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
Handling Spills of Biohazardous Materials

SOP Number: MB-13-04
Date Revised: 09-05-14


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SOP No. MB-13-04
Date Revised 09/05/14
Page 1 of 10

SOP Number

MB-13-04

Title

Handling Spills of Biohazardous Materials

Scope

The protocol presents guidelines for decontamination and cleanup of
biohazardous spills.

Application

This SOP distinguishes between large spills vs. small spills, and
spills inside vs. outside of the biological safety cabinet. Procedures
for responding to a spill may vary, depending upon the degree and
location of the spill of biohazardous material.





Approval Date

SOP Developer:



Print Name:

SOP Reviewer



Print Name:

Quality Assurance Unit



Print Name:

Branch Chief



Print Name:





Date SOP issued:



Controlled copy
number:



Date SOP withdrawn:




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SOP No. MB-13-04
Date Revised 09/05/14
Page 2 of 10

TABLE OF CONTENTS
Contents	Page Number

1.

DEFINITIONS

3

2.

HEALTH AND SAFETY

3

3.

PERSONNEL QUALIFICATIONS AND TRAINING

3

4.

INSTRUMENT CALIBRATION

3

5.

SAMPLE HANDLING AND STORAGE

3

6.

QUALITY CONTROL

3

7.

INTERFERENCES

3

8. NON-CONFORMING DATA

3

9.

DATA MANAGEMENT

4

10.

CAUTIONS

4

11.

SPECIAL APPARATUS AND MATERIALS

4

12.

PROCEDURE AND ANALYSIS

5

13.

DATA ANALYSIS/CALCULATIONS

6

14.

FORMS AND DATA SHEETS

6

15.

REFERENCES

6


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SOP No. MB-13-04
Date Revised 09/05/14
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1. Definitions

1.	Appropriate disinfectant = EPA-registered hospital disinfectant
(efficacious against S. aureus, P. aeruginosa, and S. choleraesuis),
hospital disinfectant with tuberculocidal claims (efficacious against S.
aureus, P. aeruginosa, S. choleraesuis, and M ho vis (BCG)), or EPA-
registered disinfectant towelette efficacious against spore-former
Clostridium difficile. All disinfectants must be used according to the
directions (e.g., use dilution, contact time, etc.) specified on the
labeling.

2.	Spill = A spill is defined as a biohazardous material out of control.
The quantity of the biohazardous material spilled is not the sole
determining factor in deciding whether or not an event is classified as a
spill. Rather, the essential issue is whether the biological agent, the
location, and the quantity collectively cause the situation to be beyond
the control of the laboratory worker. A major spill is one that cannot
be handled safely by laboratory employees in the immediate area. A
minor spill is one which can be handled by the laboratory workers in
the immediate area without posing a serious threat to their health and
safety, and that can be cleaned up with available absorbents and
disinfectants

Additional abbreviations/definitions are provided in the text.

2. Health and Safety

Follow procedures specified in SOP MB-01, Laboratory Biosafety. The
Study Director and/or lead analyst should consult the Material Safety Data
Sheet for specific hazards associated with any disinfectants.

3. Personnel

Qualifications and
Training

Refer to SOP ADM-04, OPP Microbiology Laboratory Training.

4. Instrument
Calibration

Not applicable

5. Sample Handling
and Storage

Refer to SOP MB-22, Disinfectant Sample Preparation.

6. Quality Control

Not applicable

7. Interferences

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.

8. Non-conforming
Data

Strict adherence to the biosafety practices is required. Nonconformance
will result in notification, retraining, or disciplinary action of laboratory
employees.


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SOP No. MB-13-04
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9. Data Management

The Branch Chief is responsible for documenting spills and accidents.

10. Cautions

1.	Lack of use or understanding of this SOP may negatively impact the
decontamination efforts of laboratory staff and hence, cause
unnecessary exposure of employees to biohazardous microorganisms.

2.	Failure to clean the ultraviolet lamps in the BSCs will reduce the
lamps' effectiveness. Periodically clean the ultraviolet lamps in the
biological safety cabinets (BSCs) with a lint-free cloth dampened with
alcohol.

3.	If a liquid bleach solution is used to decontaminate stainless steel
surfaces (e.g., BSC) following a spill, be sure to wash the surface with
water, 70% ethanol, or an EPA-registered disinfectant to remove excess
sodium hypochlorite.

11. Special Apparatus
and Materials

1.	Autoclave.

2.	Trash bags (clear in color, autoclavable) or containers inside and
outside of the biological safety cabinets for collection and storage of
biohazardous waste.

3.	Personal protective equipment (PPE) such as gloves, safety glasses, lab
coats, disposable laboratory garments, shoe covers, and temporary
clothing (i.e., scrubs).

4.	Biosafety Spill Kit containing items such as gloves and tongs for
handling broken glass, dustpan/brush, shoe covers, disposable lab coat,
and safety glasses.

5.	Signage to identify biohazardous materials and to limit access to
laboratories.

6.	Appropriate EPA-registered hospital disinfectant/tuberculocide.

7.	Bleach solutions made fresh as needed. Discard solution at the end of
the day. The container of bleach will be discarded six months from the
date of receipt or designated as use for cleaning only.

a. 1:10 diluted bleach solution at neutral pH for decontamination
purposes (spore-forming microorganisms). 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. Only
authorized personnel are permitted to enter.


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SOP No. MB-13-04
Date Revised 09/05/14
Page 5 of 10

12. Procedure and
Analysis



12.1 Guidance for
Spills of
Biohazardous
Material -
Reporting
Instructions

a.	Accidents are handled according to the practices outlined in this
subpart, as well as procedures referenced in the Occupant
Emergency Plan (OEP) and the ESC Chemical Hygiene Plan
(CHP).

b.	All spills and accidents involving a Biosafety Level 2 or 3
microorganism, regardless of how minor a spill, are reported to the
Branch Chief and the SHEM manager (or call security desk at
extension 5-2800).

c.	The Branch Chief is responsible for documenting spills and
accidents.

d.	The Branch Chief and SHEM manager will determine if additional
written documentation or follow-up is warranted.

12.2 Recommendations
for Reducing
Potential for
Spills of
Biohazardous
Material

a.	Use secondary containment (e.g., autoclave bin) when transporting
live cultures in liquid media or solid media.

b.	Use secondary containment to store biohazardous waste that is
generated during the course of an assay.

c.	Prepare the least amount of culture necessary for an assay.

d.	Maintain a clean, well-organized work environment.

12.3 Biohazardous
Organisms
Requiring
Biosafety Level 1
and 2

Containment

a. For guidance on spills outside and inside the BSC requiring
Biosafety Level 1 and 2 containment, refer to Attachment 1:
Guidance for Spills of Biohazardous Organisms Requiring
Biosafety Level 1 and 2 Containment - Spills Outside and Inside
the BSC.

12.4 Guidance for
Spills of
Mycobacterium
bovis (BCG)

a. For guidance on spills of Mycobacterium bovis, refer to

Attachment 2: Guidance for Spills of Mycobacterium bovis.

12.5 Decontamination
of Cloth Lab
Coats, Street
Clothing, and
Footwear

a.	Decontaminate clothing with an appropriate disinfectant or by
autoclaving.

b.	If using disinfection as a means of decontamination, treat area of
contamination and surrounding area with an EPA- approved
disinfectant, following the label-specified dilution and contact time.


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SOP No. MB-13-04
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c.	Clothing potentially contaminated with microorganisms in spore
form must be autoclaved according to MB-01.

d.	It is less harmful to clothing to autoclave it in a tray than it is to bag
it. Do not put water in the tray with the lab coat. Rather, put a
second tray into the autoclave and add water to this tray.

e.	After clothing is decontaminated (by disinfection or autoclaving),
immerse it in water containing detergent to aid physical removal of
decontaminated biohazardous material.

f.	Rinse lab coat and set aside to be sent out with the weekly lab coat
laundry service.

g.	Take street clothing and footwear home and launder.

13. Data Analysis/
Calculations

None

14. Forms and Data
Sheets

None

15. References

1. 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.


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SOP No. MB-13-04
Date Revised 09/05/14
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Guidance for Spills of Biohazardous Organisms Requiring Biosafety Level 1 and 2
Containment - Spills Outside and Inside the BSC

Alert workers in the laboratory that a spill has occurred and treat any injury (call 911 if
necessary).

Maintain BSC airflow: do not raise the sash or turn BSC off.

Stand the spilled container upright, and unplug any small contaminated equipment (i.e.,
vortex, timer).

Cover the spill with a paper towel (not plastic-backed material) or other appropriate
absorbent material (e.g., Isosorb 13000).

Remove any contaminated gloves, lab coat, and street clothing and place in an
autoclavable container or bag. Wash contaminated skin thoroughly with soap and water.
Replace any contaminated street clothing with temporary clothing such as scrubs (located
in B202 and B207), or jumpsuit from the shower area of the restrooms.

For small spills only on the skin, wash thoroughly with soap and water.

For large spills to a significant portion of the body and clothing, disrobe and put on
temporary clothing and shoe covers. Wash hands with soap and water. Proceed to the
restroom to shower for 10 minutes. Discard temporary clothing in autoclavable container
or bag.

Contact the SHEM Manager (or call security desk at 5-2800) and Branch Chief.

To decontaminate vegetative cells: Saturate the paper towel or other absorbent
material, starting with the edges of the spill and working towards the center, with a liquid
disinfectant (e.g. an EPA registered hospital disinfectant) and let stand for 20 to 30
minutes.

To decontaminate spore-forming bacteria: Wipe surface with a registered sodium
hypochlorite-containing towelette or a bleach solution at a neutral pH. After the contact
time wash any treated stainless steel with water, 70% ethanol, or an EPA-registered
disinfectant to remove excess sodium hypochlorite. Turn on UV light overnight.

Pick up absorbent materials and place in autoclavable container or bag. Use thick gloves,
tongs, scoop, and brush found in biosafety spill kit to clean up any broken glass and place
in autoclavable container. Carefully check the entire affected area and beyond for
remaining spill or cleanup residue.

Autoclave all contaminated materials using a 3 hour liquid cycle. Treat contaminated
gloves (for handling broken glass), tongs, scoop, and brush with disinfectant or place
under the UV light overnight. Treat contaminated PPE and any contaminated street


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SOP No. MB-13-04
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clothing with disinfectant or autoclave as described in section 12.5. Discard autoclaved
broken glass in a broken glass container.


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SOP No. MB-13-04
Date Revised 09/05/14
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Attachment 2: Guidance for Spills of Mycobacterium bovis

Follow Attachment 1 for spills inside the BSC; for spills outside of BSC proceed as
follows:

Alert workers in the laboratory that a spill has occurred and treat any injury (call 911 if
necessary).

Maintain BSC airflow: do not raise the sash or turn BSC off.

Stop all work and close all open materials.

Remove labcoat, gloves and shoe covers and discard them in a laboratory biohazard bin.
Exit the laboratory to the anteroom.

In the anteroom, remove any contaminated street clothing and place in an autoclavable
container. Replace removed street clothing with temporary clothing located in anteroom.
Wash hands.

If exposure to skin and/or clothing has occurred, proceed to the restroom to fully shower
for 10 minutes. Discard all temporary clothing in an autoclavable container. Don a new
set of temporary clothing.

Contact the SHEM Manager (or call security desk at 5-2800) and Branch Chief.

Direct everyone (via telephone, note, etc.) to stay out of the laboratory until spill cleanup
is complete.

Wait at least 30 minutes before re-entering the lab.

Re-enter the laboratory with appropriate level of PPE (including respirator for spills
outside of BSC).

Stand the spilled container upright, and unplug any small contaminated equipment (i.e.,
incubator shaker, spectrophotometer). Cover the spill with a paper towel (not plastic-
backed material) or other appropriate absorbent material (e.g., Isosorb 13000).

To decontaminate: Saturate the paper towel or other absorbent material, starting with
the edges of the spill and working towards the center, with a liquid disinfectant (e.g. an
EPA registered tuberculocide and let stand for 20 to 30 minutes).

Wearing appropriate PPE, pick up absorbent materials and place in autoclavable
container or bag. Use thick gloves, tongs, scoop, and brush found in biosafety spill kit to
clean up any broken glass and place in autoclavable container. Carefully check the entire
affected area and beyond for remaining spill or cleanup residue.


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SOP No. MB-13-04
Date Revised 09/05/14
Page 10 of 10

• Autoclave all contaminated materials using a 3 hour liquid cycle. Treat contaminated
gloves (for handling broken glass), tongs, scoop, and brush with disinfectant or place
under the UV light overnight. Treat contaminated PPE and any contaminated street
clothing with disinfectant or autoclave as described in section 12.5. Discard autoclaved
broken glass in a broken glass container.


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