Open fronted safety cabinets in ventilated laboratories

Open fronted Class I and II microbiological safety cabinets (MSCs) are required by the British Standard 5726 to provide similar levels of operator protection (viz. 10⁵). In laboratories that are naturally ventilated large numbers of both types of cabinets have been shown to exceed this requirement consistently over a number of years. The designs of some mechanically ventilated laboratories, however, produce excessive turbulence and draughts that can prejudice containment at the front aperture. On-site commissioning tests to determine operator protection factor are now well established and are recognized as being essential to the setting up of all open fronted cabinets in both ventilated and unventilated laboratories. This paper shows that where environmental conditions induce unsatisfactory cabinet containment, adjustments to air supply and exhaust systems can be made which will enable both Class I and II cabinets to produce operator protection factors in excess of 10⁵. When compatibility is achieved between the local environment and the cabinets it is demonstrated that disturbances at the front aperture, caused by operator working procedures or by disturbances due to personnel movement within the room, have similar effects on both Class I and II cabinets. Once performance levels have been satisfactorily achieved, regular containment testing has shown that consistent performance can be maintained. These aspects of open fronted safety cabinet performance are discussed in relation to ventilated laboratories suitable for work with the human immunodeficiency virus (HIV). Of paramount importance in the future is the necessity to design laboratory air systems that will be compatible with satisfactory safety cabinet performance—a relatively new requirement in ventilation system specifications.     

Containment of Microbial Aerosols in a Microbiological Safety Cabinet

A microbiological safety cabinet was evaluated to determine conditions under which microorganisms might escape. Tests were conducted under three cabinet-closure conditions, various airflow velocities, and different laboratory operations, with 10(5), 1.1 x 10(5), and 10(6) microorganisms per cubic foot of cabinet space released per min for 5 min. The data revealed that (i) escape of a human infectious dose is possible when the cabinet is used with the glove panel off; (ii) the number of organisms that escaped from the cabinet increased with a decrease in air velocity; and (iii) an increase in the number of laboratory operations resulted in an increase in the number of organisms that escaped. Thus, when the glove panel was off, the cabinet was only safe for operations that released a small number of microorganisms into the cabinet, whereas the cabinet was safe for operations of significantly greater hazard when used with the glove panel on but with the gloves unattached.     

Modified Laminar Flow Biological Safety Cabinet

Tests are reported on a modified laminar flow biological safety cabinet in which the return air plenum that conducts air from the work area to the high efficiency particulate air filters is under negative pressure. Freon gas released inside the cabinet could not be detected outside by a freon gas detection method capable of detecting 10(-6) cc/s. When T3 bacteriophage was aerosolized 5 cm outside the front opening in 11 tests, no phage could be detected inside the cabinet with the motor-filter unit in operation. An average of 2.8 x 10(5) plaque-forming units (PFU)/ft(3) (ca. 0.028 m(3)) were detected with the motor-filter unit not in operation, a penetration of 0.0%. Aerosolization 5 cm inside the cabinet yielded an average of 10 PFU/ft(3) outside the cabinet with the motor-filter unit in operation and an average of 4.1 x 10(5) PFU/ft(3) with the motor-filter unit not in operation, a penetration of 0.002%. These values are the same order of effectiveness as the positive-pressure laminar flow biological safety cabinets previously tested. The advantages of the negative-pressure return plenum design include: (i) assurance that if cracks or leaks develop in the plenum it will not lead to discharge of contaminated air into the laboratory; and (ii) the price is lower due to reduced manufacturing costs.     

A Comparison of Methods to Measure Operator Protection Factors in Open-fronted Microbiological Safety Cabinets

Comparative tests to measure operator protection factors in microbiological safety cabinets in accordance with British Standard 5726 have demonstrated good agreement in the results obtained by a microbiological method using a Collison nebulizer and the technique producing an aerosol of potassium iodide. Either method is suitable for testing for operator protection factors in Class I and Class II safety cabinets.
The Collison nebulizer should be considered as the standard aerosol generator for the microbiological method; alternative nebulizers meeting the general requirements of BS 5726 should be compared in performance with this nebulizer if they are to be used for containment tests.
Any microbiological safety cabinet specified for a new installation should have been 'type' tested to ensure compliance with BS 5726. However, in order to ensure adequate performance, on-site commissioning tests (and routine maintenance checks thereafter) are necessary to verify that air velocity, filtration and operator protection factor requirements are met.     

Continued successful operation of open-fronted microbiological safety cabinets in a force-ventilated laboratory.

The considerable refinements necessary to enable Class I and II microbiological safety cabinets to operate in a force-ventilated laboratory and to meet appropriate safety criteria have been reported previously. The continued successful operation of such cabinets without a deterioration of operator protection is described. The performance of two Class II units, one meeting and one failing the current British Standard applied to four head KI-discus testing, is compared and discussed. In addition, some further potential difficulties within the environment, which could compromise cabinet containment, are highlighted.     

Continued successful operation of open-fronted microbiological safety cabinets in a force-ventilated laboratory

R.W. OSBORNE AND T.A. DURKIN. 1991. The considerable refinements necessary to enable Class I and II microbiological safety cabinets to operate in a force-ventilated laboratory and to meet appropriate safety criteria have been reported previously. The continued successful operation of such cabinets without a deterioration of operator protection is described. The performance of two Class II units, one meeting and one failing the current British Standard applied to four head KI-Discus testing, is compared and discussed. In addition, some further potential difficulties within the environment, which could compromise cabinet containment, are highlighted.     

Airflows In and Around Linear Downflow 'Safety' Cabinets

Schlieren photography has been used to visualize the airflows in and around three re-circulating linear downflow cabinets. The results show that there is an air 'bulge' at the front opening that allows air from within the cabinet to spill out towards the operator. Hand movements within, and activity just outside, cause air to be expelled from these cabinets. Bunsen burners within the cabinets destroy the linear nature of the downflowing air streams. The results indicate that the operator protection afforded by these cabinets may be much less than is generally recognized.     

Performance of open-fronted microbiological safety cabinets: the value of operator protection tests during routine servicing

The performance of class I and II microbiological safety cabinets over 7 years, employed in a force-ventilated containment level 3 (CL-3) laboratory, is described. Operator Protection (OP) provided by the cabinets, assessed by still and latterly limited 'in-use' KI-Discus tests, showed no overall deterioration during the review period. Comparisons show that a selected class II unit, but not a second, and a new class II MSC in a recently commissioned, similar CL-3 facility, provide the same order of OP as a class I cabinet. From the experiences described, it is strongly recommended that OP tests (OPTs) should be part of the routine servicing regime to ensure that cabinets meet required performance levels, and additionally to allow detection and rectification of poor containment, particularly where induced by environmental factors. The value of OPTs is discussed with reference to certain national standards.     

Improving the biosafety of cell sorting by adaptation of a cell sorting system to a biosafety cabinet.

BACKGROUND: The jet-in-air cell sorters currently available are not very suitable for sorting potentially biohazardous material under optimal conditions because they do not protect operators and samples as recommended in the guidelines for safe biotechnology. To solve this problem we have adapted a cell sorting system to a special biosafety cabinet that satisfies the requirements for class II cabinets. With aid of this unit, sorting can be performed in conformance with the recommendations for biosafety level 2. METHODS: After integrating a modified fluorescence-activated cell sorter (FACS) Vantage into a special biosafety cabinet, we investigated the influence of the laminar air flow (LAF) inside the cabinet on side stream stability and the analytical precision of the cell sorter. In addition to the routine electronic counting of microparticles, we carried out tests on the containment of aerosols, using T4 bacteriophage as indicators, to demonstrate the efficiency of the biosafety cabinet for sorting experiments performed under biosafety level 2 conditions. RESULTS: The experiments showed that LAF, which is necessary to build up sterile conditions in a biosafety cabinet, does not influence the conditions for side stream stability or the analytical precision of the FACS Vantage cell sorting system. In addition, tests performed to assess aerosol containment during operation of the special biosafety cabinet demonstrated that the cabinet-integrated FACS Vantage unit (CIF) satisfies the conditions for class II cabinets. In the context of gene transfer experiments, the CIF facility was used to sort hematopoietic progenitor cells under biosafety level 2 conditions. CONCLUSIONS: The newly designed biosafety cabinet offers a practical modality for improving biosafety for operators and samples during cell sorting procedures. It can thus also be used for sorting experiments with genetically modified organisms in conformance with current biosafety regulations and guidelines.     

Evaluation of laminar flow microbiological safety cabinets

The microbiological control efficiency of two class 100 laminar down-flow hoods was determined by using aerosols of Bacillus subtilis var. niger spores. The first unit challenged utilized a slanted eyelid to partially enclose the front work opening. This hood showed nearly perfect control of ambient organisms in the work area. It also gave a 10(6) or greater drop in the number of organisms passing out of the exhaust system. However, when the interior work area of the hood was challenged, significant numbers of spores penetrated the air barrier and escaped into the ambient air. A redesigned laminar flow hood was built incorporating a vertical eyelid and a reduced opening to the work area. This hood showed the same excellent characteristics for controlling ambient contamination. Exhaust system leakage was also extremely low. Air barrier efficiency for the newer hood was increased with lower amounts of spore penetration into the ambient air.     

Microbiological Studies on the Performance of a Laminar Airflow Biological Cabinet

Engineering and microbiological tests indicated that a typical, commercial laminar airflow cabinet was not effective in providing either product protection or agent containment. The cabinet was modified and tested through a series of alternate configurations to establish a set of design criteria. A mock-up cabinet was developed from these design criteria. The mock-up unit was evaluated for efficiency in providing both product protection and agent containment. In these evaluations, challenge methods were developed to simulate normal, in-use laboratory operations. Controlled bacterial or viral aerosol challenges were used at higher than normal levels to provide stringent test conditions. Test results indicated that the mock-up unit was considerably better in preventing agent penetration (0.1 to 0.2 particles per 100 ft(3) of air) than the commercial cabinet (5 to 6 particles per 100 ft(3) of air) during product protection tests. Similarly, agent containment was considerably better in the new cabinet (particle escape of 2 to 3 per 100 ft(3) of air at only one of the five test sites) than in the commercial cabinet (particle escape of 2 to 14 per 100 ft(3) of air at three of the five test sites).     

Biological safety in virological field with special considerations on class II biological safety cabinets

The most critical point for the biosafety is not sophisticated devices or facilities, but education of workers and their compliance to the regulation. Appropriate devices should be carefully selected in the introduction of new devices, and they should be properly maintained. The class II biosafety cabinet is one of the delicate safety equipments. It should be kept adequately maintained throughout the lifetime of the cabinet to insure safety of the laboratory. For the maintenance, appropriate measuring equipments should be used by trained technicians. The recently enforced law for control of recombinant DNA researches should be applied for the handling of pathogens even in non-recombinant DNA researches after proper modifications.     

Mass Airflow Cabinet for Control of Airborne Infection of Laboratory Rodents

A mass airflow cabinet for handling and housing of laboratory rodents has been developed and tested. The unit consists of a high-efficiency particulate air filter and uniform distribution of air at a vertical velocity of 19 cm per s. Animals are maintained without bedding in mesh-bottomed cages that rest on rollers for rotation inside the cabinet. There is an air barrier of 90 cm per s separating the cabinet air from room air. Sampling for airborne bacteria yielded an average of 0.03 colony-forming units (CFU) per ft(3) of air inside the cabinet, whereas 28.8 CFU per ft(3) was simultaneously detected outside the cabinet during housekeeping, a reduction of almost three logs. The efficiency of the air barrier was tested by aerosolization of T3 phage. When phage was aerosolized 5 cm outside the cabinet, no phage could be detected 5 cm inside when the fans were operating; with the fans off an average of 1.6 x 10(4) plaque-forming units (PFU) per ft(3) was detected in six tests. Aerosolization of phage inside the cabinet yielded an average of 9 x 10 PFU per ft(3) outside; an average of 4.1 x 10(6) PFU per ft(3) were detected with the fans not in operation, a reduction of more than four logs. In-use studies on effectiveness showed that the cabinet significantly reduced the incidence of mice originally titer-free to Reo-3 virus. Hemagglutination inhibition antibodies to Reo-3 were detected in 9/22 (42%) mice housed in a conventionally ventilated animal laboratory while no seroconversion was detected in any of 22 mice housed in the mass air flow cabinet in the same laboratory.     

Efficacy of three microbiological monitoring methods in a ventilated cage rack

The use of individually ventilated caging (IVC) to house mice presents new challenges for effective microbiological monitoring. Methods that exploit the characteristics of IVC have been developed, but to the authors' knowledge, their efficacy has not been systematically investigated. Air exhausted from the IVC rack can be monitored, using sentinels housed in cages that receive rack exhaust air as their supply air, or using filters placed on the exhaust air port. To aid laboratory animal personnel in making informed decisions about effective methods for microbiological monitoring of mice in IVC, the efficacy of air monitoring methods was compared with that of contact and soiled bedding sentinel monitoring. Mice were infected with mouse hepatitis virus (MHV), mouse parvovirus (MPV), murine rotavirus (agent of epizootic diarrhea of mice [EDIM]), Sendai virus (SV), or Helicobacter spp. All agents were detected using contact sentinels. Mouse hepatitis virus was effectively detected in air and soiled bedding sentinels, and SV was detected in air sentinels only. Mouse parvovirus and Helicobacter spp. were transmitted in soiled bedding, but the efficacy of transfer was dependent on the frequency and dilution of soiled bedding transferred. Results were similar when the IVC rack was operated under positive or negative air pressure. Filters were more effective at detecting MHV and SV than they were at detecting MPV. Exposure of sentinels or filters to exhaust air was effective at detecting several infectious agents, and use of these methods could increase the efficacy of microbiological monitoring programs, especially if used with soiled bedding sentinels. In contemporary mouse colonies, a multi-faceted approach to microbiological monitoring is recommended.     

Opening closed arms: long-distance activation of SMC ATPase by hinge-DNA interactions

Structural maintenance of chromosomes (SMC) proteins form a V-shaped dimer in which a central hinge domain connects two coiled-coil arms, each having an ATP binding head domain at its distal end. Here, we show that the hinge domain plays essential roles in modulating the mechanochemical cycle of SMC proteins. An initial interaction of the hinge domain with DNA leads to opening of the arms by triggering hydrolysis of ATP bound to the head domains, which are located approximately 50 nm away from the hinge. This conformational change allows the inner surface of the hinge domain to stably interact with DNA by an ATP-independent mechanism and primes ATP-driven engagement between the liberated head domains either intramolecularly or intermolecularly. Consistently, a variety of hinge mutations drastically alter DNA binding properties of SMC proteins through distinct mechanisms. Our results suggest that SMC proteins possess an intrinsic property to change their own conformations upon binding to DNA.     

Lack of HLA class I and class II antigens on human preimplantation embryos

The expression of HLA Ag on polyploid early human embryonic stages, obtained in an in vitro fertilization and embryo transfer program, was investigated by indirect immunofluorescence tests using a panel of mAb. Neither HLA class I Ag, beta 2-microglobulin, nor HLA class II molecules could be detected on blastomers. The zona pellucida also lacked these Ag, but granulosa cells expressed HLA class I Ag, beta 2-microglobulin, and HLA class II Ag. These results make it likely that the absence of HLA Ag is one of the mechanisms involved in protecting the implanting embryo from rejection by the immunocompetent mother.     

The Potassium Iodide Method for Determining Protection Factors in Open-fronted Microbiological Safety Cabinets

A method for determining operator protection factors in Class I and Class II microbiological safety cabinets and for evaluating product protection factors in Class II cabinets, is described. The technique employs an aerosol of potassium iodide droplets produced by a spinning disc generator together with special centripetal air samplers detecting any aerosol escape. The method meets the requirements of British Standard (BS) 5726 and is an alternative to the microbiological technique.
The method has been used to evaluate the performance of a number of safety cabinets in relation to the requirements of BS 5726.
Working procedures and unsuitable environments have been shown to prejudice the containment performance of open-fronted cabinets by several orders of magnitude.
The relationship between inflow air velocity and protection factors in Class I safety cabinets has confirmed the optimum requirements defined in the British Standard.     

Influence of crossdrafts on the performance of a biological safety cabinet.

A biological safety cabinet was tested to determine the effect of crossdrafts (such as those created by normal laboratory activity or ventilation) upon the ability of the cabinet to protect both experiments and investigators. A simple crossdraft, controllable from 50 to 200 feet per min (fpm; 15.24 to 60.96 m/min), was created across the face of the unit. Modifications of standardized procedures involving controlled bacterial aerosol challenges provided stringent test conditions. Results indicated that, as the crossflow velocities exceeded 100 fpm, the ability of the cabinet to protect either experiments or investigators decreased logarithmically with increasing crossdraft speed. Because 100 fpm is an airspeed easily achieved by some air conditioning and heating vents (open windows and doorways may create velocities far in excess of 200 fpm), the proper placement of a biological safety cabinet within the laboratory--away from such disruptive air currents--is essential to satisfactory cabinet performance.     

Influence of crossdrafts on the performance of a biological safety cabinet.

A biological safety cabinet was tested to determine the effect of crossdrafts (such as those created by normal laboratory activity or ventilation) upon the ability of the cabinet to protect both experiments and investigators. A simple crossdraft, controllable from 50 to 200 feet per min (fpm; 15.24 to 60.96 m/min), was created across the face of the unit. Modifications of standardized procedures involving controlled bacterial aerosol challenges provided stringent test conditions. Results indicated that, as the crossflow velocities exceeded 100 fpm, the ability of the cabinet to protect either experiments or investigators decreased logarithmically with increasing crossdraft speed. Because 100 fpm is an airspeed easily achieved by some air conditioning and heating vents (open windows and doorways may create velocities far in excess of 200 fpm), the proper placement of a biological safety cabinet within the laboratory--away from such disruptive air currents--is essential to satisfactory cabinet performance.     

Conservation of Mhc class III region synteny between zebrafish and human as determined by radiation hybrid mapping

In the HLA, H2, and other mammalian MHC:, the class I and II loci are separated by the so-called class III region comprised of approximately 60 genes that are functionally and evolutionarily unrelated to the class I/II genes. To explore the origin of this island of unrelated loci in the middle of the MHC: 19 homologues of HLA class III genes, we identified 19 homologues of HLA class III genes as well as 21 additional non-class I/II HLA homologues in the zebrafish and mapped them by testing a panel of 94 zebrafish-hamster radiation hybrid cell lines. Six of the HLA class III and eight of the flanking homologues were found to be linked to the zebrafish class I (but not class II) loci in linkage group 19. The remaining homologous loci were found to be scattered over 14 zebrafish linkage groups. The linkage group 19 contains at least 25 genes (not counting the class I loci) that are also syntenic on human chromosome 6. This gene assembly presumably represents the pre-MHC: that existed before the class I/II genes arose. The pre-MHC: may not have contained the complement and other class III genes involved in immune response.