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Environment, Health, & Safety Division

Appendix D

Good Microbiological Practice

D.1    Introduction and Scope

This appendix describes 1) customary principles of good microbiological practice (GMP), and 2) explains the differences between GMP and laboratory biosafety practices defined by the Centers for Disease Control and Prevention (CDC) and the National Institutes of Health (NIH) and provided in Appendix C (Laboratory Biosafety Level 1 and 2 Criteria) of this manual. These GMP principles are guidelines that may be used to control the biosafety and research quality aspects of laboratory work. These guidelines are not biosafety requirements unless other sections of this manual describe them as biosafety requirements.

The first and most important element of control for research product protection and laboratory containment is strict adherence to 1) GMP, and 2) standard microbiological practices and special practices. These sets of practices have different main objectives, but include many overlapping practices and secondary objectives. Both sets of practices should be used when conducting work.

  • Good Microbiological Practice (GMP) is aseptic techniques and other good microbiological practices that are not uniformly defined but are necessary to prevent contamination of the laboratory with the agents being handled and contamination of the work with agents from the environment. GMP is used to keep the agents being handled inside their primary containers without any other organisms getting in and contaminating the research materials. The main objective of GMP is to ensure that contamination does not affect the research results.

  • Standard microbiological practices and special practices are defined by the CDC and NIH, discussed in Section 4.1, and listed in detail in Appendix C of this manual. Standard microbiological practices and special practices are used much like GMP to keep agents inside their primary containers. However, the main objective of these practices is to provide safety controls needed to protect workers and the environment from contamination in the event that the agents are accidentally released from their primary container.

D.2    Good Microbiological Practice

GMP involves the use of aseptic techniques and other good microbiological practices. These practices and techniques achieve two objectives:

  • Prevent handled organisms from contaminating the laboratory, and
  • Prevent organisms in a laboratory environment from contaminating the work.

Both objectives are important, but the first objective is primarily important for the safety of the worker, while the second objective is mostly important for the quality of the research.

The principles of GMP should generally be applied to all types of work involving microorganisms and tissue cultures, regardless of containment level.

D.2.1   Aseptic Technique

An aseptic technique is a procedure used to grow a microorganism or culture of interest in a clean micro-environment isolated from the outside world. This micro-environment is usually some sort of culture or holding container such as a flask, bottle, or petri dish. The organisms or cells can either be on a solid agar medium or be suspended in a broth, diluent, or other fluid medium.

Examples of aseptic techniques include ensuring all components of the system are sterile prior to use (e.g., container interior, growth medium, and any items used in manipulation) and using special care and techniques to avoid cross-contamination during the inoculation, incubation, and processing steps. They also include:

  • Keeping the container closed except for the minimum time required to introduce or remove materials.
  • Holding open containers at an angle whenever possible to prevent contaminants from entering the container.
  • Protecting sterile containers from contamination, and working with these containers inside a biosafety cabinet (BSC). When working outside a BSC, a Bunsen burner may be used to flame the opening of the container whenever tops are removed (i.e., passing the opening quickly through the Bunsen flame). The upwards current of hot air created by the Bunsen burner prevents contaminated air or particles from dropping into the culture container when the lid is open.
  • Using manipulation techniques that minimize the possibility of cross-contamination (e.g., opening lids with the little finger so that tops are not put down on the work surface).
  • Ensuring that all tools (e.g., pipette tips or loops) or other items that may come in contact with the culture are sterile and not contaminated by casual contact with the bench, fingers, or outside of the bottle. Also ensuring that these tools are disposed of or decontaminated immediately after use.

In addition to aseptic technique, GMP includes a wide range of other working methods that minimize the cross-contamination of the work and workplace. Examples of these methods are provided in the remaining sections of this appendix.

D.2.2   Personal Hygiene and Dress

  • Wash hands prior to and following manipulations of organisms or cultures and whenever contamination is suspected.
  • Wear personal protective equipment (PPE) to protect the worker and to prevent research materials from contamination. Change gloves when contaminated. Routinely clean lab coats or throw away disposable coats.
  • Tie back or confine loose or long hair.
  • Do not touch the skin, face, or unclean or nonsterile surfaces.
  • Keep fingernail tips at a length of one-quarter inch or shorter.

D.2.3   Area Cleanliness and Organization

Keep the laboratory and work area clean and organized, such as in the following examples:

  • Keep only items necessary for the task in progress on the bench or in the BSC. This practice avoids unnecessary clutter that may collect contaminants, prevent surface disinfection and spill cleanup, and increase the possibility of things getting knocked over.
  • Plan and lay out work so that everything needed for a procedure is ready to be handled in a logical order. This practice should allow the worker to sit at the BSC or bench and handle the items efficiently using aseptic techniques.
  • Use appropriate chemical antimicrobials (e.g., disinfectants) and decontamination procedures. See Appendix F of this manual.
  • Wash hands and disinfect work surfaces before and after work.
  • Immediately clean spills, and then disinfect the work surface and wash hands.
  • Organize the work area when work is complete.
  • Avoid putting items on the floor. This practice allows the cleanliness of the floor to be viewed, allows all parts of the floor to be cleaned routinely, eases spill cleanup, and prevents tripping hazards.
  • Routinely clean water baths to minimize microbial contamination of the water.
  • Routinely clean laboratory surfaces such as open shelving, benchtops, windowsills, and items on them to prevent accumulation of dust and debris. Store infrequently used items in cabinets and drawers.
  • Routinely clean floors and difficult-to-access areas to prevent buildup of dust and debris.
  • Routinely clean sink faucets and basins.
  • Identify areas and systems in the laboratory and support areas (e.g., wash and autoclave area) for storage and staging of dirty, contaminated, clean, and sterilized items that are being stored, used, or processed for eventual reuse. Ensure everyone understands and follows the system.
  • Periodically review items stored in refrigerators and freezers and on shelves and benches. Dispose of items that are no longer needed.

D.2.4   Biosafety Cabinets and Airborne Contamination

  • Use a BSC when needed to protect biological research materials and when procedures may generate biohazardous aerosols. See Appendix E, Section E.3 (Biosafety Cabinet Work Practices and Procedures) for additional GMP and containment work practices related to work in a BSC.
  • Minimize personnel traffic and unnecessary movements around the work area or BSC. Such movements cause area air turbulence that may transport contaminants into the work area and onto the biological materials that need protection. Such movements also disturb clean laminar airflows inside BSCs responsible for containing aerosols and protecting biological materials.

D.2.5   Manipulation Techniques for Minimizing Aerosols

Manipulation techniques should be used that minimize the possibility of producing aerosols. Examples include:

  • Mixing by gentle rolling and swirling rather than vigorous shaking (to avoid frothing).
  • Pipetting by putting the tip into a liquid or onto a surface prior to gently ejecting the pipette contents (to avoid bubbling and splashing).
  • Placing containers in very close proximity to each other when transferring liquids between them (to avoid drops that fall and splash).
  • Allowing loops to cool down after incineration or flaming before using the loop (to avoid sizzling).
  • Not overfilling centrifuge tubes (to avoid leakage into centrifuge).
  • Slowly removing tube caps or stoppers.
  • Not popping caps off of tubes.
  • Carrying and storing cultures (e.g., bottles and plates) in racks and spill-proof containers (to prevent dropping and breakage).

D.2.6   Worker Qualifications

Workers who handle microorganisms and cultures should have sufficient technical competence, training, and experience in GMP and containment practices. In addition, workers should use GMP and biosafety containment in anticipation of unexpected hazards when handling microorganisms and cultures (including Risk Group 1). Workers should conservatively approach their safety by assuming, for example, that an unexpected pathogen may be present or contaminate the culture; a pathogen may be unintentionally cultured; the disease potential of the agent may be altered under laboratory conditions; or exposure to an RG1 agent may cause an opportunistic infection.

D.2.7   Microbial Contamination Checks

Routine microbial contamination checks should be incorporated into protocols and undertaken at various stages of experiments. Examples of contamination checks include:

  • Taking a loopful of fluid from the vessel and plating (or streaking) it out onto a nonselective solid nutrient medium to look for single colonies.
  • Incubating culture samples at a suitable temperature (usually 30°C) to allow growth of contaminants originating from the general environment and human sources.
  • After incubation, examining plates for evidence of any contamination as indicated by colony types.

The purity of a liquid culture can also be obtained by microscopic examination. This is done by placing a loopful of the culture on a microscope slide. The slide is then examined wet either by phase contrast microscopy, or by fixed or Gram staining. Contaminant organisms should be instantly and clearly visible.

Contamination checks are particularly useful in evaluating GMP competence. Workers with poor aseptic techniques will have frequent contamination problems, while workers skilled in GMP will have problems only occasionally. It is important to recognize that poor practices not only result in contaminated cultures, but may also result in spreading biological materials and contamination to work surfaces and workers in the laboratory.

D.3 References

University of Edinburgh, Health and Safety, “Good Microbiological Practice and Containment,” Web page information from the Health and Safety Department, August 2003.