BIOSAFETY LABORATORY MANUAL
Chapter 1: Biosafety Laboratory Guidelines. 1
PART 1: INTRODUCTION.. 1
PART 2: BASIC LABORATORY – BIOSAFETY LEVELS 2 and 3. 1
Code of Practice. 1
PART 3: SAFETY MEASURES IN THE LABORATORY. 5
Pipetting Aids. 5
Personal Protective Equipment and Clothing. 5
Laboratory coats, gowns, coveralls, aprons. 5
Goggles, safety spectacles, face shields. 6
PART 4: GOOD LABORATORY SAFETY TECHNIQUES. 7
GMO sample containers. 7
Transport of GMO samples within the facility. 7
Opening of GMO packages. 7
Use of pipettes and pipetting aids. 7
Use of biosafety cabinets. 8
Avoidance of chemicals ingestion and contact with skin and eyes. 8
Use of centrifuges. 9
Care and use of refrigerators and freezers. 10
PART 5: DISINFECTION AND STERILIZATION.. 10
Cleaning of Laboratory Materials. 10
PART 6: CONTINGENCY PLANS AND EMERGENCY PROCEDURES. 12
Contingency Plan. 12
Part 7: Emergency Procedures for Biosafety Laboratories. 13
Puncture wounds, Cuts and Abrasions. 13
Ingestion of Potentially Infectious Material 13
Potentially Infectious Aerosol Release (outside a biosafety cabinet). 13
Breakage of Tubes in Centrifuges not having Sealable Buckets. 13
Breakage of Tubes inside Sealable Buckets (safety cups). 14
Fire and Natural Disasters. 14
Emergency Services: Whom to Contact. 14
Chapter 2 : Testing Protocol for GMOs. 15
PART 1 INTRODUCTION.. 15
PART2: List of Potential GMOs to Test for in Food, Feed and for Processing (FFP). 17
List of important plants that may be modified for improved productivity in the near future. 18
PART 3: TESTING METHODS AND CAPACITY IN NIGERIA.. 18
Technical Expertise. 18
Equipment/Apparatus required for Genetically Modified Organism (GMO) Testing/ Screening. 18
GMO Detection Procedure. 19
- Preparation of work place.. 19
- Sample preparation.. 19
- DNA Extraction.. 20
- Polymerase Chain Reaction (PCR). 20
- Electrophoresis. 21
Immunological (Protein) Testing. 21
- Lateral Flow Devices (LFDs)/Strip tests also known as “dipsticks”. 21
- ELISA Test (Enzyme-Linked Immunosorbent Assay). 21
PART 4: SAFETY ASSESSMENT OF PROTEINS USED IN GM CROPS. 22
Compositional Analysis. 23
Institutions with the capacity for GMO testing in Nigeria. 23
PART 5: PRECAUTIONS FOR HANDLING AND DISPOSAL OF GENETICALLY MODIFIED ORGANISMS (GMOs) 23
- Plant GMOs. 23
- Animal GMOs. 24
Things to Note. 25
Validation of inactivation. 26
Annex 2. iii
Chapter 1: Biosafety Laboratory Guidelines
PART 1: INTRODUCTION
The National Biosafety Management Agency (NBMA) recognizes safety in the practice of modern biotechnology in containment facilities. Biosafety Laboratory manual guides Scientists in the correct laboratory work ethics. Biosafety Laboratory manual serves as the national code of practice for the safe handling of Genetically Modified Organisms (GMOs) in laboratories.
This manual has been developed, with the view of reviewing on standard operational basis, for handling organisms in all BL2 laboratories which are unlikely to pose serious hazards to laboratory workers, the community, livestock or the environment. The laboratory facilities contained herein are designated as basic – Biosafety Level 2 based on a composite of the design features, containment facilities, practices and operational procedures.
All agents in Biosafety Level 2 laboratory have been assigned to Risk Group 2 and generally require Biosafety Level 2 practices and procedures for safe conduct of work. The assignment of biosafety level for specific work to be carried out in this laboratory is driven by professional judgement based on a risk assessment. This laboratory is appropriate to provide the necessary degree of work, following its provision for state of the art equipment in GMO analysis.
However, the availability of state-of-the-art equipment in a containment facility does not guarantee safety during laboratory operations. It is therefore necessary to adhere to all laboratory safety measures detailed herein to ensure safe conduct of experiments.
PART 2: BASIC LABORATORY – BIOSAFETY LEVEL 2
For the purposes of this manual, the guidance and recommendations are given as minimum requirements pertaining to Level 2 laboratories.
The guidelines for Basic Biosafety Level 2 presented here are comprehensive and detailed, as they are fundamental to this level of Biosafety laboratory.
Code of Practice
This code is a listing of the most essential laboratory practices and procedures that are basic. This laboratory has adopted a safety or operations manual that identifies known and potential hazards, and specifies practices and procedures to eliminate or minimize such hazards. Specialized laboratory equipment is a supplement to but can never replace appropriate procedures. The most important concepts are listed below:
- Only authorized persons should be allowed to enter the laboratory working areas.
- Laboratory doors should be kept closed.
- Children should not be authorized or allowed to enter laboratory working areas.
- No animals should be admitted other than those involved in the work of the laboratory.
- Laboratory coveralls, gowns or uniforms must be worn at all times for work in the laboratory.
- Appropriate gloves must be worn for all procedures. After use, gloves should be removed and hands must then be washed.
- Personnel must wash their hands after handling materials and before they leave the laboratory working areas.
- Safety glasses, face shields or other protective devices must be worn when it is necessary to protect the eyes and face from splashes, impacting objects and sources of artificial ultraviolet radiation.
- It is prohibited to wear protective laboratory clothing outside the laboratory, e.g.in canteens, offices, libraries, staff rooms and toilets.
- Open-toed footwear must not be worn in this facility.
- Eating, drinking, smoking, application of cosmetics and handling contact lenses is prohibited in the laboratory working areas.
- Storing human foods or drinks anywhere in the laboratory working areas is prohibited.
- Protective laboratory clothing that has been used in the laboratory must not be stored in the same lockers or cupboards as street clothing.
- Pipetting by mouth is strictly forbidden in the laboratory.
- Materials must not be placed in the mouth. Labels must not be licked.
- All technical procedures should be performed in a way that minimizes the formation of aerosols and droplets.
- The use of hypodermic needles and syringes should be limited. They must not be used as substitutes for pipetting devices or for any purpose other than aspiration of fluids.
- All spills and accidents or potential exposures to infectious materials must be reported to the laboratory supervisor. A written record of such accidents and incidents should be maintained.
- A written procedure for the clean-up of all spills must be developed and followed.
- Contaminated liquids must be decontaminated (chemically or physically) before discharge to the sanitary sewer. An effluent treatment system may be required, depending on the risk assessment for the agent(s) being handled.
- Written documents that are expected to be removed from the laboratory need to be protected from contamination while in the laboratory.
Laboratory Working Areas
- The laboratory should be kept neat, clean and free of materials that are not pertinent to the work.
- Work surfaces must be decontaminated after any spill of potentially dangerous material and at the end of the working day.
- All contaminated materials, specimens and cultures must be decontaminated before disposal or cleaning for reuse.
- Packing and transportation must follow applicable national and/or international regulations.
- When windows can be opened, they should be fitted with arthropod-proof screens.
- It is the responsibility of the Head of the laboratory (the person who has immediate responsibility for the laboratory) to ensure the development and adoption of a biosafety management plan and a safety or operations manual.
- The laboratory Head should ensure that regular training in laboratory safety is provided.
- Personnel should be advised of special hazards, and required to read the safety or operations manual and follow standard practices and procedures.
- The Head of the laboratory should ensure that all personnel understand these practices.
- Appropriate medical evaluation, surveillance and treatment should be provided for all personnel in case of need, and adequate medical records should be maintained.
Essential Biosafety Equipment
Equipment available in Biosafety Level 2 laboratory include the following:
- Pipetting aids – to avoid mouth pipetting.
- Autoclaves to decontaminate materials.
- Real Time Quantitative Thermal Block
- Water Bath
- Gradient Thermal Block
- Automatic Nucleic Acid Extractor and Desk Top
- PH Meter
- Vortex Mixer
- Measuring cylinders, Flasks, Reagent Bottles and Porcelain Crucibles
- Water distiller
- Biosafety cabinets, to be used whenever:
- potentially infectious materials are handled. Such materials may be centrifuged in the open laboratory if sealed centrifuge safety cups are used and if they are loaded and unloaded in a biological safety cabinet.
- there is an increased risk of airborne infection
- procedures with a high potential for producing aerosols are used. These may include centrifugation, grinding, blending, vigorous shaking or mixing, sonic disruption, opening of containers of infectious materials whose internal pressure may be different from the ambient pressure.
Equipment such as autoclaves and biosafety cabinets must be validated with appropriate methods before being taken into use. Recertification should take place at regular intervals, according to the manufacturer’s instructions.
Waste is anything that is to be discarded. In this laboratory, decontamination of wastes and their ultimate disposal are closely interrelated. In terms of daily use, all contaminated materials will require actual removal from the laboratory or destruction. Glassware, instruments and laboratory clothing may be reused or recycled. Appropriate waste containers and labelling should be maintained in the laboratory. The overriding principle is that all infectious materials should be decontaminated, autoclaved or incinerated within the laboratory.
The principal questions to be asked before discharge of any objects or materials from this laboratory should include:
- Have the objects or materials been effectively decontaminated or disinfected by an approved procedure?
- If not, have they been packaged in an approved manner for immediate on-site incineration or transfer to another facility with incineration capacity?
- Does the disposal of the decontaminated objects or materials involve any additional potential hazards, biological or otherwise, to those who carry out the immediate disposal procedures or who might come into contact with discarded items outside the facility?
Steam autoclaving is the preferred method for all decontamination processes. Materials for decontamination and disposal should be placed in containers, e.g. autoclavable plastic bags that are colour-coded according to whether the contents are to be autoclaved and/or incinerated. Alternative methods may be envisaged only if they remove and/or kill microorganisms.
PART 3: SAFETY MEASURES IN THE LABORATORY
The use of safety equipment is no assurance of protection unless the operator is trained and uses Standard Operating Procedure (SOP). Equipment should be tested regularly to ensure its continued safe performance. Some safety equipment available in Biosafety laboratory includes:
A pipetting aid must always be used for pipetting procedures. Mouth pipetting is strictly forbidden in the laboratory. The most common hazards associated with pipetting procedures are the result of mouth suction. Oral aspiration and ingestion of hazardous materials have been responsible for many laboratory-associated infections but pathogen ingestion can be prevented by the use of pipetting aids. Pipettes with cracked or chipped suction ends should not be used as they damage the seating seals of pipetting aids and so create a hazard.
Personal Protective Equipment and Clothing
Personal protective equipment and clothing may act as a barrier to minimize the risk of exposure to aerosols, splashes and accidental inoculation. The clothing and equipment selected is dependent on the nature of work performed. Protective clothing should always be worn when working in the laboratory. Before leaving the laboratory, protective clothing should be removed, and hands should be washed. Some personal protective equipment that must be used in the laboratory include:
Laboratory coats, gowns, coveralls, aprons
Laboratory coats should be worn fully buttoned. Aprons may be worn over laboratory coats or gowns where necessary to give further protection against spillage of chemicals or biological materials such as culture fluids. Laundering services should be provided at or near the laboratory.
Laboratory coats, gowns, coveralls, or aprons should not be worn outside the laboratory areas.
Goggles, safety spectacles, face shields
The choice of equipment to protect the eyes and face from splashes and impacting objects will also depend on the activity performed. Safety spectacles do not provide for adequate splash protection even when side shields are worn with them. Goggles for splash and impact protection should be worn over normal prescription eye glasses and contact lenses (which do not provide protection against biological or chemical hazards).
Goggles, safety spectacles, or face shields should not be worn outside the laboratory areas.
Contamination of hands may occur when laboratory procedures are performed. Hands are also vulnerable to “sharps” injuries. Disposable microbiologically approved latex, vinyl or nitrile surgical-type gloves are used widely for general laboratory work, and for handling infectious materials.
Gloves should be removed and hands thoroughly washed after handling infectious materials, working in a biosafety cabinet and before leaving the laboratory. Used disposable gloves should be discarded with laboratory wastes. Stainless steel mesh gloves should be worn when there is a potential exposure to sharp instruments e.g. during dissections. Such gloves protect against slicing motion but do not protect against puncture injury.
Gloves should not be worn outside the laboratory areas.
Respiratory protection may be used when carrying out high-hazard procedures (e.g. cleaning up a spill of infectious material). The choice of respirator will depend on the type of hazard(s).
It is important that the respirator filter is fitted in the correct type of respirator. To achieve optimal protection, respirators should be individually fitted to the operator’s face and t