US Environmental Protection Agency
Office of Pesticide Programs
Office of Pesticide Programs
Microbiology Laboratory
Environmental Science Center, Ft. Meade, MD
Standard Operating Procedure for
Biosafety in the Laboratory
SOP Number: MB-01-06
Date Revised: 10-19-11

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SOP No. MB-01-06
Date Revised 10-19-11
Page 1 of27
Initiated By:
EPA/OPP MICROBIOLOGY LABORATORY
ESC, Ft. Meade, MD
Standard Operating Procedure
for
Biosafety in the Laboratory
SOP Number: MB-01-06
Date Revised: 10-19-11
Date: / /
Print Name:
Technical Review:
Date: / /
QA Review:
Approved By:
Effective Date:
Print Name:
Technical Staff
Print Name:
QA Officer
Print Name:
Branch Chief
/ /
Date: / /
Date: / /
Controlled Copy No.:
Withdrawn By:
Date: / /

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SOP No. MB-01-06
Date Revised 10-19-11
Page 2 of27
TABLE OF CONTENTS
Contents	Page Number
1.0 SCOPE AND APPLICATION	4
2.0 DEFINITIONS	5
3.0 HEALTH AND SAFETY	6
4.0 CAUTIONS	7
5.0 INTERFERENCES	8
6.0 PERSONNEL QUALIFICATIONS	8
7.0 SPECIAL APPARATUS AND MATERIALS	8
8.0 INSTRUMENT OR METHOD CALIBRATION	9
9.0 SAMPLE HANDLING AND STORAGE	9
10.0 PROCEDURE AM) ANALYSIS	9
10.1	General Laboratory Practices	 10
10.2	Access to Laboratories and Placement of Signage	10
10.3	Checking Airflow to Laboratories
Equipped with Monitoring Devices	11
10.4	Using the BSC	11
10.5	PPE Requirements	12
10.6	Disinfection of Laboratory Equipment	16
10.7	Conducting Staining of
Microorganisms	17
10.8	Transport of Cultures	18

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SOP No. MB-01-06
Date Revised 10-19-11
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TABLE OF CONTENTS (continued)
Contents	Page Number
10.9	Managing Biohazardous Waste	19
10.10	Resource Management	20
11.0 DATA ANALYSIS/CALCULATIONS	21
12.0 DATA MANAGEMENT/RECORDS MANAGEMENT	21
13.0 QUALITY CONTROL	21
14.0 NONCONFORMANCE AND CORRECTIVE ACTION	 21
15.0 REFERENCES	21
16.0 FORMS AND DATA SHEETS	22

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SOP No. MB-01-06
Date Revised 10-19-11
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1.0 SCOPE AND APPLICATION:
1.1	This protocol outlines the required safety measures for working with the
microorganisms received and maintained by the OPP Microbiology Laboratory.
1.2	The scope of 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.
1.3	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
ref. 15.2).
1.3.1	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.
1.3.2	This SOP is structured so all work involving manipulation of culture
(section 2.4) 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.
1.3.3	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.
1.3.4	Recommendations set forth in Section D (Laboratory Facilities) of the
BMBL's Biosafety Level 3 criteria apply only to Biosafety Level 3
microorganisms.
1.3.4.1	Manipulation of Biosafety Level 1 and 2 microorganisms
may occur in either a Biosafety Level 2 (B201, B203,
B204, B205, B206, or D122) or a Biosafety Level 3
laboratory (B202, B207).
1.3.4.2	Manipulation of Biosafety Level 3 microorganisms may
only occur in a Biosafety Level 3 laboratory (B202, B207).

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1.4	Prior to manipulating (section 2.4) cultures of a microorganism other than those
listed in Attachment A, the laboratory staff must first determine the biosafety
level of that microorganism (see Attachment A).
1.5	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 guidelines.
DEFINITIONS:
2.1	Microorganism = includes bacteria in vegetative and spore form, bacteria in
biofilms, and viruses.
2.2	Mycobacterium bovis (BCG) is a live attenuated vaccine strain organism.
2.3	BSC = biological safety cabinet
2.4	Manipulation of culture = handling of open vessels containing microorganism.
Activities involving manipulation of culture must be performed in the BSC. The
following are examples (not an exhaustive list) of activities involving
manipulation of culture: culture transfers, inoculum or virus harvest, plating,
inoculation of carriers, sonication of inoculated carriers, recording results from
tubes and plates.
2.5	PPE = Personal Protective Equipment
2.6	CDC = Centers for Disease Control and Prevention
2.7	NIH = National Institutes of Health
2.8	"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 directions (e.g., use dilution, contact
time, etc.) specified on the labeling.

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2.9	ATCC = American Type Culture Collection
2.10	OEP = Occupant Emergency Plan
2.11	CHP = Chemical Hygiene Plan
2.12	P100 HEPA = Oil proof High-Efficiency Particulate Air Filter, 99.97% efficient
in removing particles 0.3 microns or larger
2.13	References to water mean reagent-grade water
HEALTH AND SAFETY:
3.1	To protect the laboratory worker from possible infection by microorganisms, the
health and safety guidelines provided in this protocol and in the BMBL (see ref.
15.2) 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.
3.2	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.
3.3	Laboratory workers are required to participate in the Agency's Occupational
Medical Surveillance Program as established by EPA Order 1460.1. The Branch
Chief evaluates the duties and responsibilities of the team and identifies the
employees that are subject to exposure to chemical and biological agents in the
laboratory. The names are forwarded to the ESC Safety, Health and
Environmental Management (SHEM) manager who has responsibility for
coordinating the medical monitoring program. The program is administered
through the Department of Health and Human Services/U.S. Public Health
Service.
3.4	Medical emergencies are handled according to procedures outlined in the ESC
Occupant Emergency Plan (OEP). All emergencies are reported to the Branch
Chief and SHEM manager. In the event that analysts are unable to reach the
SHEM manager, they are to call security at extension 52800 and report the
emergency. Security will locate the SHEM manager or his designee. The Branch
Chief is responsible for documenting medical emergencies or accidents.

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SOP No. MB-01-06
Date Revised 10-19-11
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3.5	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 CHP. All
spills and accidents are reported to the Branch Chief and the SHEM manager.
The Branch Chief is responsible for documenting spills and accidents.
3.6	To promote the health of exposed individuals, the Branch Chief will encourage
individuals to seek follow up, if necessary, depending upon recommendations of
the SHEM manager.
3.7	All laboratory workers must meet the requirements of the Hazard Communication
Program's Employee Training Program, as described in the CHP.
3.8	In accordance with the CDC/NIH guidelines (see ref. 15.2), the Branch Chief may
restrict access to the laboratory as specified under "special practices".
3.9	All employees required to use respirators are participants in the Agency
Occupational Medical Surveillance Program and the ESC Respiratory Protection
Program. They have been medicallycleared, fit tested for the specific respirator,
and attended initial respirator use training. They receive annual respirator fit
testing, annual use training, have documentation of training placed in the training
file, and ensure that their respirators are inspected before and after each use, or at
least monthly, (see 16.1) as specified in the CHP.
CAUTIONS:
4.1	Lack of following or understanding of this SOP may negatively impact the quality
of the microbiological practices used in the laboratory and, hence, the laboratory's
mission.
4.2	Failure to use the "STOP/DO NOT ENTER" signs to control access to the
laboratory while cultures are being manipulated (section 2.4) may result in the
inadvertent exposure of personnel to biohazardous microorganisms.
4.3	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.
4.4	Autoclaving flasks containing diluted bleach may result in pitting of the
autoclave's stainless steel interior.

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SOP No. MB-01-06
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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 disposal.
5.0 INTERFERENCES:
5.1	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.
5.2	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, Use and Maintenance of Biological Safety Cabinets.
5.3	Proper certification of the BSCs must be maintained by Facilities management.
6.0 PERSONNEL QUALIFICATIONS:
6.1	Personnel are required to be knowledgeable of the procedures in this SOP.
Documentation of training and familiarization with this SOP can be found in the
training file for each employee.
6.2	Each new analyst will complete twenty-four hours of initial safety training before
entering the laboratory to work. All analysts will complete safety re-certification
training (eight hours of training) on at least an annual basis. The facility SHEM
manager is responsible for coordinating the training program.
7.0 SPECIAL APPARATUS AND MATERIALS:
7.1	Biological Safety Cabinet (BSC)
7.2	Autoclave
7.3	Biohazard bags (clear in color, autoclavable) or containers inside and outside of
the biological safety cabinets for collection and storage of biohazardous waste.
7.4	When specified, personal protective equipment (PPE) such as gloves, safety
glasses, lab coats, disposable laboratory garments, and respiratory protection (e.g.,
respirators with PI00 HEPA filter cartridges).
7.5	Appropriate signs to identify biohazardous materials and to limit access to
laboratories.

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SOP No. MB-01-06
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7.6	Appropriate disinfectants for microorganisms listed in Attachment A.
7.6.1	For microorganisms in vegetative form, use a 1:10 bleach solution (1
part EPA-registered sodium hypochlorite product, 9 parts water) or
EPA- registered hospital disinfectant/tuberculocide/virucide.
7.6.2	For microorganisms in spore form, prepare 1:10 diluted bleach
solution at neutral pH. Using an EPA-registered sodium hypochlorite
product containing approximately 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).
7.7	Key card readers are used to limit access to testing laboratories. Only authorized
personnel are permitted to enter.
7.8	Microorganisms maintained by the laboratory are specified in Attachment A.
7.9	Secondary containment (e.g., durable, autoclavable trays and bins; containment
cart).
8.0 INSTRUMENT OR METHOD CALIBRATION:
8.1 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 QC-06,
Use and Maintenance of Biological Safety Cabinets, for additional information.
9.0 SAMPLE HANDLING AND STORAGE: Not applicable.
10.0 PROCEDURE AND ANALYSIS:
Organization of Section 10.0
(Information for quick finder)
	10.1:	General Laboratory Practices
	10.2:	Access to Laboratories and Placement of Signage
	10.3:	Checking Airflow to Laboratories Equipped with Monitoring Devices
	10.4:	Using the BSC
	10.5:	Personal Protective Equipment (PPE) Requirements
o	Safety glasses
o	Lab coats

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SOP No. MB-01-06

Date Revised 10-19-11

Page 10 of 27
o
Gloves
o
Face protection
o
Respiratory protection
10.6:
Disinfection of Laboratory Equipment
10.7:
Conducting Staining of Microorganisms
10.8:
Transport of Cultures
10.9:
Managing Biohazardous Waste
10.10:
Resource Management
General Laboratory Practices
10.1.1
Eating, drinking, smoking, handling contact lenses, chewing gum, and

applying cosmetics (including lip balm) are not permitted in the

laboratory.
10.1.2
Storing food for human consumption in laboratory areas is prohibited.
10.1.3
Laboratory workers must not wash or reuse disposable gloves.
10.1.4
Wash hands prior to leaving the laboratory.
10.1.5
Mouth pipetting is prohibited. Only mechanical pipetting devices will

be used.
10.1.6
Organism will be manipulated inside a BSC to minimize risk of

exposure and risk of contamination of lab surfaces.
10.1.7
All procedures are performed carefully to minimize the creation of

aerosols.
10.1.8	Animals and plants not related to work being conducted are not
permitted in the laboratory.
10.1.9	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.
10.1.10	For filtering Biosafety Level 3 microorganisms, protect vacuum lines
with disinfectant traps and HEPA filters.
10.2 Access to Laboratories and Placement of Signage.

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SOP No. MB-01-06
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10.2.1	Key readers are used to limit access to testing laboratories. Only
authorized personnel (e.g., laboratory staff, maintenance staff, etc.) are
permitted to enter.
10.2.2	Further limit access to the laboratory when manipulating infectious
microorganisms by posting the magnetic "STOP/DO NOT ENTER"
sign on the outside (i.e., side facing corridor) of the external laboratory
door.
10.2.3	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.
10.2.4	Remove the "STOP/DO NOT ENTER" sign once work is complete.
10.3 Checking Airflow to Laboratories Equipped with Monitoring Devices.
10.3.1 Prior to entering laboratories equipped with airflow monitoring
devices (B202 and B207), visually verify that the laboratories are
under negative pressure (airflow is into the room from the corridor).
10.3.2	Airflow monitors are located above the laboratory doors. The
laboratories have two monitors: 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 will roll from the
corridor into the laboratory or anteroom, or from the anteroom into the
lab. Monitors must indicate negative airflow for entry.
10.3.3	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).
10.3.4	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 (x4357). Work should only proceed when
proper airflow has been restored.
10.4 Using the BSC.

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10.4.1	If ultraviolet light was left on overnight for decontamination purposes,
turn it off.
10.4.2	Turn on the blowers, lights, and outlets, and allow to operate for a
minimum of 15 minutes prior to use.
10.4.3	Record the Downflow (FPM) and Exhaust (CPM) rates on the BSC
Monitoring Record Form immediately prior to use (See SOP QC-06,
Use and Maintenance of Biological Safety Cabinets).
10.4.4	BSC surfaces must be disinfected prior to and after working with
infectious material and immediately after any spill of infectious
material. 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. Halogenated materials
are not recommended for routine use on stainless steel surfaces of the
BSC.
10.4.5	As an additional step, 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. Visually verify
that UV bulb is clean. If not, wipe with alcohol and record in BSC
Monitoring Record Book.
10.4.6	See SOP QC-06, Use and Maintenance of Biological Safety Cabinets,
for more details.
10.4.7	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.
10.5 PPE Requirements.
10.5.1 Safety glasses. Safety glasses must be worn while working in the
laboratory. Safety glasses do 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 (see section 2.4) or other laboratory work is in progress.
However, safety glasses should be immediately available in the work
area.

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SOP No. MB-01-06
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10.5.2 Lab coats.
10.5.2.1	Lab coats do 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 (see section 2.4) or other
laboratory work is in progress.
10.5.2.2	For Biosafety Levels 1 and 2: Wear cloth or disposable lab
coats.
10.5.2.2.1 Remove lab coats before going to non-
laboratory areas such as the office areas,
restrooms, cafeteria, library, etc.
10.5.2.3	For Biosafety Level 3: Wear disposable lab coats.
10.5.2.3.1	Cloth lab coats may not be worn in the
Biosafety Level 3 laboratory during
manipulation of Biosafety Level 3
microorganisms.
10.5.2.3.2	Replace disposable lab coat immediately in
the event of suspected or known
contamination with infectious material.
Dispose of contaminated lab coat in a
biohazard bin.
10.5.2.3.3	All disposable lab coats worn in the
laboratory when cultures are being
manipulated may not be worn out of the
Biosafety Level 3 laboratory. This includes
lab coats worn by personnel who are present
in the laboratory but not directly involved in
culture manipulation. Prior to exiting the
laboratory and entering the double door
access zone, remove and discard lab coats
(in biohazard bin) according to the
descriptions of activities provided in
sections 10.5.2.3.4 and 10.5.2.3.5.

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SOP No. MB-01-06
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10.5.2.3.4	Disposable lab coats worn while harvesting
or homogenizing cultures of a Biosafety
Level 3 microorganism must be discarded in
a biohazard bin before the analyst leaves the
laboratory and enters the double-door access
zone. The used lab coat will not be stored in
the laboratory for re-use.
10.5.2.3.5	Disposable lab coats worn for purposes
other than harvesting or homogenizing
cultures of a Biosafety Level 3
microorganism during the day may be stored
in a designated area in the laboratory in the
event that the analyst leaves the laboratory
(e.g., to retrieve an item or take a break).
Upon return, the analyst may re-use the lab
coat to resume laboratory activities. At the
end of each day, all used disposable lab
coats should preferably be disposed of in the
biohazard bin.
10.5.3 Gloves.
10.5.3.1	Wear gloves (latex or nitrile when manipulating culture
(section 2.4) and when handling any vessel (e.g., tube, rack,
plate, biohazard bag), closed or open, containing live
organism.
10.5.3.2	Prior to beginning work, inspect gloves. Do not use gloves
that have holes, rips, or are otherwise degraded.
10.5.3.3	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.
10.5.3.4	Under certain circumstances (e.g., testing of towel ette
products), to minimize risk of contamination of the test
system while working in the BSC, analysts may opt to
periodically apply a solution of 70% ethanol over the
exterior surface of the gloves, change gloves frequently, or
use sterile gloves.

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10.5.3.5 Additional guidance for Biosafety Level 3 work: Wear a
single pair of gloves (latex or nitrile) when manipulating
culture (see section 2.4). Prior to handling materials
outside of the BSC, the analyst should surface
decontaminate used gloves (section 10.5.3.4) or discard the
used gloves and put on a new pair.
OR
Wear a double layer of gloves (latex or nitrile) when
manipulating culture (see section 2.4). 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.
10.5.4 Face protection.
10.5.4.1 Biosafety Levels 1 and 2: If a certain procedure involving
manipulation of the organism is impossible or impractical
to conduct within the BSC, face protection (e.g., a face
splash shield) must be used for protection from anticipated
or unanticipated splashes or sprays of infectious materials
to the face.
10.5.5 Respiratory protection.
10.5.5.1	Biosafety Levels 1 and 2: Respirators are not required for
manipulations of Biosafety 1 and 2 microorganisms.
10.5.5.2	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

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with mild soap and water if desired. Allow to dry and
return to storage.
10.5.5.2.1	Transporting closed petri dishes (containing
inoculated carriers) to the incubator for
drying, counting colonies on plates which
are closed and wrapped with parafilm, and
using a capped cuvette to transport culture to
the spectrophotometer and read percent
transmittance or optical density are not
considered to be aerosol-producing
manipulations of culture. Wearing
respiratory protection is not mandatory for
these activities.
10.5.5.2.2	To reduce the volume of organism during a
potential spill, use semimicro cuvettes (4
mm inside width that limits capacity to 1.5
mL) with 1.0 mL sample volume. Place the
capped cuvettes in a cuvette rack for
transport to the spectrophotometer.
10.6 Disinfection of Laboratory Equipment
10.6.1 Chillers. On a weekly basis, following testing, disinfect the water
(chillers are filled with tap water) in the recirculating chiller and
remote water bath prior to draining.
10.6.1.1	Disinfect the tap 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 16.0).
10.6.1.2	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.

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10.6.1.3	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).
10.6.1.4	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.
10.6.1.5	Rinse the unit with tap water and drain as above. Refill the
reservoir with fresh tap water on the day of testing.
10.6.2	Sonicator. If the sonicator has been used in a given week, 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 16.0).
10.6.3	Spectrophotometer. 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 spectrophotometer.
10.6.3.1 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
operation.
10.6.4	Administrative supplies (Biosafety Level 3). Use and distribution of
supplies used during testing of Biosafety Level 3 agents will be limited
to the Biosafety Level 3 laboratories.
10.7 Conducting Staining of Microorganisms

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10.7.1 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.
10.7.2 Decontamination of rinsate.
10.7.2.1 Collect the rinsate and add EPA-registered sodium
hypochlorite product full strength to the rinsate in an
approximate 1:10 ratio (one part household bleach to nine
parts rinsate) (see ref. 15.1, pg. 390) for a minimum of 60
minutes (in the event that the rinsate contains spores)
before disposal.
10.7.3 After microscopically viewing organisms, remove slides from the
microscope stage and discard them in a biohazard bin. If it is
necessary or desirable 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.
10.8 Transport of Cultures.
10.8.1	Use of secondary containment is recommended for transporting
cultures within the laboratory. For large quantities of liquid culture,
always use secondary containment to reduce potential for generating a
large spill.
10.8.2	For transporting live cultures between laboratories, place the cultures
in secondary containment; use a cart to facilitate transport of larger
volumes between laboratories.
10.8.3	Biosafety Level 3 work: Remove and discard disposable lab coat and
gloves (latex or nitrile) prior to exiting the Biosafety Level 3
laboratory and replace them with a clean disposable coat and a new set
of gloves (latex or nitrile) to be worn during the transport process.
10.8.4	Autoclave bags containing biohazardous waste should be taped shut
prior to transport.
10.8.5	Do not transport live cultures outside of the microbiology laboratory
wing.

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SOP No. MB-01-06
Date Revised 10-19-11
Page 19 of 27
10.9 Managing Biohazardous Waste
10.9.1	After manipulating culture (see section 2.4), analysts must bag
biohazardous waste and place it in a closed container (e.g., biohazard
bin with lid, biohazard bag taped shut).
10.9.2	Storage of items awaiting sterilization.
10.9.2.1	No biohazardous waste may be removed from the second
floor B-wing prior to sterilization.
10.9.2.2	Use autoclave bins. Some procedures result in the constant
generation of contaminated articles that must be removed
from the BSC work area and stock-piled on a countertop or
table for autoclaving at the completion of the procedure or
the next morning. In this case, place all contaminated
articles in autoclavable bins. Place full bins in the
autoclave to await sterilization.
10.9.2.3 Small items. Place contaminated cuvettes, homogenizers,
and other small equipment into a beaker covered with
aluminum foil prior to placing the items in the autoclavable
bin.
10.9.2.4	Close containers. 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.
10.9.2.5	Cover liquid waste with foil. All test tubes/flasks
containing liquid waste (including used micropipette tips)
must be capped or covered with aluminum foil.
10.9.2.6	Autoclave bags. Tape full autoclave bags closed.
10.9.2.7	Pipettes. 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 about an inch of liquid disinfectant or bleach

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SOP No. MB-01-06
Date Revised 10-19-11
Page 20 of 27
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.
10.9.2.8 Biohazardous waste should be autoclaved as soon as
possible (i.e., within one day).
10.9.3 Preparation of autoclave bags.
10.9.3.1 To prepare autoclave bags of biohazardous waste for
sterilization, place one bag in an autoclave bin.
10.9.3.2 Open the bag and pour approximately 250 mL of water into
the bag and 250 mL of water into the bin.
Bags containing biohazardous waste must be left open and
unsealed during autoclaving.
10.9.4 Preparation of containers of liquid waste and small items.
10.9.4.1	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.
10.9.4.2	Add approximately 250 mL of water to the bin.
10.9.4.3	Place the bin into the autoclave.
10.9.5	Use a three hour (180 minute) liquid cycle to sterilize both liquid and
solid biohazardous waste.
10.9.6	See QC-13, Performance Verification of Autoclaves, for verification
of autoclave performance and corrective actions.
10.10 Resource Management.
10.10.1 Water conservation. Laboratory personnel should be mindful of water
consumption, and whenever possible, employ practices that minimize
water use.

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SOP No. MB-01-06
Date Revised 10-19-11
Page 21 of 27
10.10.2 Specifically, laboratory personnel should run full autoclave loads
whenever possible.
11.0 DATA ANALYSIS/CALCULATIONS: None
12.0 DATA MANAGEMENT/RECORDS MANAGEMENT:
12.1	Results of the monthly respirator check will be recorded legibly and in indelible
ink on the Respirator Inspection Checklist form (see 16.0). The forms will be
kept in the Respirator Inspection Notebook.
12.2	The Respirator Inspection Notebook is kept in laboratory space rather than the file
room, D217. In addition to monthly inspections, respirators must be inspected
prior to each use. Therefore, it is crucial that the Respirator Inspection Notebook
be readily available rather than retained in a file room. Archived data is subject to
OPP's official retention schedule contained in SOP ADM-03, Records and
Archives. The SHEM manager may request copies of the Respirator Inspection
Checklist forms at any time.
12.3	Data will be recorded promptly, legibly, and in indelible ink on the BSC
Monitoring Record Form (see SOP QC-06, Use and Maintenance of Biological
Safety Cabinets) and the Sonicator Disinfection Log. Completed forms are
archived in notebooks kept in secure file cabinets in file room D217. Only
authorized personnel have access to the secure files. Archived data are subject to
OPP's official retention schedule contained in SOP ADM-03 (Records and
Archives).
12.4	The Branch Chief is responsible for documenting medical emergencies, accidents,
and spills.
13.0 QUALITY CONTROL: None
14.0 NONCONFORMANCE AND CORRECTIVE ACTION:
14.1 Strict adherence to the biosafety practices is required. Nonconformance will
result in notification, retraining, or possible disciplinary action of laboratory
employees.
15.0 REFERENCES:
15.1 Fleming, D.O. and Hunt, D.L. eds. 2000. Biological Safety: Principles and
Practices. ASM Press, Washington, D.C.

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SOP No. MB-01-06
Date Revised 10-19-11
Page 22 of 27
15.2 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.
16.0 FORMS AND DATA SHEETS:
16.1	Respirator Inspection Checklist.
16.2	Recirculating Chiller Cleaning and Disinfection Log.
16.3	Sonicator Disinfection Log.
Attachment A: Microorganisms Maintained by the OPP Microbiology Laboratory.

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SOP No. MB-01-06
Date Revised 10-19-11
Page 23 of 27
Respirator Inspection Checklist
OPP Microbiology Laboratory
Respirators must be inspected before and after each use, and monthly. A respirator kept ready for emergency use must be inspected before and
after each use and monthly to assure that it is maintained in satisfactory condition. Use this sheet to document the inspections.
Name

Type of Respirator

Storage Location

Date










Initials










Facepiece










Connections










Headbands










Valves










Cartridges










Other










Comments

Safety Office Review:
Date:

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SOP No. MB-01-06
Date Revised 10-19-11
Page 24 of 27
Recirculating Chiller Cleaning and Disinfection Log
OPP Microbiology Laboratory
INFORMATION
Chiller#
Location
Date/Init.
Action*
















































































*Record the action performed, i.e. disinfected, drained, cleaned, etc.
+ 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).

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SOP No. MB-01-06
Date Revised 10-19-11
Page 25 of 27
Sonicator Disinfection Log
OPP Microbiology Laboratory
INFORMATION
Dale/I nil.
Unit* (check a box)
Use
Treatment Performed
(product, dilution, contact, time)**
Serial Number
RKA10980258D
Serial Number
RKC100274019D





















































































*Units used for disinfectant efficacy testing.
**Units hold approximately 1500 mL of water.

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SOP No. MB-01-06
Date Revised 10-19-11
Page 26 of 27
Attachment A:
Microorganisms Maintained by the OPP Microbiology Laboratory
Organism
ATCC#
Biosafcty Level
Mycobacterium bovis (BCG)*
Not applicable
2A-3
Pseudomonas aeruginosa*
15442
2
Pseudomonas aeruginosa***
700888
2
Staphylococcus aureus*
6538
2
Salmonella enterica *
10708
2
Bacillus subtilis*
19659
1
Streptococcus equi subsp. Zooepidemicus**
43079
2
Enterococcus casseliflavus**
700327
1
Kocuria kristinae**
BAA-752
1
Listeria monocytogenes**
BAA-751
2
Staphylococcus aureus subsp. aureus**
29213
2
Staphylococcus saprophytics**
BAA-750
1
Staphylococcus sciuri subsp. sciuri**
29061
1
Streptococcus thermophilus**
19258
1
Proteus vulgaris**
6380
2
Shigella sonnei**
25931
2
Stenotrophomonas maltophilia**
17666
1
Klebsiella oxytoca**
700324
2
Acinetobacter baumannii**
BAA-747
2
Enterobacter cloacae**
700323
1
Ochrobactrum anthropi**
BAA-749
2
Aneurinibacillus aneurinolyticus**
11376
1
Bacillus circulans**
61
1
Brevibacillus agri**
51663
1
Brevibacillus laterosporus**
64
1
Geobacillus stearothermophilus**
12978
1
Paenibacillus macerans**
8509
1
Paenibacillus polymyxa**
7070
1
Virgibacillus pantothenticus**
14576
1
Bacillus licheniformis**
12759
1
Clostridium sporogenes*
3584
1
Clostridium difficile***
700057
2
Clostridium difficile***
43598
2
Clostridium difficile***
43599
2
Clostridium difficile***
700792
2
Clostridium difficile***
9689
2
Mycobacterium terrae***
15755
1
Feline Calicivirus (Strain F-9)****
VR-782
2

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SOP No. MB-01-06
Date Revised 10-19-11
Page 27 of 27
Organism
ATCC#
Biosafety Level
Influenza A Vims (H3N2)****
VR-544
2
Biosafety Level 2 with Biosafety Level 3 practices and equipment
*	Used as a Test Microbe in the Antimicrobial Testing Program
** Used in Quality Control of the VITEK 2 Compact
*	* * Foruseinresearch
****Used as Test Microbe for the Virucidal Testing Program

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