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BIOSAFETY
BIOSAFETY IN MICROBIOLOGICAL
AND BIOMEDICAL LABORATORIES
BMBL is the “Bible” for Biosafety matters
 Published by US Dept. of Health and Human Services
and CDC and NIH
 Does not have the force of law, BUT
 The standard for biosafety. Must comply if receiving
certain grants, etc.

HISTORY

Published data regarding Laboratory Acquired
Infections – most are aerosol
Lab workers are infected by the agents they work with.
(The good news – “not been shown to represent a
threat to the community.”)
1979 Pike concludes “the knowledge, the techniques,
and the equipment to prevent most laboratory
infections are available.”
The “Biosafety in Microbiological and Biomedical
Labortories” (BMBL) is born
LAI’S CONTINUED,
(MMWR JAN 6, 2012 SUPPLEMENT/VOL 61)
Recent MMWR reports indicate bacteria account for
>40%
 >37 species as etiologic agents
 Brucella, Shigella, Salmonella and Staph aureus
are common
 2005 CDC Neisseria meningitidis
 General population 13/100,000
 30-59 year population 0.3/100,000
 30-59 year old microbiologists 20/100,000


April 2012, 25 yr old lab tech dies from infection same serotype as lab strain (no vaccine available for this strain)
5 PREDOMINANT
ROUTES OF INFECTION
Parenteral inoculation with “sharp”
 Spill/splash on skin and mucous membranes
 Ingestion or exposure via touching mouth or eyes with
fingers or contaminated objects
 Inhalation of infectious aerosols
 Animal bites/scratches (zoonotic)

RISK CLASSIFICATION
Many agencies have classified infective
microorganisms by Risk Group
 CDC/NIH
 World Health Organization
 Canadian Laboratory Safety Guidelines
 European Union
 Australia/New Zealand

RISK GROUPS

RG 1
Organisms not known to cause disease in healthy
adult humans and pose minimal hazard to people and
the environment. (ATCC)
 Individual risk: low
 Community risk: low

RISK GROUPS

RG 2
Organisms that pose a moderate risk and are
associated with human disease through skin breaks,
ingestion or mucous membrane exposure (ATCC)
 Individual risk: moderate, potential hazard
 Community risk: low, limited, unlikely

RISK GROUPS

RG 3
Indigenous or exotic agents with potential for
aerosol transmission/inhalation route of
exposure and have the potential for serious and
even lethal effects. (ATCC)
 Treatment usually available
 Individual risk: high, serious
 Community risk: Low, may be present

RISK GROUPS

RG 4
Dangerous/exotic agents which pose high risk of life
threatening disease, aerosol-transmitted lab
infections; or related agents with unknown risk of
transmission.
 Preventive or therapeutic interventions not usually
available
 Individual risk: high, serious
 Community risk: high

RISK GROUP VS. BIOSAFETY LEVEL
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Risk Group is a stable comparative descriptor of the
inherent pathogenic nature of a given microorganism;
RG does not change based on how or where the agent is used.
Biosafety Level is a variable comparative descriptor of the
facility, equipment and practices that serve to "contain" a
microorganism while it is being handled; BSL is based on risk
assessment and technical judgment and may vary with the use
of the agent.
(Glenn Funk, ABSA)
PRINCIPLES OF BIOSAFETY:
CONTAINMENT
Laboratory practice and technique
 Safety Equipment
 Facility Design
 Biosafety Levels
Combination of lab practices & techniques, safety
equipment and facilities specific for each of the 4
biosafety levels

PRIMARY VS SECONDARY CONTAINMENT
Primary – protect workers in the immediate area
of the lab.
 Secondary – external to the lab. Usually facility
design.

BIOSAFETY LEVEL 1
Level 1 for agents that are “defined and
characterized strains of viable microorganisms
not known to consistently cause disease in
healthy adult humans.”
B. subtilis, E. coli K12, S. cervasiae
 Containment relies on “standard microbiological
practices, no special…barriers other than a sink
for handwashing.”

BIOSAFETY LEVEL 2
 Level
2 for the “broad spectrum of indigenous
moderate-risk agents that are present in the
community and associated with disease of varying
severity.” S. aureus, B. anthracis, HIV, Hep B

S. aureus is a common LAI
 Containment
relies primarily on “good
microbiological technique…”provided the potential
for producing splashes and aerosols is low.”
 Primary
hazard is by accidental percutaneous or
mucous membrane exposure. Careful with
sharps!!!
BIOSAFETY 2, CONTINUED
Aerosols. If manipulations will produce aerosols,
then “primary containment” such as PPE and
other safety devices such as Biological Safety
Cabinet (BSC) must be used.
 Handwashing facilities
 Waste decontamination

Aerosols
AEROSOL – TWO CONSIDERATIONS
Respirable-size particles that remain airborne for
protracted period of time. Source of infection if
inhaled.
 Formation of droplets that settle rapidly on
surfaces – clothing, hands, benchtops, etc. Large
size droplet can contain multiple copies of the
agent.


(BMBL 5th edition)
Gross Contamination On Horizontal
Surface Near Pipetting Operation
Volume of
Pipetting Aid
Microliter
3
5
10
25
50
200
1000
1500
6000
Total CFU
On All
Trial 1
4
0
3.5
27
6
49.5
205.5
138
315
Collected
Plates
Trail 2
0
3
6
11
21
38
1225
588
2025
Aerosol and Surface Recovery from Ten
Pipetting Operations of 109/ml. B. subtilis
Run
1
2
3
4
5
6
Average
Airborne
CFU
2,040
657
2,050
388
5,110
649
1,820
Settled
Hands
35,800
22,000
14,800
9,300
6,900
228,000
CFU
Area
3,700
860
1700
550
2100
2900
52,800
1,970
(average time 3 minutes; 1 ml. Pipette; ca. 2ml. bulb. Chatigany et. al. 1979
CFU Recovered From Operator’s
Glove
Trail #
1
2
3
4
Average
Before Pouring After Pouring Into
Centrifuge Tubes
0
48,000
0
48,000
0
7,900
0
8,900
0
28,000
(suspension poured contained 109/ml. Flavobacterium.)
Aerosols From Lab Equipment
(1010/ml culture - 10 min. use)
Blender, opened at once
Sonicator with bubbling
Pipetting, vigorous
Dropping culture
Splash on a centrifuge rotor
Blender, opened after 1 minute
Pipetting, carefully
Dimmick, et. al. 1973
106
106
106
3 X 105
105
2 X 104
104
OTHER AEROSOL GENERATING ACTIVITIES:
Flaming loops
 Cooling loops in culture media
 Subculturing and streaking culture media
 Expelling the last drop from a pipet
 Setting up cultures, inoculating media
 Preparing smears, performing heat fixing,
staining slides


(MMWR Jan 6, 2012 Supplement/Vol 61)
BIOSAFETY 3 AND 4
Level 3 for “indigenous or exotic agents with a
potential for respiratory transmission and
which may cause serious and potentially lethal
infection.” M. tuberculosis.
 Much higher level of secondary containment
 Level 4 – the really nasty stuff. Ebola, Marburg.

STANDARD MICROBIOLOGICAL
PRACTICES
BSL 1
Access to laboratory limited when work with
cultures is in progress.
 Handwashing
 No eating, drinking, applying lip balm, makeup, etc.
 No mouth pipetting
 Policies for handling sharps
 Minimize splashes and aerosols
 Work surface decontaminated at least once per day
 Decontamination of cultures and lab wastes
 Biohazard sign must be posted at lab entrance
 Pest management program
 Supervisor ensures that personnel are trained

BSL 1, SAFETY EQUIPMENT
Safety Equipment – not usually required
 PPE such as lab coats recommended
 Gloves worn when skin is broken, rash
 Eye protection when splashes likely
 No special facility considerations

Calvin College
SB 210
BIOSAFETY LEVEL 1
ACCESS: FACULTY, STAFF AND APPROVED/BIO 321 & 336 STUDENTS
PRECAUTIONS: Standard microbiological practices. Keep doors closed when
working with microbial agents.
WASH HANDS BEFORE LEAVING ROOM!!
RESPONSIBLE INVESTIGATORS:
Arlene
Elizabeth
Ext: 6-8668
Home: 1
SECONDARY CONTACT: Lori Keen
Extension: 6-6080
Home
Ext: 6-7085
Home:
AGENTS USED: E.coli, E. faecalis, B. subtilis, M. luteus, M. smegmatis,
M. chloropheniclum, S. marcescens, S. cerevasie
BSL 2
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Standard Microbiological practices PLUS
Access limited especially to those at increased risk of infection
Biosafety manual specific to the lab including SOP’s
PI’s ensure training of personnel regarding hazards,
prevention.
Gloves and lab coats must be worn when working with
infectious agents.
High degree of caution regarding sharps.
Disinfection of work surfaces.
Spills and accidents resulting in exposure are immediately
reported
Biohazard sign MUST be posted stating the name of the
agent(s), the biosafety level, PI’s name and phone number,
PPE required to enter, special procedures for exit.
Immunizations, medical surveillance if appropriate.
Appropriate furniture/chairs – non-porous, easily cleaned.
BSL 2, SAFETY EQUIPMENT
 PPE
and equipment (BSC) used when
procedures with a potential for significant
aerosols and/or splashes are conducted or
high concentrations or large volumes of the
agent are in use.
 Lab coats routinely worn in the laboratory
 Gloves worn when hands may come in
contact with agent, contaminated equipment
or surfaces.
 PPE remains in lab and is removed before
leaving the lab!!!
 Facility requirements
Calvin College
SB 210
Biosafety Level 2
ALL UNAUTHORIZED PERSONS KEEP OUT!
ACCESS: Allowed for faculty, staff and Bio 207 students.
PRECAUTIONS: Standard microbiological practices; lab coats must be worn during all
procedures; additional precautions may apply. WASH HANDS AND REMOVE LAB
COATS AND GLOVES BEFORE LEAVING ROOM!!
RESPONSIBLE INVESTIGATORS:
Amy
Anding
Ext. 6-7620
Ext. 6-6025
AGENTS IN USE: may include S.aureus, P.aeruginosa, P. vulgaris, S. choleraesius
Also RG1 agents - E.coli, E. faecalis, B. subtilis, M. luteus, M. smegmatis,
S. marcescens, S. cerevasie
RISK ASSESSMENT
Agent Hazards
 Lab Procedure Hazards
 Staff (student) proficiency

RISK ASSESSMENT
 BSL
assessments need to be done on case by case
basis. Consider the following:
 Procedures to be performed
 Pathogenicity
 Route of transmission
 Agent stability
 Infectious dose
 Concentration
 Experience and skill level
 Effective prophylaxis
 Health status of the worker
 Always err on the side of caution!
AGENT HAZARDS
 Pathogenicity

HepB virus is 50-100 times more infectious
than HIV (World Health Organization)
 Route

Inhalation, ingestion, mucous membrane?
 Agent


of transmission
stability
HBV- at least 7 days and still be capable of
causing infection.
HIV- evidence suggests only hours of viability
http://www.cdc.gov/hiv/resources/qa/transmission.htm
 Infectious
dose
RISK OF INFECTION
FROM PERCUTANEOUS EXPOSURE
(PATHOGENICITY )

Virus
HBV
 HCV
 HIV

Virus/mL Serum
Risk
102-108
100-106
100-103
30%
2%
0.3%
TRANSMISSION RATES
EXPOSURE TO HIV INFECTED BLOOD
(ROUTE OF EXPOSURE)
Percutaneous
 Mucocutaneous

0.31%
0.03%
LAB PROCEDURE HAZARDS
Safety equipment available
 Complexity of procedure
 Agent concentration and volume
 Aerosol generating
 Animals

STAFF (STUDENT) CONCERNS
Proficiency/competency of laboratorian
 Medical status

HOST FACTORS AFFECTING RISK
 Deficiencies
in host defenses
Skin – eczema, chronic dermatitis, psoriasis
Mucosa – antimicrobial therapy; bowel
pathology
Immune system deficiencies
Asplenia
Other medical conditions such as viral
infections, poorly controlled Type I diabetes,
pregnancy, asthma, cancer, connective tissue
diseases
(treatment often causes immunosuppresion)
SUMMARY STEPS TO RISK
ASSESSMENT…START TODAY
Identify the hazards associated with the agent
 Identify the activities that might cause exposure
 Consider competency/experience of personnel
 Evaluate and prioritize risks and severity of
consequences
 Develop and IMPLEMENT controls to minimize the
risk for exposure

LESSONS FROM SALMONELLA
Aug 2010-June 2011 109 individuals in 38 states
infected with strain X of Salmonella
Typhimurium. Ages 1-91, median 21.
 Exposure to clinical and teaching microbiology
labs was a possible source. 60% of ill persons had
exposure to a microbiology lab in the week prior
to illness. Some specifically worked with
Salmonella in micro labs.
 Several children living in households with a
person who worked or studied in micro labs
became ill with the outbreak strain.
 http://www.cdc.gov/salmonella/typhimuriumlaboratory/011712/index.html

CONCLUSIONS
Investigated two groups of laboratories – labs
associated with illness and labs w/out illness
 Lab practices and safety policies were similar BUT
 Labs associated with illness had less knowledge of
biosafety training materials
 Those free of illness were more likely to train
employees regarding signs and symptoms of illness
 Enforcement of policies may be difficult to enforce or
monitor

TAKE HOME LESSONS
“If you work in a laboratory it is possible to bring
bacteria home through contaminated lab coats, pens,
notebooks, and other items…”
 “Leave food, drinks or personal items like car keys, cell
phones and mp3 players outside of the laboratory.”
 Wear a lab coat, leave the lab coat in the lab.
 Train for signs and symptoms (illness) of agents in use.
