1 Molecular Biology Laboratory Practices

• Biosafety is the discipline addressing the safe handling and containment of infectious microorganisms and hazardous biological materials
• Biosafety Risk assessment allows a laboratory to determine the relative level of risks and should consider activities and procedures in a laboratory that involves infectious disease agents
• Biosafety Level 2 is suitable for work involving agents of moderate hazards to personnel and environment

  • it includes microorganisms that causes diseases varying severity to humans, and are difficult to contract via aerosol in a laboratory setting
  • some of these infectious agents have therapeutics or vaccines available
• Biosafety Level 3 is applicable to facilities where work is done with indigenous/exotic agents which may cause serious or potentially lethal disease after inhalation

  • it includes microorganisms that causes severe, often fatal diseases in humans because preventive or therapeutic interventions may or may not be readily available
• Safety equipment minimizes or eliminates contact between laboratory personnel and biological hazards and provides product protection and prevents contamination of biological samples from laboratory workers and the environment
• Clothing protection such as disposable and non-disposable laboratory coats should be made of flame-retardant material and prevent contamination of personal clothing
• Gloves selection depends on the application and the material characteristics; it is important to change it between assays or specimens
• Shoe covers provide protection from reagent or sample splashes
• Eye and Face protection protects your eyes and face from specimen or reagent splashes
• In separate working areas, Clean area includes:

  • Reagent preparation area
  • Nucleic acid extraction area
• In separate working areas, Dirty area includes:

  • Nucleic acid amplification area
  • Post-amplification area
• Area A in working area is the PCR reagent mix preparation
• Area B in working area is the Nucleic acid extraction area
• Area C in working area is the PCR amplification area
• Area D in working area s the Post-amplification area
• PCR reagent mix preparation is the no template laboratory area; it is the reagent preparation area which is the cleanest work area in the molecular bio laboratory
• Nucleic acid extraction area is the specimen processing area which is also a clean area
• PCR amplification area is a dirty area where large amount of nucleic acid is generated
• Post-amplification area is the dirtiest area in the molecular bio laboratory; samples that enter in this room contains millions of amplified DNA fragments
• Thermal cycler is a machine used for PCR in PCR amplification area, since PCR is temperature regulated
• Biological agents are a major concern especially for specimen processing area
• Nucleic acid may produce inaccurate quantitative data or false positive during contamination in the molecular laboratory
• Nucleases may produce false negative results during contamination in the molecular laboratory
• Barrier pipette tips are used to prevent sample contamination
• Nuclease-free water, tubes, and pipet tips for reagent preparation is also used to prevent contamination
• A unidirectional workflow is always ensured that it is properly followed to prevent contamination
• In decontamination of Area B, disinfection methods should be used to inactivate infectious agents such as bacteria, viruses, and other microorganisms
• In decontamination of Areas A, C, D, reduce possible introduction of unwanted nucleic acids or nuclease enzymes to your assay
• Removing nucleic acid and nuclease in Area B is also required in decontamination of the area
• Disinfectants are a variety of liquid disinfectants can be used to decontaminate surfaces and equipment
• Heat and pressure such as autoclaving or steam sterilization is used in decontamination of biological agents
• In decontamination of nucleic acid, 10% household bleach is used in wiping laboratory working surfaces or soaking equipment
• In decontamination of nucleic acid, a UV light is used to decontaminate surfaces and equipment such as small vortexers and pipettes
• A commercial DNA removing reagents are also used in decontamination of nucleic acid
• DNase can be destroyed by autoclaving
• RNase is a heat tolerant enzyme and requires additional treatments to completely inactivate
• It is the responsibility of the Laboratory Supervisor to conduct a biosafety risk assessment before conducting any procedure in the laboratory that involves pathogenic materials
• All laboratory staff members must be familiar with the risk assessments relevant to their work
• Between the clean and dirty areas, the workflow should be unidirectional and the relative air pressure and direction should differ