NSF/ANSI Standard 49 Certification Testing of Biosafety Cabinets

Abstract

NSF International maintains NSF/ANSI Standard 49 Class II (laminar flow) biosafety cabinetry and also certifies that biosafety cabinets conform to the requirements of the standard. More than 20 tests are required during initial qualification testing; of these, the biological challenge tests are the most critical to ensuring that the model tested will provide personnel, product, and cross contamination protection.

Testing of biosafety cabinets at NSF International (formerly the National Sanitation Foundation) began in 1976 with the adoption of NSF Standard 49 Class II (laminar flow) Biohazard Cabinetry. Earlier, the U.S. Department of Health and Human Services' National Institutes of Health (NIH) oversaw a similar testing program, but NIH made the strategic decision to entrust the standard maintenance responsibilities to NSF. Class II biosafety cabinets are suitable for use when working with Biosafety Level 1, 2, or 3 contaminants. Most of the test equipment initially used by NSF's testing laboratory to determine whether biosafety cabinets conformed to the requirements of Standard 49 was donated by NIH. Since then, all of the test equipment has been replaced and Standard 49 has gone through eight revisions, in addition to becoming an American National Standard. The biosafety cabinets tested by NSF have also changed over the years. Although the basic concept of an open front protected by an air curtain remains, many refinements to cabinet design have taken place since 1976, particularly with respect to ergonomics, the use of on board computers, more efficient motors, airflow sensors, and digital display panels (airflow velocity, motor speed setting, and so on).

Biosafety cabinets operate by drawing air in through an open front that is normally set at the industry standard access opening height of either 8 or 10 in. NSF has also certified biosafety cabinets with access openings of 9 and 12 in. The front opening is as wide as the work area inside the cabinet. Air entering the cabinet through the front opening must be exhausted. Exhaust air always passes through a high-efficiency particulate air (HEPA) filter. Filtered exhaust air may be released back into the laboratory or exhausted remotely, out of the building. A second HEPA filter located above the entire work area is used to provide clean downflow air to the work area. A front intake grille captures all of the air coming in through the front of the cabinet and some of the air from the downflow filter. This split between intake air, and downflow air is what creates an air curtain that prevents contamination from exiting the cabinet or from entering the cabinet work area. The most critical element in containment is maintaining the balance between the intake (inflow) and downflow air within the manufacturer's specified velocity ranges. Modern cabinets use the concept of “negative surround.” This means that all parts of the cabinet that are in direct contact with the environment and are potentially contaminated are under negative pressure relative to the environment at all times during cabinet operation. Figure 1 is a schematic of a biosafety cabinet.

NSF's certification program for biosafety cabinets covers two areas. The first is an initial qualification of each certified cabinet model. This includes an extensive battery of tests completed in the NSF engineering laboratory at NSF's headquarters in Ann Arbor, Michigan, and auditing of the biosafety cabinet manufacturing facility by NSF personnel. Cabinets undergo retesting in the NSF engineering laboratory every five years to retain certification, and manufacturing facilities are inspected on an annual basis to ensure that the production models are equivalent to the certified model. The second area covered by Standard 49 is an annual field certification of each installed cabinet, following an abbreviated list of test procedures that can be performed on-site. NSF personnel do not perform field certification but NSF does administer an accreditation program for biosafety cabinet field certifiers.

Figure 1.

Example of a type A2 biosafety cabinet.

NSF certification testing includes tests that are designed to enhance user comfort and safety, such as work area light intensity, cabinet noise, and vibration. Electrical safety is ensured by conforming to the requirements of UL 61010A–1. Structural integrity tests, such as work surface deflection, overturn resistance, and resistance to distortion, assure a solid cabinet that will last for many years. However, the core of the testing program is the biological testing, which focuses on protecting both the user from hazardous samples and samples inside the work area from environmental contamination. Before initiating any biological testing, each cabinet must first be successfully balanced within ± +3 fpm of the nominal downflow and inflow airflow velocity set points and at a minimum, the following three tests should be completed: (1) the soap bubble leak test, (2) the HEPA filter leak test, and (3) the airflow smoke patterns test. If a biosafety cabinet is likely to demonstrate poor biological containment, these three tests act as bellwethers to the NSF test engineer, before the point at which poor performance would contaminate the test facilities with bacterial spores, necessitating a time-consuming and labor-intensive cleanup.

The biological testing required for NSF Certification under Standard 49 is divided among three tests: personnel protection, product protection, and cross contamination protection. The personnel and product protection tests are also repeated with the cabinet inflow and downflow air balance set outside of the manufacturer's specified nominal ranges, to assure an additional level of protection. The biological testing is completed using a suspension of bacterial spores that are disseminated into the air using an aerosol nebulizer that is calibrated to deliver a single spore per droplet (monodispersion). Because the droplets are so small, the water evaporates a few inches from the outlet of the nebulizer, meaning that dry spores are delivered to the air on either side of the cabinet window opening (the position of the nebulizer either inside or outside of the air curtain is dependent on the biological test being performed). The organism used for the certification testing is Bacillus subtilis. While this is a common bacteria, the strain used for testing forms distinct colonies that allow the test laboratory to differentiate between the test organism and background contamination.

Personnel protection

The most important function of a biosafety cabinet is the protection of the operator using it. Operator safety is not only dependent on the functionality of the equipment, but on proper use, including using slow, steady movements that do little to disturb the air curtain. Proper use of personal protective equipment, including gloves and laboratory coats is also important. Operators of biosafety cabinets should avoid using the cabinet to store equipment and supplies that are not needed for a particular operation. When an operation has the potential to generate aerosols or particulates, working near the rear of the work area provides additional protection. Care should also be taken with respect to locating biosafety cabinets away from regular foot traffic and windows that may be opened, creating air currents in the room. If an operator uses the appropriate techniques, follows good laboratory practices, and ensures that the cabinet is checked annually by an accredited field certifier, they should feel assured that an NSF-certified biosafety cabinet will effectively protect them. This is verified during the qualification testing period via the personnel protection test.

The personnel protection test is run using a suspension concentration of 5–8 × 10⁸ CFU/mL. The nebulizer is positioned inside the cabinet facing the front opening, above an aluminum tube penetrating the air curtain and representing a user's arm. Air sampling impingers are positioned around the tube just outside the cabinet to capture spores that pass through the air curtain. Two slit-type air samplers are positioned at the sides of the cabinet opening because the corners can be weak spots in the air curtain. The nebulizer is calibrated to deliver more than 2 × 10⁸ viable spores during the 6.5 min of nebulization for the personnel protection test. Three replicate tests are run. If more than 10 spores are captured by the impingers or more than five by the slit samplers during any one of the three replicate tests, the cabinet fails the personnel protection test. The personnel protection test is run with the cabinet airflows set at the manufacturer's specified set points, and is repeated twice, with both the inflow and downflow air velocities reduced by 10 fpm from those set points and with the inflow velocity reduced by 10 fpm and the downflow velocity increased by 10 fpm. Figure 2 is a photograph of the personnel protection test.

Figure 2.

Personnel protection test.

In addition to personnel protection, the NSF testing also evaluates protection of the sample from environmental contamination and cross contamination within the cabinet. Operators can do their part by performing a thorough surface decontamination inside the cabinet work area before and after any operation inside the cabinet, in addition to observing good laboratory practices.

Product protection

The product protection test is run using a suspension concentration of 5–8 × 10⁶ CFU/mL. The aerosol nebulizer is positioned outside the cabinet facing in, above the aluminum tube penetrating the air curtain. Plates containing trypticase soy agar (TSA) agar are arranged to cover the work surface, with the intent of capturing any spores that penetrate the air curtain. Three replicate tests are run with the nebulizer operating for 5 min per replicate, followed by a 5-min settling period. If more than five spores are captured by the plates during any one of the three replicate tests, the cabinet fails the product protection test. The product protection test is run with the cabinet airflows set at the manufacturer's specified set points and again with the inflow velocity increased by 10 fpm and the downflow velocity reduced by 10 fpm. Figure 3 is a photograph of the product protection test.

Figure 3.

Product protection test.

Cross contamination protection

In a busy laboratory, it is not uncommon for two separate operations to take place inside a biosafety cabinet at the same time or for a single operation to use more than one biological component that should remain separate from another biological component. Cross contamination protection evaluates the ability of a cabinet to keep contaminated air from moving laterally within the work area.

The cross contamination protection test is run using a suspension concentration of 5–8 × 10⁴ CFU/mL B. subtilis. The aerosol nebulizer is positioned against each side wall facing the opposite side wall. Plates containing TSA agar plates are centered on a line 14 in. from the side wall with two additional rows of plates nested behind them to capture any spores moving laterally within the cabinet. Three replicate tests are run from each side of the cabinet. If more than two spores are captured by the agar plates located beyond the 14” line during any one of the six replicate tests, the cabinet fails the cross contamination protection test.

For more information about NSF, its biosafety cabinet certification, and field certifier accreditation programs, or to obtain a list of Certified products, please refer to www.nsf.org.

About nsf

NSF International, The Public Health and Safety Company, a not-for-profit, nongovernmental organization, is the world leader in standards development, product certification, education, and risk-management for public health and safety. For 60 years, NSF has been committed to public health, safety, and protection of the environment. While focusing on food, water, indoor air, and the environment, NSF develops national standards, provides learning opportunities through its Center for Public Health Education, and provides third-party conformity assessment services while representing the interests of all stakeholders. The primary stakeholder groups include industry, the regulatory community, and the public at large.