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UNITED STATES DEPARTMENT OF AGRICULTURE  

ANIMAL AND PLANT HEALTH INSPECTION SERVICE  

PLANT PROTECTION AND QUARANTINE  

CONTAINMENT GUIDELINES  

FOR PLANT PATHOGENIC NEMATODES  

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(Revised: Dec.2009) (Nematodes)  

UNITED STATES DEPARTMENT OF AGRICULTURE  

  ANIMAL AND PLANT HEALTH INSPECTION SERVICE  

PLANT PROTECTION AND QUARANTINE  

   CONTAINMENT GUIDELINES  

FOR PLANT PATHOGENIC NEMATODES  

(Revised: Feb., 2010) (Nematodes)  
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CONTAINMENT GUIDELINES FOR NEMATODES  

CONTENTS  

I. PURPOSE OF THIS DOCUMENT  

II. CONSTRUCTION STANDARDS FOR THE ENTIRE CONTAINMENT STRUCTURE

A. Location and Identification

B. Floor Plan  

C. Walls, ceilings, and floors

D. Windows

E. Doors

F. HVAC system

G. Electrical system

H. Plumbing system

I. Vacuum Cleaning systems and Vacuum Aspiration system

J. Communication system

III. CONSTRUCTION STANDARDS FOR SPECIALIZED ROOMS

A. Greenhouses  

B. Vestibules  

C. Showers and Restrooms  

IV. EQUIPMENT STANDARDS

A. Benches, tables and other furniture  

B. Sterilization or Decontamination equipment  

C. Cages and Containers  

D. Biosafety Cabinet  

V. OPERATIONAL STANDARDS  

A. Containment Director  

B. Authorized Personnel  

C. Apparel  

D. Personal Cleanliness  

E. Facility Cleaning and disinfecting  

F. Opening and Handling packages  

G. Starting, Growing, and Storing cultures  

H. PPQ Regulatory Requirements  
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CONTAINMENT GUIDELINES FOR NEMATODES  

I. PURPOSE OF THIS DOCUMENT:  

These guidelines are a reference to help you (a scientist of a state, federal, or commercial entity) design,

build, maintain, and operate a containment facility for plant pathogenic nematodes that are non‐

indigenous or of limited distribution. Widely distributed indigenous nematodes may not require a

containment facility. These guidelines are not intended for nematodes of medical and veterinary

importance. If there is doubt as to the applicability of guidelines to specific organisms, contact

Containment Facilities Staff (CF), or Permits and Risk Assessment (PRA) scientific personnel for

appropriate guidance.  

During inspections or reinspections of your facility, USDA, APHIS, PPQ personnel will review these

guidelines and any risk mitigation instructions that may accompany your permit. When your facility

meets containment standards and risk mitigation instructions it will be considered “adequate" from a

containment standpoint; then permits may be issued to you.

The inspection and permitting procedures of the USDA, APHIS, PPQ are intended to prevent the release

of nonindigenous plant pests to the environment of the United States. Accidental or purposeful release

of these organisms is a violation of the PLANT PROTECTION ACT and is subject to civil and/or criminal

penalties, loss of permits, and destruction of valuable cultures.  

Components of this Document:  

Your containment facility must meet the “Standards” listed in the grey, shaded boxes. To help you meet

these standards, ask biotechnology industries, university Biosafety committees, and/or contractors for

research hospitals and other research institutions to recommend specialized professional contractors.

Professionals know state, local and federal laws that regulate construction, including the installation of

emergency doors, incinerators, air intake and exhaust ducts, emergency lighting, plumbing, and many

other features. PPQ’s permit specialists have little or no knowledge of these laws.  

N. B.   Notify PPQ of any structural changes prior to implementation, the development of blueprints,

signing of construction contracts, start of construction, etc.

The “Suggestions” listed under each Standard are methods or equipment that are commonly used at

this time to accomplish each containment standard. The design, construction and operation of your

containment facility may vary, depending on the organisms you wish to contain, your research

objectives, the functionality of your equipment and structural components, and your location. Again, we

recommend professional advice on all design issues. [Some other useful containment references (non‐

USDA‐APHIS) are: 1) "Containment Facilities and Safeguards for Exotic Plant Pathogens and Pests ".  

1999.   R.  P. Kahn and S. B Mathur. (eds).  APS Press, St. Paul, MN.  2) " A Practical Guide to Containment

(Plant Biosafety in Research Greenhouses ) ".  Adair, D. and R. Irwin. 2008 (revised edit.). Information
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Systems for Biotechnology, Virginia Tech, Blacksburg (http:// www.isb.vt.edu);  3) Containment

Standards for Facilities Handling Plant Pests.  2009.(1st edition) Canadian Food Inspection Agency, Office

of Biohazard Containment and Safety,

(http://www.inspection.gc.ca/english/sci/bio/plaveg/placone.shtml)]).  

Safety of facility personnel should not be compromised by containment requirements.  

Emergency exits should not be blocked by equipment; their operation should not be obstructed with

tape or caulk.  

We encourage Good Laboratory Practices (GLP) in all facilities. The standards and suggestions listed here

only cover containment. Cross contamination of cultures within the containment may or may not signify

poor containment.  

USDA, APHIS, PPQ welcomes alternatives that are proven to meet or exceed the standards. To insure

timely permitting, please review this document and research design alternatives. Once design options

are narrowed, call or fax PPQ’s Containment Facility staff at (301) 734‐5592 or FX (301) 734‐5392 and

continue discussions as the facility is planned and built.   
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II. CONSTRUCTION STANDARDS FOR THE ENTIRE STRUCTURE

SUGGESTIONS:  

1. Locate the facility in areas relatively free of agricultural zones, high‐risk microclimates (e.g..

known flood zones) or other high‐risk areas.

2. If possible design the containment facility as a separate, dedicated building. If this is not

possible, design and build to prevent pest escape.

3. Install a 15 foot‐ wide buffer strip of a material that doesn’t support plant growth   outward

from the foundation to surround the containment building(s) ;  (no soil or host plants should be

in this area )

4. Install barriers to prevent unlawful entry into the facility.

5. Post  a sign with the following information:

• Containment director/ containment officer name and contact numbers.

• A sign stating: “ACCESS IS BY AUTHORIZED PERSONNEL ONLY”.

• Emergency telephone numbers.

SUGGESTIONS:  

1. Install one primary entry/exit.

2. The entry/exit should have a vestibule with self closing doors with at least 6 ft. separating them.

3. There should be a foot bath inside the vestibule constructed to decontaminate feet upon exiting

the facility. Booties and disposal container and a rack for dedicated footwear would be useful.

4. Design the facility with laboratories and rearing rooms connected to a main laboratory.

5. Build restrooms outside of containment rooms. However, if restrooms must be built inside a

containment room, use the same construction standards used throughout the facility and place

the restroom next to low risk areas.

6. Build offices outside of containment areas.

7. Install self‐closing doors throughout the containment structure(s).

8. Install exterior doors that lock.

9. Install a central closet for cleaning supplies.

CONSTRUCTION STANDARD A. Locate the facility in areas with minimal human, agricultural and

environmental risk. Identify the facility as dedicated and secure.

CONSTRUCTION STANDARD B. Design the floor plan to prevent escape of the enclosed organism(s).
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SUGGESTIONS:  

1. Construct the walls and ceilings with building materials that resist moisture and withstand

repeated decontaminations with bleach or other caustic solutions.

2. Walls and ceilings should be painted a light color for detection of dirt or debris which may

harbor organisms.

3. Flooring cannot have any porous elements;

Monolithic (in one‐piece) floors, e.g. poured concrete, sheet linoleum, chemically resistant

paint, etc. are desirable. Wood floors are not acceptable.  Floor should be able to withstand

repeated cleanings

4. Install floor drains to collect liquid wastes for sterilization.

5. Seal junctions, holes or penetrations of walls, ceilings, and floors with plaster, caulk, or

equivalent materials.

SUGGESTIONS:  

1. Install breakage resistant glazing (Plexiglas, Lexan, etc).

2. Install windows that do not open.

3. Seal joints between the windowsills, frames, etc. and walls with appropriate materials.

4. Store extra transparent panels nearby for emergency use.

SUGGESTIONS:  

1. Install exterior doors that lock.

2. Install thresholds and gaskets that seal the exterior and interior doors with their frames. The

space between seal and doorframe should not exceed 6.3mm (0.250 inch).

3. Emergency doors

 Insure emergency doors are not commonly used as an entrance ( may  use “ panic bars “ on

the interior  side )

CONSTRUCTION  STANDARD  C.  Construct walls, ceilings and floors that are impenetrable to the

enclosed organisms, and withstand repeated cleaning and decontamination

CONSTRUCTION STANDARD D.  If windows are necessary, install Windows impenetrable to the

enclosed organisms.

CONSTRUCTION STANDARD E. Install Doors that contribute to the security of the facility.
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SUGGESTION:

While an HVAC system is not required for use with many nematode species, if for example, the

nematode (such as Bursaphelenchus  spp.) is vectored by an aerial organism an HVAC system may be

required.  Contact PPQ if necessary.

SUGGESTIONS:  

1. Install an alarm to indicate power failure. (optional)

2. Install an alternative power source (generator, battery bank, etc.) for use when normal power is

lost or interrupted. (optional )

3. Install weatherproof electrical boxes, receptacles, light fixtures, switches, etc.

4. Seal electrical boxes, lighting, switches, wiring, conduit, etc, with appropriate materials (caulk,

foam, etc,) that are impenetrable to the contained organisms and withstand repeated

decontaminations with bleach or other caustic solutions.

SUGGESTIONS:  

1. Seal sewer or drains (sink, floor, shower, etc.) with suitable filters (and perhaps prefilters) to

prevent escape of infected host material, soil, and cysts or dormant stages of nematodes.

2. Sterilize effluents from sinks, floor drains, etc with steam or its equivalent before releasing them

into the sewer system.

3. Install inline sink traps for laboratories and greenhouses.

4. In the alternative that large quantities of effluent cannot be handled, develop and submit an

SOP that describes how effluent will be handled to reduce pest risk prior to disposal.

CONSTRUCTION STANDARD G. Design and install an Electrical System that maintains containment

features under normal and emergency situations and is impenetrable to the contained organisms.

CONSTRUCTION STANDARD H. Design and install a Plumbing System to contain the organisms and

remove liquid wastes.

CONSTRUCTION STANDARD F. Design and install an HVAC System (Heating, Ventilation and Air

Conditioning) that prevents escape of the contained organisms.
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SUGGESTIONS:  

1. Use vacuum appliance only in facility.  

2. Autoclave or sterilize vacuum filters and waste before disposal.  

1. Install a telephone(s) or intercom system.  

2. Install a computer (LAN, modem, etc.) or Fax machine to allow for communication and data

transfer to and from the facility.  

3. Electronic devices (i.e., cell phones, etc.) may not be removed from containment area without

suitable decontamination.

III. CONSTRUCTION STANDARDS FOR SPECIALIZED ROOMS.  

1. Inoculated plants may be confined in walk‐in large growth chambers and growth rooms with  

HEPA filtered HVAC systems. This is preferable and more secure than greenhouses for containing

organisms.  

2. Construct the foundation of concrete, concrete block, brick, or similar material.  

3. Extend the foundation below the soil line to insure a permanent and stable structure. Build the

foundation at least 3 ft. above the soil line.  

4. Construct greenhouse floors of materials that are impervious to the contained organisms and

can withstand repeated disinfecting with caustic liquids.  

CONSTRUCTION STANDARD I. Install Vacuum Systems and Vacuum Aspiration Systems that prevent

the escape of the contained organisms

CONSTRUCTION STANDARD J. Install Communication System that allows communication between the

interior and exterior of the facility and prevents organism escape.

CONSTRUCTION STANDARDS FOR SPECIALIZED ROOMS A. Build Greenhouses with security and

containment features.
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5. Install a frame strong enough to support the translucent walls and ceilings.

• Install translucent wall and ceiling materials strong enough to guarantee the security of the

facility. Plexiglas, lucite, lexon, safety glass, and wire‐reinforced glass are acceptable.

Polyethylene, vinyl or plastic sheeting are NOT acceptable.

• Seal the translucent panels to the frame with caulk or appropriate materials on the inside

and outside surfaces.

• Consider the installation of screens over the roof to protect it from hail.

6. Seal joints between the greenhouse and other containment rooms with caulk or other suitable

material.

7. If containment greenhouse is detached from the primary containment facility, install a vestibule

at each door. (See specialized room section on Vestibules.)

8. Install doors between the greenhouse and the rest of the facility that close completely, and seal

to their frames. Use doors that are windowless or cover windows with blinds.

9. Ensure any HVAC system can be turned off to allow greenhouse fumigation.

SUGGESTIONS:  

1. Install a vestibule in each entrance and /or exit. (Have contractor check local construction codes

on vestibules at emergency exits, as they may be prohibited.)

2. Shower rooms can count as a vestibule for the main entrance (see restroom construction).

3. Design vestibule to be at least 6 feet long from door threshold to door threshold.

4. Insure vestibule doors interlock, so that only one door can be opened at a time.

5. Insure thresholds and gaskets of doors seal to their frames.

6. Shoe racks useful if dedicated footwear is used.

7. Provide bin for discarded disposable footwear.

8. If no vestibule, then have a walkway with mats or shaker capability.

SUGGESTIONS:  

1. Place showers/ restrooms near lowest risk rooms.

2. Ensure thresholds and gaskets of restroom doors seal to their frames.

CONSTRUCTION STANDARDS FOR SPECIALIZED ROOMS B. Vestibules

CONSTRUCTION STANDARDS FOR SPECIALIZED ROOMS C. Install Showers and Restrooms to prevent

organism escape.
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IV. EQUIPMENT STANDARDS

SUGGESTIONS:  

1. Install work surfaces and laboratory furniture (bench tops, etc.) impervious to water, and

resistant to chemicals and heat.

2. Insure spaces between benches, wall cabinets,  and  equipment  are easy to clean and inspect.

3. Dedicate cleaning equipment (mops, brooms, buckets, etc.) for use only in the containment

area, and store it in the containment area.

4. Repair or replace dented, broken or cracked benches and tables if soil or dirt can become

imbedded in the surfaces.  Disposable blotter material maybe required as a liner between the

work surface and the bench or table.

SUGGESTIONS:  

1. Install an autoclave. A double‐door pass‐through model is recommended. Conduct tests to

evaluate effectiveness of autoclave on a regular basis.

2. Install a gas sterilizer for articles that would be damaged by steam. A double‐door pass‐through

model is recommended.

3. If you are considering an incinerator within the facility, consult your contractor about state,

local, and federal laws and ordinances.

4. Since large quantities of soil may be used in nematode research, rooms for sterile and

contaminated should be kept separate, and should be equipped with a high pressure steam

decontamination system to decontaminate the inside and outside of the rooms including walls,

ceilings, and floors.

5. Clean potting soil and contaminated soil with other solid wastes must be subjected to a

minimum temperature of 104°C (220°F) for three hours. [SEE USDA‐APHIS TREATMENT MANUAL

for soil‐‐online].  The efficacy of the treatment must be verified by test organisms (such as

Geobacillus stearothermophilus endospores teststrips/kits).

EQUIPMENT STANDARD A. Use Benches, Tables and Other Furniture that are easy to inspect and clean.

EQUIPMENT STANDARD B.  Use equipment to sterilize contained organisms, soil, plant growing

medium, plant material, and solid waste.  Decontaminate solid waste and contaminated or infested

articles before removing it from the facility.
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SUGGESTIONS:  

1. Install a biosafety cabinet, Class II, type A/B3 in which to open foreign source packages; it

should be tested/certified annually by a professional technician.

2. Have access to a biosafety cabinet for initial opening of packages to ensure contents don’t

contain unpermitted materials and/or contaminants.

V. OPERATIONAL STANDARDS  

SUGGESTIONS:  

A Containment Officer is responsible for the organisms contained in the facility. He/she also maintains a

copy of the Standard Operating Procedures (SOP) Manual for the facility. SOPs contain directions for

normal use, maintenance, and disinfection of the facility and its equipment.  

SOPs also describe how to:  

• Respond to a typical emergency event (power outage, fire, glass breaks in containment area

(flood, hurricane, earthquake, tornadoes, etc.). Maintain biosecurity for unauthorized removal

of materials from containment area.  

• Replace translucent panels in greenhouse.  

• Monitor visitors.

• Clean the facility.

Copies of SOPs should be available to workers within the containment areas. Each revision must be

dated.  

Containment Officer:

• Implements the SOPs and conditions listed in permits for organisms held by the facility.  

• Trains employees and/or authorized personnel in the SOPs.  

EQUIPMENT STANDARD C. Install a Biosafety cabinet to work with organisms.

OPERATIONAL STANDARD A. A Containment Director is responsible for the daily operation of the

facility and its physical integrity.
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• Updates copies of construction records (blueprints) for the facility.

• Maintains daily, weekly and monthly maintenance records of the facility.

The Containment Officer updates these lists:  

• The names and phone numbers of those to call during emergencies as changes occur.

• The plant species in facility, as changes occur.

• Authorized personnel, as changes occur.

• Incoming and outgoing shipments of permitted organisms, including dead or destroyed

incoming organisms, and submits list as per permit conditions to USDA, APHIS, PPQ, PRA

(Permits & Risk Assessment)  or by January 31 of each year (see address on last page.)

Once your facility is adequate to receive plant pests, the behaviors of people who access your facility

will have far more impact on the containment of the organism than any containment feature. Your

selection of individuals to work in this facility is critical to maintenance of plant pest containment. In

addition to picking good personnel, please consider the suggestions below.  

SUGGESTIONS:  

1. Ensure only authorized personnel have keys.

2. Train authorized personnel in the SOPs.

3. List the personnel authorized to enter the facility.

4. Require that visitors sign a logbook upon entry and exit.  Authorized personnel must accompany

visitors at all times to insure they do not bring in or carry out pests.

5. Insure emergency exit doors are not used routinely as an entrance.

SUGGESTIONS:  

1. Insure visitors and employees wear laboratory coat in the containment area and remove it prior to

leaving the containment area. Foot covers, rubber aprons, and/or gloves may be appropriate.

OPERATIONAL STANDARD B. Only Authorized Personnel have routine access to the facility.

OPERATIONAL STANDARD C.  Wear, sterilize, and handle personal Apparel to minimize the risk of

organism escape.
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2. Prohibit entry of overcoats, hats, backpacks, purses, etc. into the containment areas, as these

articles may allow infested soil to adhere and organisms escape containment.

SUGGESTION:  

1. Ensure authorized personnel and visitors clean and disinfect shoes before exiting.

2. Ensure laboratory coats are periodically washed in hot water.

SUGGESTIONS:  

1. Clean and disinfect the facility, its furniture, and its equipment regularly with bleach or a similar

disinfectant.

2. Eliminate undesired pests , pathogens  and weeds from the facility.

3. Autoclave or sterilize solid wastes (cultures, plant materials, soil, trash, etc.) prior to disposal.

SUGGESTIONS:  

1. Establish one enclosed area that is easy to disinfect, and use this area to open packages received

from foreign sources if a biosafety cabinet is not available.

2. Place foreign source packages in the biosafety cabinet before opening.

3. Autoclave or incinerate packing materials immediately after the removal of specimens and

cultures.

4. Establish procedures to eliminate hitchhiking pests (insects, entomopathogens, fungi, bacteria,

etc.)

OPERATIONAL STANDARD D. Use Personal Cleanliness to contain organisms.

OPERATIONAL STANDARD E. Clean and Disinfect the interior of the facility and its equipment regularly.

OPERATIONAL STANDARD F. Open and Handle packages of permitted organisms to prevent organism

release.
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SUGGESTIONS:  

1. List all arthropod and/or plant materials used to maintain nematode populations.

2. Sterilize/destroy all packing materials from shipments and contaminants shortly after receipt.

3. 3. Autoclave or sterilize used   media prior to disposal.  

4. Destroy contaminated populations as soon as the contamination is detected.

5. Store tissue cultures in screw‐ top vials, sealable tissue culture containers or the equivalent.

SUGGESTIONS:  

1. Meet all PPQ requirements or conditions as listed in permits for organisms kept in the facility.

Permits from other Federal and State Agencies may also be required for certain plant pests.

Receipt of USDA Plant Pest Permits does not relieve applicants from the responsibility of

obtaining other permits. USDA Permits may be withheld or revoked if other Federal and State

requirement are not satisfied.

2. Send SOP and blueprints to:

Containment Facilities Program

USDA, APHIS, PPQ, PPB, CF, Unit 133

4700 River Road, Riverdale, MD 20737

Phone (301) 734‐5592

FAX (301) 734‐5392

3. Obtain permission from PPQ prior to shipping regulated organisms outside of the facility. Only

ship regulated organisms to permit holders who supply a copy of their valid permit to receive

these organisms.  Follow all permit conditions that specify shipping requirements in their

permit.

4. Maintain a log of all organisms that leave and enter the facility under permit.

OPERATIONAL STANDARD G.  Maintain nematode populations with as few contaminants as

possible. Cross contamination indicates poor laboratory practice. However, it may not indicate

containment problems.

OPERATIONAL STANDARD H. Follow all PPQ Regulatory Requirements for organisms received,

reared in, or released from the containment facility.
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Submit the above list to USDA APHIS PPQ (at address below) by January 31 of every year  

USDA, APHIS, PPQ, PPB, Unit 133  

4700 River Road, Riverdale, MD 20737  

Phone (301) 851-2046

FAX (301) 734‐4300  

5. If the facility stops operating as a containment facility, either temporarily or permanently,

immediately notify PPQ.