US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Mic ro bio lo gy La bo ra to ry
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Biosafety in the Laboratory
SOP Number: MB-01-07
Date Revised: 11-24-14

SOP No. MB-01-07
Date Revised 11-24-14
Page 1 of 14
SOP Number
Biosafety in the Laboratory
This protocol outlines the required safety measures for working with
the microorganisms used by the OPP Microbiology Laboratory.
This SOP is based largely on the guidance provided in the Centers
for Disease Control and Prevention/National Institutes of Health
(CDC/NIH) publication "Biosafety in Microbiological and
Biomedical Laboratories," 5th ed. (BMBL; see Section 15 #2).

Approval Date
SOP Developer:

Print Name:
SOP Reviewer

Print Name:
Quality Assurance Unit

Print Name:
Branch Chief

Print Name:

Date SOP issued:

Controlled copy

Date SOP withdrawn:

SOP No. MB-01-07
Date Revised 11-24-14
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The Laboratory recognizes the biosafety levels set forth in the BMBL, and the need to provide
different degrees of protection (i.e., ascending biosafety levels) depending upon the danger of the
microbe to the worker, community, and the environment. The majority of the microorganisms
contained in-house fall within Biosafety Level 2.
This SOP is structured so all work involving manipulation of culture of all microorganisms,
regardless of the biosafety level, is performed in the BSC and not on the open bench. This is due
to the availability of BSCs within the laboratory, the ease and practicality of working within the
BSC, and the ease of containing spills of chemical or biohazardous materials that may occur
within the BSC.
This SOP provides additional practices and procedures to be followed when working with
Biosafety Level 3 microorganisms in order to provide analysts added protection from disease.
For new microorganisms, laboratory staff must first determine the biosafety level of that
microorganism by referring to the BMBL.
The BMBL (see ref. 15.2) manual presents recommended guidelines for working with
microorganisms assigned to Biosafety Levels 1 through 4. Although these guidelines are not
currently legally enforceable guidelines, they are considered to be international standards of
practice. Should an exposure event occur, the CDC/NIH guidelines could take on the force of
law in that the laboratory management could be held legally responsible for not following
accepted standards of practice. Consequently, the laboratory will comply with the CDC/NIH

SOP No. MB-01-07
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Contents	Page Number

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1. Definitions
1.	Microorganism = includes bacteria in vegetative and spore form, fungus,
bacteria in biofilms, and viruses. Refer to Attachment A for a list of
organisms used by MLB.
2.	Manipulation of culture = handling of open vessels containing
microorganism. Activities involving manipulation of culture including
culture transfers, virus harvest, plating, inoculation of carriers, sonication
of inoculated carriers, recording results from tubes and plates, must be
performed in the BSC. The following are examples of activities involving
manipulation of culture: culture transfers, virus harvest, plating,
inoculation of carriers, sonication of inoculated carriers, recording results
from tubes and plates, etc.
3.	"Appropriate" disinfectant = EPA-registered hospital disinfectant (have
label claims for S. aureus, P. aeruginosa, and S. choleraesuis) or hospital
disinfectant with virucidal or tuberculocidal claims (efficacious against M.
bovis [BCG]). All disinfectants must be used according to the label
directions (e.g., use dilution, contact time, etc.) specified on the labeling.
4.	PI00 HEPA = Oil proof High-Efficiency Particulate Air Filter, 99.97%
efficient in removing particles 0.3 microns or larger.
5.	References to water mean reagent-grade water.
2. Health and
1.	To protect the laboratory worker from possible infection by
microorganisms, the health and safety guidelines provided in this protocol
and in the BMBL manual must be followed. All laboratory personnel are
required to read and familiarize themselves with this protocol and sections
on Biosafety Levels 2 and 3 in the BMBL.
2.	Laboratory workers must familiarize themselves with the laboratory's
biosafety spill clean-up procedures (see SOP MB-13), and the facility's
Chemical Hygiene Plan (CHP) prior to performing any laboratory work.
Biosafety spill clean-up procedures are posted in the laboratories.
3.	Laboratory workers are required to participate in the Agency's
Occupational Medical Surveillance Program as established by EPA Order
4.	Medical emergencies are handled according to procedures outlined in the
ESC Occupant Emergency Plan (OEP).
5.	Spills and accidents are handled according to the practices outlined in this
SOP and SOP MB-13, as well as procedures referenced in the OEP and the
6.	To promote the health of exposed individuals, the Branch Chief will
encourage individuals to seek follow up, if necessary, depending upon

SOP No. MB-01-07
Date Revised 11-24-14
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recommendations of the SHEM manager.
7.	All laboratory workers must meet the requirements of the Hazard
Communication Program's Employee Training Program, as described in
the CHP.
8.	In accordance with the CDC/NIH guidelines, the Branch Chief may
restrict access to the laboratory as specified under "special practices".
9.	All employees required to use respirators are participants in the Agency
Occupational Medical Surveillance Program and the ESC Respiratory
Protection Program.
3. Personnel
and Training
Refer to SOP ADM-04, OPP Microbiology Laboratory Training.
4. Instrument
Performing maintenance and repairs on the BSCs and maintaining annual
certification of the BSCs are the responsibilities of the Facility Manager or his
designees (e.g., operations, maintenance and repair contractors). See SOP
EQ-11, Use and Maintenance of Biological Safety Cabinets.
5. Sample
Handling and
Not Applicable
6. Quality Control
7. Interferences
1.	Failure to become familiar with and to put into practice the procedures set
forth in this SOP will result in analysts who are a danger to themselves,
others, and the environment.
2.	Improper maintenance and/or sudden power failures may result in failure
of the BSCs to operate properly. Refer to proper use and maintenance
procedures in SOP QC-06.
8. Non-
Strict adherence to these biosafety practices are required. Nonconformance
will result in notification, retraining, or possible disciplinary action of
laboratory employees.
9. Data
1. Completed forms are archived in notebooks in D217, with the exception of
the Respirator Inspection Notebook which is kept in laboratory space.
10. Cautions
1.	Lack of following or understanding of this SOP may negatively impact the
quality of the microbiological practices used in the laboratory.
2.	Failure to use the "STOP/DO NOT ENTER" signs to control access to the
laboratory while cultures are being manipulated (section 12.2) may result
in the inadvertent exposure of personnel to biohazardous microorganisms.

SOP No. MB-01-07
Date Revised 11-24-14
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3.	Failure to clean the ultraviolet lamps in the BSCs will reduce the lamps'
effectiveness. Periodically clean the ultraviolet lamps in the BSCs with a
lint-free cloth dampened with alcohol (200 proof ethanol), and record the
cleaning on the BSC Monitoring Record Form.
4.	Autoclaving flammable and caustic liquids (e.g., alcohols or highly acidic
disinfectants) can present an explosion or exposure hazard. Seek advice
from the SHEM Manager for appropriate method of decontamination and
5.	Halogenated materials are not recommended for routine use on stainless
steel surfaces of the BSC.
6.	This protocol does not include working with select agents in the
laboratory. For biosafety measures associated with working with select
agents, consult the Biosafety Plan for Bacillus anthracis.
11. Special
Apparatus and
1.	Biological Safety Cabinet (BSC)
2.	Autoclave
3.	Biohazard bags or containers inside and outside of the biological safety
cabinets for biohazardous waste.
4.	Personal protective equipment (PPE) such as gloves, safety glasses, lab
coats, disposable laboratory garments, and respiratory protection.
5.	Signs to identify biohazardous materials and to limit access to
6.	For microorganisms in vegetative form, use an EPA-registered hospital
7.	For microorganisms in spore form, prepare 1:10 diluted bleach solution at
neutral pH. Using an EPA-registered sodium hypochlorite product
containing at least 6% sodium hypochlorite, dilute as follows: 1 part
bleach, 8.4 parts water, and 0.6 parts 5% white vinegar or 5% lab grade
acetic acid).
8.	Key card readers are used to limit access to testing laboratories.
9.	Microorganisms maintained by the laboratory are specified in the file MB-
10.	Secondary containment (e.g., durable, autoclavable trays and bins;
containment cart).
12. Procedure and

SOP No. MB-01-07
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12.1 General
a.	Eating, drinking, smoking, handling contact lenses, chewing gum,
and applying cosmetics (including lip balm) are not permitted in the
b.	Storing food for human consumption in laboratory areas is
c.	Laboratory workers must not wash or reuse disposable gloves.
d.	Wash hands prior to leaving the laboratory.
e.	Mouth pipetting is prohibited. Only mechanical pipetting devices will
be used.
f.	Organism will be manipulated inside a BSC to minimize risk of
exposure and risk of contamination of lab surfaces.
g.	All procedures are performed carefully to minimize the creation of
h.	Animals and plants not related to work being conducted are not
permitted in the laboratory.
i.	No material suspected or known to be contaminated with
biohazardous material (e.g., latex gloves, pipet wrappers, paper
towels, etc.) is to be placed in the trash cans. These items are to be
placed in an appropriate biohazardous waste bag.
j. For filtering Biosafety Level 3 microorganisms, protect vacuum lines
with disinfectant traps and HEPA filters.
12.2 Access to
and Placement
of Signage
a.	Key readers are used to limit access to laboratories. Only authorized
personnel are permitted to enter.
b.	When manipulating infectious microorganisms, post the magnetic
"STOP/DO NOT ENTER" sign on the outside (i.e., side facing
corridor) of the external laboratory door.
c.	Only Microbiology Laboratory Branch (MLB) staff are authorized to
enter the laboratory while the "STOP/DO NOT ENTER" sign is
posted. Non-MLB personnel must be escorted into a laboratory while
the sign is posted.
d.	Remove the "STOP/DO NOT ENTER" sign once work is complete.
12.3 Checking
Airflow to
Equipped with
a. Airflow monitors are located above the B202 and B207 laboratory
doors. The laboratories have two monitors each: one above the door
leading from the corridor to the anteroom, and one above the door
leading from the anteroom to the lab. The orange ball in the tube rolls
in the direction of the airflow. For negative airflow, the orange ball

SOP No. MB-01-07
Date Revised 11-24-14
Page 8 of 14
will roll from the corridor into the laboratory or anteroom, or from the
anteroom into the lab. Monitors must indicate negative airflow for
b.	Prior to entering laboratories visually verify that the laboratories with
monitoring devices are under negative pressure (airflow is into the
room from the corridor).
c.	Do not enter the laboratory if airflow is positive for either the
laboratory or anteroom. Report positive airflow status to the Branch
Chief and facilities hotline (x54357).
d.	In the event that airflow becomes positive during work, cease work
immediately and close all open materials. If working inside a BSC,
back out and pull down the sash. Report the problem to the Branch
Chief and facilities hotline (x54357). Work should only proceed when
proper airflow has been restored.
12.4 Using the BSC
a.	Turn off ultraviolet light.
b.	Turn on the blowers, lights, and outlets, and allow to operate for a
minimum of 15 minutes prior to use.
c.	Record the Downflow (FPM) and Exhaust (CPM) rates on the BSC
Monitoring Record Form immediately prior to use
d.	Spray the surface of the BSC with the use dilution of an appropriate
disinfectant or with 70% ethanol. Allow the surface to remain wet for
the label-specified contact time.
e.	At the conclusion of activities involving bacteria in spore form (e.g.,
Bacillus subtilis spore suspensions, spore strips), turn on the ultraviolet
light and leave it on overnight.
f.	In the event that the BSC alarms during work, cease work
immediately, pull down the sash, and call the facilities hotline
(x54357) to report the problem.
12.5 PPE
a.	PPE does not have to be worn while doing paperwork in the laboratory
or when entering the laboratory solely to retrieve an item such as a
document, provided that no manipulation of cultures or other
laboratory work is in progress.
b.	Safety Glasses
i. Safety glasses must be worn while working in the laboratory.
c.	Lab Coats
i. For Biosafety Levels 1 and 2: Don cloth or disposable lab

SOP No. MB-01-07
Date Revised 11-24-14
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coats. Remove lab coats before going to non-laboratory areas
such as the office areas, restrooms, cafeteria, library, etc.
ii. For Biosafety Level 3 (e.g. M. bovis [BCG]): Don solid-
front disposable lab coat before entering the lab.
Replace disposable lab coat immediately in the event of
suspected or known contamination with infectious material.
d.	Prior to exiting the laboratory and entering the double door access
zone, remove and discard lab coats in a biohazard bin or bag.
i.	Wear gloves (latex or nitrile) when manipulating culture and
when handling any vessel (e.g., tube, rack, plate, biohazard
bag), closed or open, containing live organism.
ii.	Inspect gloves prior to use. Do not use gloves that have holes,
rips, or are otherwise degraded.
iii.	Replace gloves immediately in the event of overt
contamination (e.g., visible drops of liquid) with infectious
material. Dispose of contaminated gloves in the biohazard
bin only.
iv.	To minimize risk of contamination of the test system while
working in the BSC, analysts may periodically apply a
solution of 70% ethanol over the exterior surface of the
gloves, change gloves frequently, or use sterile gloves.
v.	Additional guidance for Biosafety Level 3 work: Wear a
single pair of gloves (latex or nitrile) when manipulating
culture. Prior to handling materials outside of the BSC, the
analyst should discard the used gloves and put on a new pair.
Wear a double layer of gloves (latex or nitrile) when
manipulating culture. Prior to leaving the BSC to conduct
other activities (e.g., open the incubator, record data, retrieve
supplies, etc.), the outer pair of gloves must be discarded in
the biohazard bin. Replace the outer gloves upon returning to
the BSC.
e.	Respiratory Protection
i. Inspect respirators prior to use and on a monthly basis.
Record the information on the Respirator Inspection Checklist
form (see Section 14). The forms will be kept in the
Respirator Inspection Notebook.

SOP No. MB-01-07
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ii.	Biosafety Levels 1 and 2: Respirators are not required for
manipulations of Biosafety 1 and 2 microorganisms.
iii.	Biosafety Level 3: If a certain procedure involving
manipulation of the organism is impossible or impractical to
conduct within the BSC (e.g., reading the percent
transmittance or optical density of a culture using an
uncapped cuvette), respiratory protection (respirators with
PI 00 HEP A filter cartridges) must be worn while working
with the organism outside of the BSC. Once work is
complete, remove filter cartridges from respirator and place in
a biohazardous waste bag. Respirators may be washed with
mild soap and water if desired. Allow to dry and return to
12.6 Disinfection of
a. Chillers
i.	On a weekly basis, following testing, disinfect the water in
the recirculating chiller and remote water bath prior to
ii.	Disinfect the water in the chiller by adding the appropriate
amount (i.e., to achieve the product's use dilution in the
chiller water) of a disinfectant labeled for use against the test
organisms to the recirculating chiller and remote water bath.
Follow label directions for use. Record information on the
Recirculating Chiller Cleaning and Disinfection Log (see
Section 14).
iii.	During use, the recirculating chiller/remote water bath system
circulates approximately 23.5 L of water (20.5 L in chiller
reservoir + 3 L in remote water bath). Use this value as the
volume of diluent in calculations to determine the amount of
disinfectant to add to the chiller water.
iv.	After the disinfectant is added, the unit should be turned on
and run thoroughly. Turn off the unit and allow the
disinfectant to remain in the unit for the specified contact
time (e.g., 10 minutes).
v.	Each recirculating chiller is equipped with a drain valve and a
drain hose located on the back of the unit. With the unit off,
open the valve and allow the reservoir contents to drain into
an appropriately sized container or directly into a sink.
vi.	Rinse the unit with tap water and drain as above. Refill the

SOP No. MB-01-07
Date Revised 11-24-14
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reservoir with fresh tap water on the day of testing.
b.	Sonicator
i. Disinfect the tap water in the sonicator bath at the end of the
testing week by adding appropriate disinfectant to the tap
water in the bath to achieve the disinfectant product's use
dilution. Let the disinfectant remain in the sonicator bath for
the contact time stated on the disinfectant labeling. Once the
contact time is achieved, discharge the treated water
appropriately, rinse the unit with tap water, and dry the
sonicator bath with paper towels. Record cleaning and
disinfection of sonicator on the Sonicator Disinfection Log
(see Section 14).
c.	Spectrophotometer
i.	Periodically and prior to shipping the unit out for calibration,
remove the cell holder from the instrument and disinfect it
with an appropriate disinfectant. Allow the surface to remain
wet for the label-specified contact time. Thoroughly rinse
with tap water, allow to dry, and replace cell holder in the
ii.	Disinfectant is not to be sprayed or wiped on the inner
surfaces of the spectrophotometer as disinfectant residue may
remain on the optics, negatively impacting instrument
d.	Administrative supplies (Biosafety Level 3)
i. Supplies such as pens, clipboards, etc., that are used during
testing of Biosafety Level 3 agents will be limited to the
Biosafety Level 3 laboratories.
12.7 Conducting
Staining of
a.	While staining and viewing slides, wear gloves and a lab coat, and
conduct any steps involving manipulation of the organism (e.g.,
smear preparation) in the BSC.
b.	After microscopically viewing organisms, remove slides from the
microscope stage and discard them in a biohazard bin. If it is
necessary to keep a prepared slide, store it in a sealed petri dish or a
microscope slide case to which a biohazard label has been affixed.
c.	To decontaminate stain rinsate, collect the rinsate and add an EPA-
registered sodium hypochlorite product full strength to the rinsate in
an approximate 1:10 ratio (one part household bleach to nine parts
rinsate) for a minimum of 60 minute before disposal.

SOP No. MB-01-07
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12.8 Transport of
a.	Use of secondary containment is recommend for transporting
cultures within the laboratory. For large quantities of liquid culture,
always use secondary containment to reduce potential for generating
a large spill.
b.	For transporting live cultures between laboratories, place the cultures
in secondary containment; use a cart to facilitate transport of larger
volumes between laboratories.
c.	Biosafety Level 3 work: Remove and discard disposable lab coat
and gloves prior to exiting the Biosafety Level 3 laboratory and
replace them with a clean disposable coat and a new set of gloves to
be worn during the transport process.
d.	Autoclave bags containing biohazardous waste should be taped shut
prior to transport.
12.9 Managing
a.	After manipulating culture, analysts must bag biohazardous waste
and place it in a closed container (e.g., biohazard bin with lid,
biohazard bag taped shut).
b.	Storage of items awaiting sterilization
i.	No biohazardous waste may be removed from the second
floor B-wing prior to sterilization.
ii.	Place all contaminated articles in autoclavable bins. Place full
bins in the autoclave to await sterilization.
iii.	Place contaminated cuvettes, homogenizers, and other small
equipment into a beaker covered with aluminum foil prior to
placing the items in the autoclavable bin.
iv.	Keep all biohazardous waste-containing articles (e.g.,
autoclave bags, containers, tubes, flasks, homogenizers,
cuvettes, etc.) closed, covered, or in the BSC while awaiting
sterilization in order to prevent the generation and release of
infectious aerosols into the laboratory environment.
v.	All test tubes/flasks containing liquid waste (including used
micropipette tips) must be capped or covered with aluminum
vi.	Tape full autoclave bags closed.
vii.	It is recommended that used pipettes be collected in a waste
container (e.g., bag, bin, stainless steel beaker) inside the
BSC rather than discarded in the autoclave bag outside of the
BSC. If using a waste bin or beaker to collect pipettes, place

SOP No. MB-01-07
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about an inch of liquid disinfectant or bleach in the container
prior to collecting the pipettes. Transport the waste container
to the autoclave for sterilization and discard autoclaved
pipettes with other biohazardous waste.
viii. Biohazardous waste should be autoclaved as soon as possible
(i.e., within one day).
c.	Preparation of Autoclave Bags
i.	Place one bag in an autoclave bin.
ii.	If an autoclave bag contains only dry material, open the bag
and pour approximately 250 mL of water into the bag and 250
mL of water into the bin.
d.	Preparation of containers of liquid waste and small items.
i.	To prepare containers of liquid waste and materials such as
contaminated micropipette tips, homogenizers, racks,
cuvettes, and glassware for autoclaving, place the items in an
autoclavable bin.
ii.	If an autoclave bin contains only dry material, add
approximately 250 mL of water to the bin.
iii.	Use a three hour (180 minute) liquid cycle to sterilize both
liquid and solid biohazardous waste.
iv.	See QC-13, Performance Verification of Autoclaves, for
verification of autoclave performance and corrective actions.
12.10 Resource
a.	Laboratory personnel should be mindful of water consumption, and
whenever possible, employ practices that minimize water use.
b.	Laboratory personnel should run full autoclave loads whenever
13. Data Analysis/

SOP No. MB-01-07
Date Revised 11-24-14
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14. Forms and Data
1. Data Sheets and Attachments are stored separately from the SOP under
the following file names:
Respirator Inspection Checklist. MB-01-07 Fl.docx
Recirculating Chiller Cleaning
and Disinfection Log. MB-01-07 F2.docx
Sonicator Disinfection Log MB-01-07 F3.docx
Attachment A: Microorganisms Used
by the OPP Microbiology Laboratory MB-01-07 F4.docx
15. References
1.	Fleming, D.O. and Hunt, D.L. eds. 2000. Biological Safety: Principles and
Practices. ASM Press, Washington, D.C.
2.	Centers for Disease Control and Prevention and National Institutes of
Health, 2007. Biosafety in Microbiological and Biomedical Laboratories,
5th edition. U.S. Department of Health and Human Services. U.S.
Government Printing Office, Washington, D.C.