BACTERIOLOGY
The consistent absence of colonies recovered after nebulisation of the spore suspension indicates that the filter in the workstation was highly effective in the removal of spores from the atmosphere
The Whitley A135 GMP laminar flow anaerobic workstation can achieve physical isolation of samples within the workspace and provide EU GMP Grade A air cleanliness.
filters and ventilation, safety features and testing, and certification. By adhering to the requirements outlined, workspace and sample handling can be protected from particulate and microbial contamination which compromises quality assurance. To validate and verify maintenance of workspace conditions within the Whitley A135 GMP Workstation, the removal of Bacillus atrophaeus ATCC 9372 spores from the internal work area was evaluated. The methodology used to achieve this was based on that set out in BS EN 12469:2000.
Materials and methods A stock culture of Bacillus atrophaeus DSM 675 (ATCC 9382) was subcultured onto 15 plates of Campden Sporulating Agar,2
(used when an operator is present in the workstation) and Eco Mode (used when the workstation is not in use and the glove ports are closed). On each test day, the workstation fan was allowed to run for half an hour prior to the start of each experiment to ensure that stable operating conditions had been achieved. Before the commencement of each
test, B. atrophaeus spore suspension was diluted in 0.85% saline to achieve a theoretical density of 1.1 × 108
which exceeds the density of 5 × 106 to 8 × 106
spores/mL specified in EN 12469:2000. Diluted suspensions were enumerated (as described above) to confirm the spore density and were found to be in the range of 1.0 × 108 to 1.2 × 108
cfu per mL. In each test,
to encourage spore formation, and incubated for 24 hours at 37°C. Characteristic orange pigmented colonies were removed from the agar using sterile spreaders, suspended in 100 mL of PBS and vortex-mixed thoroughly to achieve a homogeneous suspension. This bacterial suspension was heat shocked at 65°C for 15 minutes in a water bath. The spore density of each suspension used in the investigation was determined by serial dilution and plating duplicate 100 µL linear plates using a Whitley Automatic Spiral Plater (WASP) onto Tryptone Soya Agar. All plates were incubated for 24h at 37°C. Spore counts determined in this way fell between 1.4 × 109
and 4.6 × 109 (cfu) per mL.
The Whitley A135 GMP Workstation was used for all the experiments. The workstation was tested on three separate occasions and was tested in Active Mode
38 colony forming units
50 mL of diluted spore suspension was placed in a six-jet Collison nebuliser supplied with compressed air at a pressure of 140 kPa. Each run consisted of the nebuliser being run for five minutes and ejected approximately 2 mL aerosolised suspension (exact volume
spores/mL,
was determined by weighing the vessel before and after nebulisation). On each occasion, four repeats of the test were carried out: two runs were performed with the nebuliser placed at the front of the cabinet facing the front grill (Active mode and Eco mode) and two runs were performed at the rear of the cabinet with the nebuliser facing the rear grill (Active mode and Eco mode). Throughout the five-minute nebulisation period, eight TSA plates were positioned across the work area (floor tray) of the cabinet, as shown in Figure 1, with lids removed to expose the agar surface to the downward atmospheric flow.
During each run, two positive control samples were taken, after two minutes and four minutes of nebulisation, by exposing an open agar plate to the nebuliser outlet for 10 seconds. After five minutes of nebulisation, the nebuliser air supply was turned off and plates were left to stand for a further minute to allow complete circulation of atmosphere through the workstation’s HEPA filter. Plate lids were then replaced. All plates were removed from the workstation and incubated at 37°C for 24 hours.
BS EN 12469:2000 outlines the performance criteria for microbiological safety cabinets to ensure the correct handling of samples and the maintenance of environmental integrity. By adhering to the requirements outlined, workspace and sample handling can be protected from particulate and microbial contamination which compromises quality assurance
MAY 2024
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