Standard Operating Procedures for the Handling of Transgenic Plant Pathogenic Fungi


Michael Thon           July 19, 2006

Laboratory Handling Procedures:

1.    All work with transgenic isolates of Colletotrichum spp. are done in strict adherence to NIH BL1/BL2-P containment procedures (see excerpts at the end of this document.)

2.    The Laboratory (Lab N 1) should be locked whenever it is unoccupied.  Visitors should be greeted by a lab member upon entering the lab.  Lab members working alone in a back portion of the lab and not near the main door should lock the door to prevent anyone from entering without your knowledge.

3.    All cultures will be handled (transfer of cultures, preparation of spore suspensions etc and other manipulations) in the class II-A biological safety cabinet in Lab 1.Cultures will be disposed of in (covered) bucket lined with plastic, autoclave-safe bags.  When the bags are full, they will be sealed with tape and autoclaved.

4.    Disposable materials that come into contact with any form of fungal propagule will be and placed in the waste culture buckets.


  1. Spills should be cleaned up with paper towels (wear latex gloves) and 70% ethanol.  Used towels should be placed in the waste culture buckets.

Plant Inoculation Studies:

1.    Inoculation will be performed in a designated growth chamber only.

2.    Conidial suspensions will be prepared in the flow hood and transported to the inoculation chamber in sealed disposable sample tubes. The tubes and will be autoclaved after use.

3.    Plants will be inoculated in the growth chamber.  They will be placed in plastic bags and sprayed with fungal conidia through a small opening in the top of the bag.  The plants will be removed from the bags after 24h and the bags will then be placed in biohazard bags and autoclaved.

4.    Infected plants will be grown in the inoculation chamber until disease assessment and will then be transferred to biohazard bags and disposed of by autoclaving.

5.    All materials that are routinely used for inoculation experiments (trays, sprayers etc.) will not be removed from the inoculation chamber unless it is necessary.  In this case they will be removed in closed containers and autoclaved.

6.    All disposable items that come into contact with inoculum will be placed in sealed plastic bags and placed within biohazard bags. The biohazard bags will be autoclaved immediately.




Excerpts from



Section III-E-2.  Experiments Involving Whole Plants

This section covers experiments involving recombinant DNA-modified whole plants, and/or experiments involving recombinant DNA-modified organisms associated with whole plants, except those that fall under Section III-A, III-B, III-D, or III-F.  It should be emphasized that knowledge of the organisms and judgment based on accepted scientific practices should be used in all cases in selecting the appropriate level of containment.  For example, if the genetic modification has the objective of increasing pathogenicity or converting a non-pathogenic organism into a pathogen, then a higher level of containment may be appropriate depending on the organism, its mode of dissemination, and its target organisms.  By contrast, a lower level of containment may be appropriate for small animals associated with many types of recombinant DNA-modified plants.

Section III-E-2-a.  BL1-P is recommended for all experiments with recombinant DNA-containing plants and plant-associated microorganisms not covered in Section III-E-2-b or other sections of the NIH Guidelines.  Examples of such experiments are those involving recombinant DNA-modified plants that are not noxious weeds or that cannot interbreed with noxious weeds in the immediate geographic area, and experiments involving whole plants and recombinant DNA-modified non-exotic (see Section V-M, Footnotes and References of Sections I-IV) microorganisms that have no recognized potential for rapid and widespread dissemination or for serious detrimental impact on managed or natural ecosystems (e.g., Rhizobium spp. and Agrobacterium spp.).

Section III-E-2-b.  BL2-P or BL1-P + biological containment is recommended for the following experiments:

Section III-E-2-b-(1).  Plants modified by recombinant DNA that are noxious weeds or can interbreed with noxious weeds in the immediate geographic area.

Section III-E-2-b-(2).  Plants in which the introduced DNA represents the complete genome of a non-exotic infectious agent (see Section V-M, Footnotes and References of Sections I-IV).

Section III-E-2-b-(3).  Plants associated with recombinant DNA-modified non-exotic microorganisms that have a recognized potential for serious detrimental impact on managed or natural ecosystems (see Section V-M, Footnotes and References of Sections I-IV).

Section III-E-2-b-(4).  Plants associated with recombinant DNA-modified exotic microorganisms that have no recognized potential for serious detrimental impact on managed or natural ecosystems (see Section V-M, Footnotes and References of Sections I-IV).

Section III-E-2-b-(5).  Experiments with recombinant DNA-modified arthropods or small animals associated with plants, or with arthropods or small animals with recombinant DNA-modified microorganisms associated with them if the recombinant DNA-modified microorganisms have no recognized potential for serious detrimental impact on managed or natural ecosystems (see Section V-M, Footnotes and References of Sections I-IV).


Appendix G-II-A.    Biosafety Level 1 (BL1) (See Appendix G-III-M, Footnotes and References of Appendix G)

Appendix G-II-A-1.   Standard Microbiological Practices (BL1)

Appendix G-II-A-1-a.  Access to the laboratory is limited or restricted at the discretion of the Principal Investigator when experiments are in progress.

Appendix G-II-A-1-b.  Work surfaces are decontaminated once a day and after any spill of viable material.

Appendix G-II-A-1-c.  All contaminated liquid or solid wastes are decontaminated before disposal.

Appendix G-II-A-1-d.  Mechanical pipetting devices are used; mouth pipetting is prohibited.

Appendix G-II-A-1-e.  Eating, drinking, smoking, and applying cosmetics are not permitted in the work area.  Food may be stored in cabinets or refrigerators designated and used for this purpose only.

Appendix G-II-A-1-f.  Persons wash their hands:  (i) after they handle materials involving organisms containing recombinant DNA molecules and animals, and (ii) before exiting the laboratory.

Appendix G-II-A-1-g.  All procedures are performed carefully to minimize the creation of aerosols.

Appendix G-II-A-1-h.  In the interest of good personal hygiene, facilities (e.g., hand washing sink, shower, changing room) and protective clothing (e.g., uniforms, laboratory coats) shall be provided that are appropriate for the risk of exposure to viable organisms containing recombinant DNA molecules.


Appendix P-II-B-1.   Standard Practices (BL2-P)

Appendix P-II-B-1-a.   Greenhouse Access (BL2-P)

Appendix P-II-B-1-a-(1).  Access to the greenhouse shall be limited or restricted, at the discretion of the Greenhouse Director, to individuals directly involved with the experiments when they are in progress.

Appendix P-II-B-1-a-(2).  Personnel shall be required to read and follow instructions on BL2-P practices and procedures.  All procedures shall be conducted in accordance with accepted greenhouse practices that are appropriate to the experimental organisms. 

Appendix P-II-B-1-b.     Records (BL2-P)

Appendix P-II-B-1-b-(1).  A record shall be kept of experimental plants, microorganisms, or small animals that are brought into or removed from the greenhouse facility.

Appendix P-II-B-1-b-(2).  A record shall be kept of experiments currently in progress in the greenhouse facility.

Appendix P-II-B-1-b-(3).  The Principal Investigator shall report any greenhouse accident involving the inadvertent release or spill of microorganisms to the Greenhouse Director, Institutional Biosafety Committee, NIH/OBA and other appropriate authorities immediately (if applicable).  Reports to the NIH/OBA shall be sent to the Office of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD  20892-7985 (20817 for non-USPS mail), 301-496-9838, 301-496-9839 (fax).  Documentation of any such accident shall be prepared and maintained.

Appendix P-II-B-1-c.     Decontamination and Inactivation (BL2-P)

Appendix P-II-B-1-c-(1).  Experimental organisms shall be rendered biologically inactive by appropriate methods before disposal outside of the greenhouse facility.

Appendix P-II-B-1-c-(2).  Decontamination of run-off water is not necessarily required.  If part of the greenhouse is composed of gravel or similar material, appropriate treatments should be made periodically to eliminate, or render inactive, any organisms potentially entrapped by the gravel.

Appendix P-II-B-1-d.     Control of Undesired Species and Motile Macroorganisms (BL2-P)

Appendix P-II-B-1-d-(1).  A program shall be implemented to control undesired species (e.g., weed, rodent, or arthropod pests and pathogens) by methods appropriate to the organisms and in accordance with applicable state and Federal laws.

Appendix P-II-B-1-d-(2).  Arthropods and other motile macroorganisms shall be housed in appropriate cages.  If macroorganisms (e.g., flying arthropods or nematodes) are released within the greenhouse, precautions shall be taken to minimize escape from the greenhouse facility.

Appendix P-II-B-1-e.     Concurrent Experiments Conducted in the Greenhouse (BL2-P)

Appendix P-II-B-1-e-(1).  Experiments involving other organisms that require a containment level lower than BL2-P may be conducted in the greenhouse concurrently with experiments that require BL2-P containment provided that all work is conducted in accordance with BL2-P greenhouse practices.

Appendix P-II-B-1-f.    Signs (BL2-P)

Appendix P-II-B-1-f-(1).  A sign shall be posted indicating that a restricted experiment is in progress.  The sign shall indicate the following:  (i) the name of the responsible individual, (ii) the plants in use, and (iii) any special requirements for using the area. 

Appendix P-II-B-1-f-(2).  If organisms are used that have a recognized potential for causing serious detrimental impacts on managed or natural ecosystems, their presence shall be indicated on a sign posted on the greenhouse access doors.

Appendix P-II-B-1-f-(3).  If there is a risk to human health, a sign shall be posted incorporating the universal biosafety symbol.

Appendix P-II-B-1-g.     Transfer of Materials (BL2-P)

Appendix P-II-B-1-g-(1).  Materials containing experimental microorganisms, which are brought into or removed from the greenhouse facility in a viable or intact state, shall be transferred in a closed non-breakable container.

Appendix P-II-B-1-h.     Greenhouse Practices Manual (BL2-P)

Appendix P-II-B-1-h-(1).  A greenhouse practices manual shall be prepared or adopted.  This manual shall:  (i) advise personnel of the potential consequences if such practices are not followed, and (ii) outline contingency plans to be implemented in the event of the unintentional release of organisms.

Appendix P-II-B-2.     Facilities (BL2-P)

Appendix P-II-B-2-a.   Definitions (BL2-P)

Appendix P-II-B-2-a-(1).  The term “greenhouse” refers to a structure with walls, a roof, and a floor designed and used principally for growing plants in a controlled and protected environment.  The walls and roof are usually constructed of transparent or translucent material to allow passage of sunlight for plant growth.

Appendix P-II-B-2-a-(2).   The term “greenhouse facility” includes the actual greenhouse rooms or compartments for growing plants, including all immediately contiguous hallways and head-house areas and is considered part of the confinement area.

Appendix P-II-B-2-b.     Greenhouse Design (BL2-P)

Appendix P-II-B-2-b-(1).  A greenhouse floor composed of an impervious material.  Concrete is recommended, but gravel or other porous material under benches is acceptable unless propagules of experimental organisms are readily disseminated through soil.  Soil beds are acceptable unless propagules of experimental organisms are readily disseminated through soil.

Appendix P-II-B-2-b-(2).  Windows and other openings in the walls and roof of the greenhouse facility may be open for ventilation as needed for proper operation and do not require any special barrier to exclude pollen or microorganisms; however, screens are required to exclude small flying animals (e.g., arthropods and birds). 

Appendix P-II-B-2-c.   Autoclaves (BL2-P)

Appendix P-II-B-2-c-(1).  An autoclave shall be available for the treatment of contaminated greenhouse materials.

Appendix P-II-B-2-d.   Supply and Exhaust Air Ventilation Systems (BL2-P)

Appendix P-II-B-2-d-(1).  If intake fans are used, measures shall be taken to minimize the ingress of arthropods.  Louvers or fans shall be constructed such that they can only be opened when the fan is in operation.

Appendix P-II-B-2-e.   Other (BL2-P)

Appendix P-II-B-2-e-(1).  BL2-P greenhouse containment requirements may be satisfied by using a growth chamber or growth room within a building provided that the external physical structure limits access and escape of microorganisms and macroorganisms in a manner that satisfies the intent of the foregoing clauses.


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