sharing eating utensils, cups, etc.
 Casual contact
 Saliva, tears, perspiration, urine, feces (when there is
no blood present)

36. How is HIV Transmitted?
 Blood and body fluids
 serum
 semen
 vaginal secretions
 fluids around internal organs/systems
 IV drug use
 vaginal or anal intercourse
 mother to child in utero
* There are no documented cases of HIV being transmitted through saliva, tears or perspiration, saliva.
35

37. Source: https://www.cdc.gov/hiv/workplace/healthcareworkers.html
36

38. Diagnoses of HIV Infection among Adults and Adolescents, by Transmission
Category, 2018—United States and 6 Dependent Areas
N = 37,741
Note. Data for the year 2018 are considered preliminary and based on 6 months reporting delay. Data have been statistically adjusted to account for
missing transmission category. “Other” transmission category not displayed as it comprises less than 1% of cases.
a Heterosexual contact with a person known to have, or to be at high risk for, HIV infection.

39. HIV Transmission in Healthcare
Occupational HIV transmission is extremely rare.
As of December 31, 2013, only 58 confirmed
occupational transmissions and 150 possible
transmissions of HIV to healthcare workers had
been reported in the United States. Of these, only
1 confirmed case has been reported since 1999.
Source: https://www.cdc.gov/hiv/workplace/healthcareworkers.html
38

40. HIV Exposure Risk
 Rate of seroconversion after needlestick exposure
to infective material from HIV+ person is 0.23% or
about 2.3 of every 1000 such injuries will result if
infection, if untreated.
 HIV in high concentration during period prior to
antibody development.
 Much less infective than HBV, HCV, Herpes
Source: https://www.cdc.gov/hiv/pdf/workplace/cdc-hiv-healthcareworkers.pdf
39

41. HIV Transmission in
Healthcare Workers
 Factors associated with HIV transmission in healthcare:
 Deep injury
 Device visibly contaminated with source patient’s blood
 Procedures involving a needle placed directly in a vein or artery
 Terminal illness in source patient
 No zidovudine (AZT) prophylaxis
40

42. Hepatitis B Virus
 Inflammation of the liver – most common
bloodborne disease
 Symptoms range from flu-like to none at all
 No symptoms – person can still be
infectious and can spread the disease
 Approximately 95% of adults recover
completely and have no chronic infection
41

43. Hepatitis B Virus
 Incubation period from 28-160 days
 Symptoms may include:
 loss of appetite
 fatigue
 fever
 possible jaundice and dark urine
 HBV is a much greater risk on the job than
HIV, especially if you are not vaccinated.
42

44.  Fluids that pose risk of infection:
 blood and blood products
 body fluids containing visible blood
 semen and vaginal secretions
 breast milk
 saliva (bite that breaks skin)
Hepatitis B Virus
43

45. Occupational HBV transmission
 Needlestick/cut with sharp, contaminated object
 Splash to eyes/nose/mouth
 Contact with broken skin
 HBV can survive outside of the body for at least 7
days and still be capable of causing infection.
44

46. Figure 3.3. Rates of reported acute hepatitis B, by age
group — United States, 2002–2017
Source: CDC, National Notifiable Diseases Surveillance System.
0
1
2
3
4
5
6
Reportedcases/100,000
population
Year
0-19 yrs
20-29
yrs
30-39
yrs

47. Hepatitis B in Healthcare Workers
(HCWs) in the U.S.
 Prior to vaccination, it was estimated that more than
12,000 HCWs were occupationally infected with HBV
annually, resulting in 250 deaths.
 1983 – Incidence of HBV among HCWs was 3 times higher
than in the general population.
 By 1995 it was 5 times lower.
 Advent of HBV vaccine was a major advance in preserving
health and lives of HCWs.
Source: Arch Intern Med 1997; 157:2601-2605
46

48.  Administered in 3 shots over 6 months.
 Engerix-B Vaccine is yeast derived - no chance of infection
from vaccination
 Provides long term protection against HBV for 96% of
healthy adults: no booster recommendation by CDC at this
time.
 Post exposure vaccination is 70-88% effective when started
within one week.
47
Hepatitis B Vaccination

49. Hepatitis B Vaccination
 Vaccination is given at 4K University Health Center (Detroit
Receiving Hospital), Monday – Friday 9:00 – 4:30.
 Must present certificate of training to be vaccinated.
 Must sign declination form if you choose to not be
vaccinated.
 Call 313-577-1200 for more
information.
48

50. Hepatitis C Virus
 Spread primarily through
blood/blood products. Less
likely to be spread sexually.
 If left untreated, 80-85% of
cases become chronic
 There is no vaccine for HCV.
Based on limited studies, risk for infection after
needlestick is approximately 1.8%.
49

51. Hepatitis C Virus
 Identified in 1988, formerly called non-A non-B
hepatitis - called “silent epidemic”
 Blood supply not tested until early 90s.
 Incubation period from 2-26 weeks. Most people
never have symptoms
 Most prevalent among those born during 1945–1965,
who were likely infected during the 1970s and 80s
when rates were highest.
50

52. Recent Increases in Hepatitis C Infections
 In 2014, an estimated 30,500 new HCV infections
occurred in the United States.
 Between 2010 and 2017, the CDC reported that acute
HCV infections quadrupled after several years of relatively
stable rates of new infections. The CDC has determined
that this increase is linked to the ongoing opioid abuse
epidemic in the United States.
 High rates of new infections were predominantly among
White adolescents and young adults with histories of
injection drug use and previous use of prescription
opioids such as oxycodone, mostly in nonurban areas.
Source: https://www.hhs.gov/hepatitis/learn-about-viral-hepatitis/hepatitis-c-basics/index.html 51

53. Figure 4.3. Rates of reported acute hepatitis C, by age
group — United States, 2002–2017
Source: CDC, National Notifiable Diseases Surveillance System.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Reportedcases/100,000
population
Year
0-19
yrs
20-29
yrs

54. Figure 4.1. Actual number of acute hepatitis C cases
submitted to CDC by states and estimated* number of
acute hepatitis C cases — United States, 2010–2017
Source: CDC, National Notifiable Diseases Surveillance System.

55. Generation of Aerosols
 If aerosols may be generated, work must be performed
in Class II biosafety cabinet.
 Procedures that may generate aerosols include:
 Pipetting
 Centrifugation
 Tissue culture
 Sonication
 Blending of tissues
 Animal innoculations
54

56. Biological Safety Cabinets
these cabinets provide:
 Product protection
 Personal protection
 Environmental protection
55

57. Laminar Flow “Clean Benches”
 This is not a Biosafety Cabinet –
it does NOT protect the
worker!
 Filtered air flows from back of
cabinet, across work surface,
and towards user.
 Protects samples and work but
NOT the user
56

58. Biological Safety Cabinets
 Contain HEPA Filters (high efficiency
particulate air). These trap particulates
as small as 0.3u.
 Does not protect from chemicals:
fumes and vapors pass through
 Chemicals and heat may damage filter.
57

59. Biological Safety Cabinets
 May use for non-volatile toxic
chemicals or low-level radioactive
materials.
 May use for “minute” amounts of
volatile chemicals.
 Ensure annual certification from
OEH&S.
 Place BSC away from high traffic
areas, airflow ducts and lab
entrance doors.
58

60. Biological Safety Cabinets
Working Safely
 Disinfect work surfaces with iodine based
disinfectant (e.g. Wescodyne).
 Load cabinet with all needed supplies.
 Allow cabinet to run 10-15 minutes
before beginning work.
 Check inward airflow with a piece of
tissue.
59

61. Biological Safety Cabinets
Working Safely
 Always enter straight into
cabinet – no sweeping
motions.
 Perform work in a slow,
methodical manner.
 Place materials well within the
cabinet – don’t block grills.
 Place discard pan within
cabinet – discard pipettes
inside in disinfectant tray
60

62.  When finished, decontaminate all
items to be removed from
cabinet.
 Decon work surfaces again.
 Allow cabinet to run 10-15
minutes before turning off.
 Only use UV light as secondary
disinfection.
61
Biological Safety Cabinets
Working Safely

63. Natural Gas in a BSC
 Natural gas/Bunsen Burner use in a BSC represents a significant
fire risk.
 Use of flames & excessive heat can compromise the integrity of
the BSC and increase exposure risks.
 Explore safer options, including:
 Disposable loops/spreaders
 Electric Bunsen Burners
 Electric incinerators
 Hot bead sterilizers
62

64. Safe Centrifuge Use
 Check tubes for cracks/chips.
 Use matched sets of tubes,
buckets, etc.
 Tightly seal all tubes and
safety cups.
 Ensure that rotor is locked to
spindle and bucket seated.
 Close lid during operation.
 Allow to come to complete stop before opening.
63

65. Proper vacuum system set-up
A. Primary flask – type of disinfectant
B. Secondary flask – overfill – should be cleaned immediately
C. Hepa filter – component that is frequently missing!
HEPA filters: Whatman HEPA-Vent Filter Inlet/Outlet: Fisher Scientific Catalog #: 09-744-79

66. Emergency Response
Surface Decontamination
 Alert co-workers
 Define/isolate contaminated area
 Put on appropriate PPE
 Remove glass with forceps or scoop
 Apply absorbent towel(s) to spill;
remove bulk & reapply if needed
 Apply disinfectant on top of towels.
65

67. Emergency Response
Surface Decontamination
 If using bleach, mix FRESH 1:9 solution.
 Allow adequate contact time (20 minutes).
 Remove towel & mop up.
 Clean again with soap/water or alcohol.
 Properly dispose of materials in biohazard bins.
 Wash your hands last.
 For larger spills or questions, contact OEHS.
66

68.  Minimum strength of cleaners must be tuberculocidal
(kills M. tuberculosis var. bovis and all vegetative
bacteria, fungi, and most viruses)
 Require minimum 20 minute exposure
 Examples: quaternary ammonium detergents,
phenolics, iodophores, chlorine compounds, alcohols
67
Emergency Response
Surface Decontamination – other acceptable disinfectants

69. Emergency Response
responding to exposures
 Occupational exposure is contact with blood or other
potentially infectious materials:
 needlestick or cut with sharp, contaminated object
 contact with broken skin through cuts, rashes, other
breaks in skin
 splashes to eyes, nose, mouth
68

70.  Thoroughly wash affected area. Do not wash with bleach or
other strong cleaners.
 If eyes/face exposed, flush in eyewash immediately.
 Report incident to supervisor: Get medical attention! Henry
Ford Medical Center – Harbortown Occupational Health
includes assessment, blood tests, titer, counseling, follow-up
visits…all confidential.
 Complete a WSU Report of Injury form: Office of Enterprise
Risk Management & Insurance Programs 313-577-3112.
69
Emergency Response
responding to exposures

71. 70

72. Biological Waste Disposal
Request Waste Pick-Ups & Replacement Containers:
http://research.wayne.edu/oehs/bio-safety/biological-waste.php
SHARPS containers
Red Bins
71
21 gal bin (M-64)
31 gal bin

73.  Must be used for all SHARPS
(contaminated or not)
 Don’t overfill containers!
 Locate containers conveniently.
 Never recap needles:
#1 cause of needlesticks!
Correct SHARPS Disposal
Get free containers from OEH&S
 Request SHARPS pick-up and replacement containers
on OEH&S website:
 http://research.wayne.edu/oehs/bio-safety/biological-waste.php 72

74.  Hypodermic needles, with syringe
 IV tubing with needles attached
 Contaminated Pasteur pipettes
 Razors and microtome blades
 Scalpels
 Lancets
73
Correct SHARPS Disposal
What goes into SHARPS container?

75. NEVER dispose of SHARPS
in glass waste boxes or in the trash!
74

76. Biological Waste (Red) Bins
 Free 28 gallon red plastic bins and a
liner bag are provided by OEH&S.
 Labs must purchase their own
autoclavable biohazard bags
 Some materials should be autoclaved
before placing into bins. Check with
the Biosafety Officer for more
information.
 Always place bags in bins or other
leak-proof secondary containers.
 DO NOT autoclave bin or the liner bag!
75

77.  Items contaminated with human or animal blood, body
fluids or tissue.
 Cultures/stocks of infectious agents: including waste from
production of biologicals, discarded vaccines, and culture
dishes.
 Materials/microorganisms used
in recombinant DNA research.
 NO SHARPS
(needles, razor blades, etc!)
76
Biological Waste (Red) Bins
what goes into bins?

78.  When bin is full:
 close & tie liner bag shut
 place lid on securely
 do not overfill bins. They shouldn’t weigh more than 35 lbs.
 request pick-up and replacement containers from OEH&S
website:
http://research.wayne.edu/oehs/bio-safety/biological-waste.php
Only properly prepared bins
will be accepted!
77

79. All biohazard waste must be in
secondary containment
 Biohazard bags must always
be stored in leakproof
secondary containers
78

80. Improperly stored waste

81. Properly stored waste
• Closeable
• Constructed to contain all
contents and prevent leakage
of fluids
• Puncture resistant
• Labeled with biohazard symbol

82. Waste glass & plastic disposal
 Decontaminate items with
bleach or autoclave before
putting into box.
 Use a sturdy, durable box.
 Label the box “broken glass”
or “glass waste”
 Line the box with plastic bag.
 Keep weight reasonable – 25
lbs. or less! 81

83. 82
 When box is full, tape shut
securely.
 Place box in an area for the
custodial staff to dispose.
 Custodians reserve the right to
refuse pick up of boxes that
aren’t prepared properly.
Waste glass & plastic disposal

84. Autoclave Decontamination
 Place items in secondary containers:
stainless steel or autoclavable
plastic bins.
 Temps of 121 degrees C for 20
minutes for most recombinant
organisms and pathogens.
 Larger loads require more time.
 Autoclaves are usually in shared
areas – clean up after yourself!
83

85.  Do not cap or plug vessels.
 Do not add excessive amounts of liquid
to load.
 Usually steam remains in the chamber
at end of a liquid cycle, so be careful!
 Wear eye and face protection.
 Stand behind door when opening it –
keep face away from door.
 Slowly open door only a crack to allow
residual steam to escape.
84
Autoclave Decontamination

86. Liquid Biological Waste Disposal
 Aqueous biological solutions such as blood, urine, cells, microbial
cultures must be autoclaved or chemically disinfected (treated
with bleach)
 1 part bleach to 9 parts contaminated liquid – let stand for 30
minutes.
 After treating, dispose down
drain with lots of H2O.
 Do not autoclave bleach
treated waste.
85

87. Working with research animals
 Animals release airborne allergens which can cause
allergy symptoms in some people.
 Mild symptoms include: itchy/watery eyes, runny nose,
skin rash/itchy skin, nasal congestion, and cough.
 Reduce your exposure by using PPE, including:
Gloves
Lab Coat
N-95 Mask for your nose/mouth (requires a fit-test)
 For more information on WSU’s Animal Contact program,
call the OEH&S Occupational Health Nurse at 577-5917.
86

88. Shipping Dangerous Goods
• Common materials shipped include: human
and animal samples, bacteria, DNA, proteins,
chemicals (solids or liquids), dry ice, etc.
• Shipping ANY material on dry ice by air is
considered a Dangerous Good/Hazardous
Material
• OEH&S is the designated authority for
shipping Dangerous Goods from WSU.
• Contact OEH&S at 577-1200 to ship any
Dangerous Goods.

89. Take the Quiz!
To verify your completion of this course through WSU,
you must return to the CITI site and take the quiz.
88
If you are unable to take the quiz on CITI, email fracassa@wayne.edu.