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Biosafety Guidelines for Handling Microorganisms in Microbiology Laboratories

by Tony Cundell, PhD, Microbiological Consulting, LLC

What guidance is available to help us design and operate our labs? USP General Informational Chapter <1117> Microbiological Best Laboratory Practices, revised in 2022, has a number of answers.

USP Chapter <1117> states, “Layout and design should carefully consider the requirements of good practices in a microbiology laboratory and laboratory safety. It is essential that cross-contamination of microbial cultures or DNA/RNA samples for polymerase chain reaction testing (PCR) testing be minimized to the greatest extent possible. It is also important that microbiological samples be handled in an environment that makes contamination highly unlikely.”

The documents go on to mention that a laboratory should be divided into clean or aseptic areas and live culture areas, which both environmental and other microbiological test samples are handled and incubated and should be completely free of live cultures, if possible…

This is all good advice from the USP Microbiology Committee of Experts. It is notable that USP Chapter <1117> now addresses the aseptic manipulation of both viable cultures and nucleic acid extracted from microbial cells.

Another key publication is the 2020 CDC and NIH Biosafety in Microbiological and Biomedical Laboratories, which addresses the safe handling and containment of infectious microorganisms and hazardous biological materials. A worldwide literature search concerning lab-acquired infection by Harding and Byers (2006) revealed 1,267 overt infections with 22 deaths between 1978 and 2000, with clinical (45%) and research (51%) labs accounting for most of the documented infections. The 10 most common infectious agents, typically not encountered in a QC pharmaceutical microbiology laboratory, were Mycobacterium tuberculosis, Coxiella burnetii, hantavirus, arboviruses, hepatitis B virus, Brucella spp. Salmonella spp. Shigella spp., hepatitis C virus, and Cryptosporidium spp. Some people may be surprised by the claim that Salmonella is rarely encountered, but the author only isolated Salmonella once in his long career managing QC labs, and that was an intrinsic factor that aids the absorption of Vitamin B12, which was derived from porcine intestine.

The frequency of exposure, infection, and death from handling microorganisms in United States pharmaceutical QC laboratories is largely unknown because, unlike BSL-3 and -4 facilities that handle serious pathogens, lab exposures that occur in BSL-1 and -2 facilities are not reportable in the United States.

In contrast, our northern neighbor, Canada, monitors laboratory exposure to human pathogens. The Laboratory Incident Notification Canada (LINC) surveillance system monitors laboratory incidents reported under the Human Pathogens and Toxins Act and the Human Pathogens and Toxins Regulations. Forty-two incidents involving 57 individuals were reported to LINC in 2020 from 999 licensed laboratories. There were no suspected or confirmed laboratory-acquired infections. The annual incident exposure rate was 4.2 incidents per 100 active licenses. Most exposure incidents occurred during microbiology activities (52%) and were reported by the hospital sector (45%). Moreover, most affected individuals were exposed via inhalation (49%) and worked as technicians or technologists (63%). Issues with standard operating procedures (SOPs) (inadequate SOPs and not following SOPs) were the most common root cause (27%), followed by human interactions (24%).

Brief Summary of the CDC/NIH Biosafety Level Requirements

BSL-1 represents a basic level of containment that relies on standard microbiological practices with no specific primary or secondary barriers recommended other than a sink for hand washing. A summary of the protection recommended for BSL-1 includes:

  • Discretionary limited access to the lab when tests are in progress.
  • Surfaces must be easily cleaned, imperious to water, and resistant to acid, alkali, organic solvents, and moderate heat.
  • Work surfaces are decontaminated daily and after spills.
  • Mechanical pipetting is required, mouth pipetting is prohibited.
  • No eating, drinking, or smoking is permitted.
  • Protective laboratory clothing, safety glasses as needed.
  • Hands must be washed before leaving the lab.
  • Insect and rodent control programs are to be effect.
  • Contaminated liquid or solid waste must be decontaminated before disposal by an approved decontamination method or treated a biohazardous waste.

My interest in the design and operation of pharmaceutical quality control (QC) microbiology laboratories goes back to the invitation from my late colleague and friend Scott Sutton to contribute a chapter on laboratory design and layout for microbial identification in a little-read but informative 2010 PDA book he edited entitled Laboratory Design — Establishing the facility and Management Structure.

The recent publication of veteran science journalist Alison Young's book Pandora's Gamble: Lab Leaks, Pandemics, and a World at Risk rekindled my interest in the topic, which provoked many questions from the microbiology community. Young's book documents misadventures in lab safety in the United Kingdom, Singapore, Hong Kong, Russia, China, and the United States, with emphasis on the lab safety breaches and leaks in Centers for Disease Control (CDC), United States Department of Agriculture, National institutes of Health (NIH), and U.S. Army Medical Research Institute of Infectious Disease laboratories working with restricted microorganisms and viruses in biosafety level (BSL)-3 and -4 facilities. The book does not answer the more difficult question of whether the SARS-CoV-2 virus spilled over naturally from exotic animals sold in the Wuhan fresh market rather than having originated from lab safety breaches and leaks at the Wuhan Virus Institution but dispels the much-held belief that highly infectious agents are always handled safely.

In the absence of consensus amongst pharmaceutical microbiologists on the details, my take-home message will be that CDC BSL-1 is applicable in the general laboratory area, and BSL-2 guidelines are applicable when making aseptic manipulation, handling live cultures, and conducting microbial identifications.

Biosafety Level 2

BSL- 2 represents containment for handling agents associated with human disease that pose moderate hazards to personnel and the environment. BSL-2 requires the same guidelines as BSL1, but it also requires the following also:

  • Access to the lab is limited, especially when work is being conducted.
  • Personnel are trained in handling pathogenic agents and biologic materials and are directed by a competent microbiologist.
  • Biological safety cabinets are used when aerosols or splashes are generated.
  • A universal biohazard warning sign must be posted on the lab door, identifying the infectious agent, giving specific precautions to be taken, and listing the names and numbers of people to be contacted in case of emergency.
  • Gloves are to be worn when handling potential infectious agents and materials.
  • Needle-locking syringes or disposable syringe-needle units are used and disposed of in sharp containers.
  • Autoclaves are available to decontaminate potentially hazardous materials.
  • An eyewash facility is readily available.
  • Lab coats designated for lab use are worn while in the lab and removed and left before leaving for non-laboratory areas.
  • Spills and exposures must be reported. Medical treatment is provided when necessary and written records of events maintained.
  • Although there are no specific requirements for ventilation systems, when planning new facilities, consider a design that provides an inward flow of air without recirculation to spaces outside the laboratory.
  • A biosafety manual must be prepared and available. Personnel are advised of special hazards and are required to read and follow instructions. Document these requirements.

Recommendations for Laboratory Design and Operation

I am frequently asked the following questions from lab managers who are designing a new facility:

  • Does the general laboratory area need to meet Grade D air cleanliness standards?
  • Which laminar flow hoods should I purchase?
  • What personal protective equipment should be worn?
  • Are there special facility requirements for PCR-based microbial identification and sterility testing?

The two most important concerns are lab personnel safety and inadvertent contamination of the test materials. Drug products made under good manufacturing practice (GMP) conditions typically have low bioburdens or are sterile and are unlikely to be contaminated with the CDC/NIH-selected microorganisms that require BSL-3 and -4 containments.

The following recommendation are made:

  • General laboratory areas may be non-controlled with separate rooms with a Grade B area surrounding a Grade A laminar flow hood for sterility testing.
  • A laminar flow hood with horizontal airflow is most appropriate for pharmaceutical-grade water testing involving open filtration of up to 200 mL volumes. Tests that utilize live cultures (e.g., antimicrobial preservative effective tests) are typically conducted in a Class II, Type A2 biological safety cabinet. If testing drug substances in early development where their toxicology is not fully known or involves organic solvents, use a Class II, Type B2 BSC, which exhausts to the outside.
  • The routine use of lab coats, hairnets, safety glasses, and gloves are recommended. Use a facemask when handling live cultures and cleanroom uniforms when conducting sterility tests.
  • As PCR amplification is susceptible to DNA contamination, the extraction, amplification, and sequencing steps should be conducted in separate rooms. The room for DNA extraction should have Grade C conditions, positive space pressurization, and BSC equipment with ultraviolet light sources to decontaminate DNA.

Conclusion

As supervisors and managers in pharmaceutical microbiology, we are responsible for operating our laboratory in a GMP-compliant manner and ensuring the safety of the lab personnel. The information mentioned is intended to relate the CDC/NIH biosafety guidelines to the pharmaceutical QC microbiology laboratories and reconcile the guidelines with major GMP requirements and industry practices.

About the Author

  • Tony Cundell, PhD, Microbiological Consulting, LLC
    Cundell is the Principal Consultant at Microbiological Consulting, LLC, Rye, New York, and a member of the PEMM Working Group. Cundell consults with a number of pharmaceuticals, consumer health, and dietary supplement companies, microbiology instrument manufacturers, contract testing laboratories, and sterile compounding pharmacies in the areas of microbial risk assessment, regulatory affairs, and microbiological testing.