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Class III biological safety cabinets are highly specialized laboratory gloveboxes, but are not interchangeable with just any glovebox.


Class III biological cabinets offer the highest level of personnel, product, and environmental protection for total containment of high risk agents and potent compounds, and have specific requirements they must meet.  They add an extra level of safety not available in Class II cabinets or typical gloveboxes.

Class III Biosafety Cabinet Defined


A Class III cabinet is a negative-pressure, totally enclosed, gas-tight cabinet.  Work within the cabinet is conducted through a series of arm-length rubber sleeve / glove systems that are secured to fixed port openings.  Personnel protection, which is of utmost importance for Class III applications, is accomplished by placing a primary physical barrier between the laboratory investigator and the biological agent.  Gas-tight construction and utilization of a pass-thru chamber to move materials into and out of the main cabinet further ensures containment within the primary work area.

Product protection is provided within a Class III biosafety cabinet through HEPA-filtered air.  In the Baker IsoGARD design, the entire volume of air within the cabinet is replaced (rather than relying on dilution of cabinet air to provide cleanliness).  Bathing the interior of the cabinet with HEPA-filtered air, increases entrainment of generated particles and keeps the work free of contaminants. Some Class III cabinet designs utilize air dilution, which may be appropriate for certain applications.

In Class III cabinets, 100% of the air is exhausted.  Environmental protection is provided because all exhaust air is passed through dual HEPA filters before being removed by the facility exhaust system.  This protects lab personnel and others from potential particulate exposure.

Typical Uses for a Class III Biological Safety Cabinet


A Class III cabinet is typically used for applications with the highest risk, including vaccine research, aerosolization studies, processing suspected terrorism samples (biological and chemical), infectious disease diagnostic procedures and research, and handling potent pharmaceutical compounds in an aseptic environment.

Applications for Class III Biosafety Cabinets include:

  • Working with emerging diseases or diseases marked for near eradication.
  • Weighing and diluting chemical carcinogens.
  • Working with high concentrations and/or large amounts of low to moderate risk agents.
  • Use of equipment or instrumentation generating high aerosol volumes.
  • Maximum containment of highly infectious or hazardous experimental materials.
  • An extra level of safety not available in Class I or II cabinets.

Class III Biosafety Cabinet Performance


Performance requirements of a Class III biosafety cabinet are not covered by a currently active standard body (like the NSF/ANSI Standard 49 covers Class II biosafety cabinets).  Therefore Class III cabinets are tested in accordance with the Laboratory Safety Monograph: A supplement to the NIH guidelines for recombinant DNA research (LSM), published by the National Cancer Institute in 1979.  The LSM is still the most recent federally-accepted authority in the United States to publish testing requirements for Class III cabinets.

Class III biosafety cabinets are designed to ensure no exposure to infectious doses of biohazards.  The LSM states that Class III biosafety cabinets must maintain a negative air pressure of at least 0.5 inches water gage (“ w.g.) and are considered gastight when they have a leak rate of not more than 1 x 10-5cubic centimeters per second (cc/sec) with 100% tracer gas when pressurized to 3” w.g.

Since it is difficult, and impractical, to assure that 100% tracer gas is in the cabinet during the test, the LSM suggests testing the cabinet with 1% tracer gas in the cabinet at 3” w.g. pressure.  At that concentration, an acceptance leak rate of 1 x 10-7 should be used because of the dilution of the tracer gas in the cabinet – the leak meter is only seeing 1/100 of the total leak concentration, so the acceptance criterion is adjusted by a factor of 100.

In order to ensure that a Class III biosafety cabinet will truly prevent operator exposure, it must be validated by the LSM cabinet integrity test.  Other tests, like the cabinet integrity (“pressure decay”) test from the American Glovebox Society (AGS), are not reasonable tests to validate a Class III cabinet.

Dig deeper!


For a more in-depth and technical look at this topic, please see Analysis of the Class III Biological Safety Cabinet Integrity Testby David Stuart, David Eagleson, Robert Lloyd, Christopher Hersey, and Dennis Eagleson published in the Applied Biosafety: Journal of the American Biological Safety Association (Vol. 17, No. 3, 2012, pp. 128 – 131).

It addresses some of the information covered in this post, as well as:

  1. Why use the Laboratory Safety Monograph as the reference for testing Class III BSCs?
  2. Why is the cabinet integrity test so important to the Class III BSC?
  3. Why are there two criteria in the literature for the tracer gas cabinet integrity test?
  4. Is the acceptance criterion for the Class III BSC cabinet integrity test meaningful and appropriate?
  5. Since Freon R-12 can no longer be used as a tracer gas because of its impact on the ozone layer, how can the Class III BSC cabinet integrity test now be performed?

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