Non-Tariff Barriers

Non-Tariff Measure

NTM classification

B82: Testing requirement 

Date when the measure came into force

05 April 2001 

Publication where the measure is specified

Government Notice R93 (Government Gazette 22014) Of 2 February 2001 

Regulation where the measure is specified

Compulsory Specification For Microbiological Safety Cabinets (Classes I, II And III) 

Country/Region applying the measure

South Africa 

The rationale of the measure

1.1 This specification covers requirements for the construction, fittings and pre-installation and post- installation performance of class I, class li and class III microbiological safety cabinets (also known as biological safety cabinets) intended to protect the operator and the environment from hazardous microbiological materials and (if so required by the customer or user or both), organic toxins and non-corrosive volatile organic agents.

NOTE - Microbiological safety cabinets are not intended to provide protection against corrosive chemical or radioactive materials.

1 .2 The specification does not cover the actual design of a safety cabinet and in no way restricts new design, provided that a microbiological safety cabinet of a new design complies with the requirements for materials, reliability, performance and safety given in this specification.

NOTE - Microbiological safety cabinets of class I, class II and class III should not be confused with laminar flow
clean workstations that usually discharge horizontally and vertically towards the operator and that do not provide
protection for an operator, but can even increase exposure to airborne hazards. 

Coded list of objectives

X: For purposes n.e.s. 

Description of the measure

5.2 Installation

After installation, the following tests shall be performed to ensure that the cabinet complies with the relevant performance and safety requirements.
a) Gastightness of the outer shell (class III cabinets)
When tested in accordance with 6.1, a class III cabinet shall comply with the requirements of 4.3.1.3.
b) HEPA-filter and HEPA-filter installation Integrity
When tested in accordance with 6.4, class I, class II and class III cabinets shall comply with the requirements of 3.5.1 .3.1 .

c) Integrity of the viewing window seal
When tested in accordance with 6.5, a class II cabinet shall comply with the requirements of 3.4.3.4.
d) Flow and distribution of air, average velocity and uniformity and rate of airflow
Class I cabinets: When determined in accordance with 6.6.3.1, the flow and distribution of air through the work-access aperture shall comply with the requirements of 4.1 .3.2.
When a smoke test is carried out in accordance with 6.5.3.3. the airflow shall comply with the requirements of 4.1 .3.1 .
Class 11 cabinets: When determined in accordance with 6.6.3.2.1, the velocity and uniformity of airflow in the work space shall comply with the requirements of 4.2.4.2.1 .
When determined in accordance with 6.6.3.2.2, the inward airflow velocity through the work-access aperture shall comply with the requirements of 4.2.4.3.3.
Class III cabinets: When determined in accordance with 6.6.3.3, the airflow shall comply with the requirements of 4.3.4.1 and 4.3.4.2.


6 Methods of test
6.1 Determination of gastlghtness of outer shell (class ill cabinets)
6.1.1 Principle
The cabinet is sealed and positively pressurized with hydrofluorocarbon gas. All surfaces and joints are scanned with the detector probe for leakage of the gas.
6.1.2 Apparatus

6.1.2.1 Gas detector, adjusted and calibrated to detect, at a reference leak source, the loss of hydro- fluorocarbon gas at a maximum rate of 16,5 g per annum.
6.1.2.2 Manometer, with scale divisions not exceeding 10 Pa and that is capable of
registering pressures m the range 200 Pa to 300 Pa.

6.1.2.3 Cylinder of hydrofluorocarbon gas (1.1.1.2 tetra-fluoro-ethane), commercially available as a refrigerant, R1 34(a), with a regulator valve, nozzle and connecting hose.

6.1.3 Procedure
6.1 .3.1 Prepare the cabinet for testing as a closed system by sealing all openings such as the exhaust opening, removable panel and other penetrations by any convenient means. Remove all external covers that are not essential for the operation of the cabinet.
6.1.3.2 Attach the manometer to the relevant test area of the cabinet to indicate interior pressure.
6.1.3.3 Suitably connect the gas cylinder to the test area and release the gas to positively pressurize the cabinet interior to a pressure of 250 Pa ± 10 Pa.
6.1.3.4 Prepare, calibrate and operate the gas detector in accordance with the manufacturer's instructions.
6.1.3.5 Move the probe of the instrument over the seams, joints, utility penetrations, gaskets and other locations of possible leakage, keeping the probe 7 mm to 12 mm from any surface and moving it at a rate of approximately 0,013 m/s.
6.1.4 Evaluation
Deem the cabinet to be gaslight if at no location a gas leak in excess of 1 6,5 g per annum is detected.
6.2 Determination of illuminance
6.2.1 Principle
Measurements of illuminance are taken at evenly spaced locations at a specified work level.
6.2.2 Apparatus
Illuminance meter (calibrated, cosine and vision-corrected), of such range that the illuminance measured is at least one-fifth of the full-scale value.
6.2.3 Procedure
6.2.3.1 Operate the lamps in the cabinet for at least 2 h.
6 2.3.2 Take eight illuminance measurements at eight evenly spaced locations at a height not exceeding 25 mm from the surface of the work floor but not within 1 50 mm of the perimeter of the work space. Record the results obtained at each location.

6.3 Determination of vibration
6.3.1 Principle
Measurements of the vibration velocity are made with a simple vibration meter at the geometric centre of the work surface, both with and without the cabinet in operation, to permit comparison of the vibration levels under these two conditions. Determination of the net vibration, i.e. vibration attributable to the cabinet alone, would require vibration frequency analysis.
6.3.2 Apparatus
Vibration meter, capable of measuring steady-state vibration velocities in the range
0,05 mm/s-1 ,0 mm/s (r.m.s.) in the frequency range 10 Hz ± 1 Hz to 250 Hz ± 25 Hz.
6.3.3 Procedure
6.3.3.1 Attach the vibration meter to the geometric centre of the work surface.
6.3.3.2 Ensure that the airflow is as specified.
6.3.3.3 With the cabinet in normal operation, measure the gross vibration velocity in the vertical, horizontal front to rear, and horizontal side-to-side axes.
6.3.3.4 Turn off the mechanical system and with the sensing element positioned and attached as in 6.3.3.1 , measure the ambient vibration velocity.
NOTE - The vibration frequency components of the ambient vibration are usually quite different from those of the mechanical cabinet system and hence the derivation of the net r.m.s. velocity (that attributable to the cabinet equipment) from measurements of gross and ambient vibration is not necessarily a simple mathematical subtraction.
6.3.4 Report
The following information shall be reported:
a) maximum value of the gross r.m.s. vibration velocity; and
b) maximum value of the ambient r.m.s. vibration velocity.
6.4 Determination of HEPA-filter and HEPA-filter installation integrity and
integrity of gaskets and construction joints in the vicinity of the HEPA-filter
installation
6.4.1 Principle
A polydisperse aerosol at ambient temperature is fed into the upstream side of the HEPA-filter installation at a specified flow rate and the downstream surface sides of the filters, seals, gaskets and construction joints in the vicinity of the filter installation is scanned with a probe nozzle to determine the percentage of penetration.
6.4.2 Apparatus

6.4.2.1 Vane anemometer or thermo-anemometer, as appropriate, accurate to within 2 %.
6.4.2.2 DOP generator
6.4.2.2.1 A cold DOP aerosol generator fitted with suitable nozzles and using filtered, compressed air at a pressure of 140 kPa ± 14 kPa. with the free airflow adjusted to not less than 30 e/min per nozzle producing an aerosol of particles with a median diameter of less than 0,8 pm.
6.4.2.2.2 A flexible aerosol delivery hose or tubing of nominal internal diameter 50 mm.
NOTES
1. A hot DOP aerosol generator that uses compressed nitrogen gas may be used to
determine the HEPA-filter installation integrity, provided that the aerosol particles produced should comply with the provisions in 6.4.2.2.1.

2 Liquids other than DOP may be used to generate an aerosol of particles, provided that the aerosol generator and the photometer have been suitably adjusted and calibrated for the alternative liquid. The aerosol produced should have a similar particle size distribution to that given in 6.4.2.2. 1.
6.4.2.3 Aerosol photometer
A light-scattering mass concentration indicator fitted with a probe nozzle. The probe nozzle or tip used for filter integrity testing is of internal diameter (d) not exceeding 30 mm. Any transition from initial inlet diameter to final inlet diameter is gradual. A maximum excluded angle  of 20° is recommended. Photometers that have a threshold sensitivity of at least 10^3µg/l for DOP particles of diameter 0,3 pm, and that are capable of measuring concentrations in the range of 80 µg /l to 120 µg /l are suitable. The test photometer has a sample flow rate of 30 C/min ± 3 «/min. The probe inlet is of sufficient size to maintain the probe inlet rate at or slightly higher than a test flow rate of 27,5 «/min through the filter.
6.4,3 Procedure
NOTE - The test operator should avoid inhalation and exposure to heavy concentrations of the test aerosol. It is recommended that a suitable mask or respirator be worn for the duration of the test and thereafter, if necessary.
6.4.3.1 By using the method given in 6.6, determine the airflow and ensure that the flow through the air filter bank is within the operating limits of the cabinet design flow (see 4.1.3.2, 4.2.4.2. 4.3.4.1, or 4.3.4.2, as applicable). Ensure that the cabinet is operating normally while this procedure is being carried out.
6.4.3.2 Regulate the generator pressure and the gas flow rate in accordance with the manufacturer's instructions or as specified in 6.4.2.2.1 , as appropriate.

Introduce the aerosol via a sparge arrangement, if necessary, so that it is evenly distributed across the air entry.
6.4.3.3 For photometers that have
a) a linear readout, establish the upstream concentration by introducing the least amount of DOP aerosol required to produce a 100 % reading, thus allowing the instrument to be stray light adjusted to zero on the lowest scale range when the sample air stream is filtered free of aerosol, and
b) a logarithmic readout, adjust the upstream concentration (as determined from the instrument calibration curve) by introducing the least amount of DOP aerosol required to produce a concentration of 1 X 1 0^4 above that concentration required to give a reading of one scale division. Avoid prolonged exposure of filters to DOP.
6.4.3.4 With any removable filter guard removed, scan the entire filter media face in slightly overlapping strokes at a distance between the probe and the filter media face of approximately 25 mm and at a traverse rate not exceeding 50 mm and ensure that each filter pleat is scanned parallel to the direction of the pleat. Also scan the entire periphery of the filter at the bond between the filter media and the frame at the seal between the filter frame and the cabinet, and any construction joints
downstream of the filter installation. Record any local areas or points where a reading exceeding 0,03 % is obtained.
6.5 Method for the detection of leaks Into the work space of, and demonstration
of the integrity of a class II cabinet and its air barrier
6.5.1 Principle
6.5.1.1 While air-generated DOP aerosol is directed at joints in the vicinity of the work space or at the work-access aperture (air barrier), measurements are made using an aerosol photometer. Any meter readings in excess of 0,03 % penetration are an indication of seal or joint leakage or induction of contaminants into the clean work zone.

6.5.1.2 Smoke is released on the work space side of the work-access aperture.
Escape of smoke to the ambient air indicates an ineffective air barrier.
6.5.2 Apparatus
6.5.2.1 DOP generator, as in 6.4.2.2.
6.5.2.2 Aerosol photometer, as in 6.4.2.3.
6.5.2.3 Air current tube (smoke generating tube).
6.5.2.4 Barrier test fitting. (For barrier integrity testing, the aerosol delivery hose is fitted with a smooth parallel-bore fitting of internal diameter 50 mm ± 1 mm at the point of discharge. The fitting is of overall length 250 mm ± 5 mm, incorporates flow straighteners at its inlet and has a square cut end as illustrated in figure 2.)
6.5.3 Procedure
NOTE - The test operator should avoid inhalation and exposure to heavy concentration
of the tests aerosol. It is recommended that a suitable mask or respirator be worn for the duration of the test and thereafter if necessary.
6.5.3.1 Joints and seals
6.5.3.1 .1 Using the aerosol photometer, measure the ambient aerosol level of the work room/environment and of the work space of the cabinet. If the reading of the aerosol photometer is less than 10^ 3 above the filter face reading, discharge sufficient aerosol at the exterior of the joint or seal from a distance of approximately 150 mm to ensure the challenge is maintained at 0,1 % concentration or more, with the photometer setting as that used to establish the 100 % baseline during the integrity testing of the HEPA-filter.
6.5.3.1 .2 Use the photometer to scan all construction joints bordering the work space Hold the probe nozzle inside the cabinet, not more than 25 mm away from the joint and move it along the joint at not more than 5 cm/s (see figure 3).
6.5.3.1.3 Start scanning approximately 3 s after the aerosol cloud has been directed at the joint.
6.5.3.2 Air barrier
6.5.3.2.1 Discharge sufficient cold aerosol at a distance of approximately 1 50 mm in front of each test position and scan the lower edge of the viewing glass with the probe inlet held inside the work space at a distance of 25 mm away from the glass and at 100 mm centres. Start scanning at a point 25 mm from each of the work surface boundaries (see figure 4).
6.5.3.2.2 Direct the probe inlet towards the work-access aperture and position its centre approximately 1 mm above the lower edge of the viewing window.
6.5.3.2.3 Operate the aerosol photometer at each test position for at least 1 5 s. Where an intermittent penetration reading greater than 0,03 % above the ambient concentration is obtained, continue for at least a further 30 s.
6.5.3.2.4 Scan the front edge of the work floor (not the front edge of the cabinet) at a distance of 25 mm and at 1 00 mm centres. Commence scanning at a point 25 mm from each of the work surface boundaries (see figure 5). Direct the probe inlet towards the work-access aperture and ensure that its centre is positioned approximately 25 mm above the work floor.
6.5.3.2.5 Operate the photometer at each test position for at least 15 s and record any photometer reading, and its location, in excess of 0,03 % aerosol penetration, relative to the 100 % measured upstream. Where an intermittent penetration reading greater than 0,03 % above the ambient concentration is obtained, continue for at least a further 30 s.
6.5.3.3 Smoke test
6.5.3.3.1 In addition, carry out a simple smoke test to determine the direction of airflow near tile work- access aperture. Generate aerosol or any other smoke on the ambient side of the aperture so that the smoke cloud is within 150 mm of the entire area of the opening and note the direction of the airflow.

6.5.3.3.2 Using the air current tube (smoke generating tube) (see 6.5.2.3), release an even stream of smoke at a distance of 70 mm ± 5 mm from the inside plane of the work-access aperture at a series of positions with the tip of the air current tube approximately 25 mm below the bottom edge of the viewing window. Test positions at approximately 50 mm from both inner side walls of the work space of the cabinet, and at intervals of approximately 100 mm between these points.

6.6 Determination of flow and distribution of air, average velocity and uniformity and rate of airflow
6.6.1 Principle
Airflow velocity readings are taken at selected locations, using an anemometer to determine the average airflow velocity, the uniformity of airflow and the airflow rate.
6.6.2 Apparatus
6.6.2.1 Vane anemometer, free-standing, where applicable, of appropriate vane diameter and accurate to within ± 2 %.
6.6.2.2 Thermo-anemometer, where applicable, and accurate to within ± 2 %.
6.6.2.3 Barometer.
6.6.2.4 Thermometer.
6.6.3 Procedure
6.6.3.1 Class I cabinets
Ensure that the cabinet is operating normally. With the vane anemometer in the plane of the work- access aperture, take and record velocity readings of the air flowing into the aperture for at least 1 min and at least five locations evenly distributed across the plane of the work-access aperture.

6.6.3.2 Class II cabinets
6.6.3.2.1 Down flow
6.6.3.2.1.1 Ensure that the cabinet is operating normally. Take and record velocity readings in the horizontal plane 100 mm above the top edge of the work-access aperture using a free-standing vane anemometer.
6.6.3.2.1.2 Take velocity readings at 200 mm to 225 mm intervals in both directions, starting at a location 75 mm to 1 00 mm from the inner edge of the work surface. Record each reading and its location.
6.6.3.2.1 .3 Record the pressure drop across the filter system as indicated by the manometer or gauge fitted to the cabinet.
6.6.3.2.2 inflow

6.6.3.2.2.1 Ensure that the cabinet is operating normally.
6.6.3.2.2.2 Using a suitable vane anemometer or thermo-anemometer (as applicable), take readings at multiple points on a plane bounded by the perimeter of the exhaust aperture or duct. Record each reading and calculate the mean airflow velocity.
6.6.3.2.2.3 Multiply the area of the exhaust aperture or duct by the mean velocity obtained to yield the volumetric discharge rate of effluent air.
6 6 3 2.2.4 Obtain the average inward airflow velocity at the work-access aperture by dividing the volumetric effluent air volume by the cross-sectional area of the work-access aperture.
6.6.3.3 Class III cabinets
6.6.3.3.1 Inflow through open glove ports
6.6.3.3.1.1 Ensure that the cabinet is operating normally.
6.6.3.3.1 .2 Remove the gloves. Using the vane anemometer placed at the centre of each open glove port, take and record the airflow velocity for at least 1 min.
6.6.3.3.2 Inflow through the inlet filter
6.6.3.3.2.1 Ensure that the cabinet is operating normally with the gloves attached.
6.6.3.3.2.2 Using the thermo-anemometer, take multiple measurements within the exhaust duct along two axes perpendicular to each other, and record the mean airflow velocity.
6.6.3.3.2.3 Multiply the area of the exhaust duct by the average velocity obtained to yield the volumetric discharge rate of effluent air which is equal to the inflow through the inlet filter.
6.6.4 Report
6.6.4.1 Class I
Report
a) each velocity reading and its location;
b) the average of the velocity readings taken;
c) maximum and minimum velocity readings; and
d) percentage variations from the average of the maximum and minimum readings.
6.6.4.2 Class II
6.6.4.2.1 Downflow
Report
a) the pressure drop across the filter system;
b) each velocity reading and its location;
c) the average of the velocity readings taken;
d) maximum and minimum velocity readings; and

e) percentage variations from the average of the maximum and minimum readings.
6.6.4.2.2 Inflow
Report
a) the pressure drop across the exhaust filter system;
b) exhaust airflow velocity (m/s);
c) dimensions of the exhaust duct and work-access aperture; and
d) mean inward airflow velocity (m/s).
6.6.4.3 Class III
Report
a) inflow velocity, right glove port (m/s);
b) inflow velocity, left glove port (m/s);
c) exhaust airi'low velocity (m/s);
d) dimensions of exhaust duct; and
e) inlet airflow rate (m^/min).


6.7 Determination of noise level
6.7.1 Principle
Noise levels are measured at selected locations near the cabinet under normal operating conditions
and the background ambient conditions are also recorded.
6.7.2 Apparatus
6.7.2.1 Sound level meter
Use an integrating sound level meter configuration, that complies at least with the accuracy requirements specified for a type 1 instrument in SABS IEC 60651:1979, Sound level meters, and SABS IEC 60804: 1 985, Integrating-averaging sound level meters, as published by Government Notice No. 399 of 1 April 1999. Use a windscreen of a type specified by the sound level meter's manufacturer as being suitable for the particular microphone and that does not detectably influence the accuracy of the meter under the ambient conditions of the test.
NOTE - In principle, no time weighting other than l-time weighting is allowed during Integration: S-time weighting in particular should be disabled when L is measured since it could introduce errors over short integration intervals.

6.7.2.2 Calibration source
As the calibration source, use a sound calibrator that complies with the requirements prescribed for a type 1 calibrator in SABS IEC 60942:1997, Electroacoustics - Sound calibrators, as published by Government Notice No. 399 of 1 April 1999.
6.7.3 Procedure
6.7.3.1 Ensure that the cabinet is operating normally. For class 1 and class 11 cabinets, measure and record the noise level with the sound level meter situated 0,3 m from, and 0,3 m above, the top edge of the work-access aperture, at the vertical centre line of the cabinet and 1 m from any other part of the cabinet including the duct work and from the discharge point of the extraction system, if fitted. For class III cabinets, take measurement with the sound level meter situated 0,6 m above the work surface of the cabinet and 0,3 m from the cabinet front, at the vertical centre line of the cabinet and 1 m from any duct work and from the discharge point of the extraction system, if fitted.
6.7.3.2 Ensure that the airflow of the cabinet is as specified. Take all measurements with the sound level meter set to use the A-weighted network and fast response. Using the acoustic calibration source, check the acoustic sensitivity of the sound level meter before and immediately after the measurements are made and discard the results If the two checks do not coincide to within 1,0 dB.
6.7.4 Report
Include the following details in the report:
a) all operating noise level measurements and their location;
b) the identified maximum noise level and its location; and
c) the ambient noise level measurements at locations where indicated.
6.8 Determination of the protection factor for class I and class II cabinets
6.8.1 Principle
6.8.1 .1 Tests which are performed to assure that aerosols will be contained within open-fronted micro- biological safety cabinets, are specified in terms of an operator protection factor. This factor expresses the leakage from the open front of a cabinet, of a given aerosol that was released within the world space.
6.8.1 .2 The transfer index defines the exposure experienced at a given point as a result of the release of a known amount of tracer substance (bacterial spores or potassium iodide particles), within the cabinet. This exposure is defined as n/(Ns), where N is the number of particles released and n is the number of particles recovered at a sampling rate of s, the sampling being continued to completion. The transfer ratio index with and without the cabinet defines the protection factor and it is necessary to
define the reference situations which represent the open bench conditions, that is the exposure that an operator is subjected to by working in a ventilated room without the use of a safety cabinet. The reference open-bench conditions are defined as a room with a ventilation rate V, of 10 m^3/min, with complete mixing. The transfer index of the reference room is equal to 1/V = 1/10 and the protection factor then becomes (Ns)/(10n) if the sampling rate s is expressed in cubic metres per minute, or
(Ns)/(10^4n), if s is expressed in litres per minute. The minimum value of the protection factor that can be determined depends on the sensitivity and selectivity of the test, i.e. the magnitude of the challenge, N, the sampling rate s, and the least number of particles recovered that can be readily distinguished from background contamination. Practical values for these are N at least 3 x 10^8, s at least 50 ?/min and n not exceeding 10, which leads to a minimum verifiable value of not less than 1,5 x10^5 for the protection factor.
6.8.1.3 An "artificial arm" in the form of a cylinder of diameter between 60 mm and 65 mm, is used to mimic the turbulence produced by the worker's arm at the front aperture.
6.8.1.4 Containment tests on safety cabinets can be performed
a) with a microbiological aerosol that consists of a fine spray of microorganisms produced by a nebulizer charged from an aqueous suspension, or
b) with an airborne challenge of potassium iodide particles produced by a spinning disk aerosol generator.
6.8.2 Microbiological method
NOTE - In a room where cross-contamination, external contamination or protection factor tests have recently resulted in considerable leakage of the bacterial challenge into the ambient air, it is particularly advisable to perform a background test for the presence of the test organism 24 h before performing protection factor tests. A count of more than five test organisms on one of the culture plates following a 1 min test should be regarded as unsatisfactory, and the protection factor tests should be postponed until the ambient air is no longer contaminated with the test organisms. It is advisable to perform the protection factor tests before the cross-contamination and
external contamination tests.
6.8.2.1 Apparatus and materials
6.8.2.1.1 Spore suspension
A suspension of spores of a non-pathogenic microorganism, for example Bacillus subtills var. globigii (SABS Type Culture Collection (SABSTCC) Sac 35) in sterile distilled water, standardized to contain approximately 10^8 spores to 10^9 spores per millilitre
6.8.2.1.2 Culture plates
Petri dishes of diameter 90 mm that contain 15 mHo20 mi of nutrient agar (see 6.8.2.1.5).
6.8.2.1 .3 Slit air samplers
Two slit air samplers, each being able to operate at between 25 i and 30 H of air per minute.
6.8.2.1.4 Nebulizer
A Collison six-jet nebulizer with an internal outlet of diameter 14 mm, operated from a pressure line at 70 kPa, that sprays approximately 0,2 ml/min and discharges not more than 10l/min of free air at a velocity of 0,8 m/s.

6.8.2.1.5 Nutrient agar
6.8.2.1.5.1 Ingredients
Agar………..15,0 g
Peptone……… 10, Og
Beef extract …….5,0 g
Sodium chloride…….. 5,0 g
Water ………1 000 ml
6.8.2.1.5.2 Procedure
Dissolve the ingredients in the water by heating. Adjust the pH value to 7,2. Sterilize in bulk by autoclaving at 1 21 °C for 1 5 min. Cool to 45 °C and aseptically dispense 1 5 m5 in sterile Petri dishes. Ensure that the surfaces of the Petri dishes are dry before use.
6.8.2.1.6 Ml agar
6.8.2.1.6.1 Ingredients
Nutrient broth 3,125 g
Manganese sulphate tetrahydrate 0,03 g
Dipotassium hydrogen phosphate 0,25 g
Agar (Oxoid no. 3) 12,0 g
Water sufficient to produce 1 000 mH
6.8.2.1.6.2 Procedure
Dissolve the ingredients in the water by heating. Dispense 30 mH volumes in medical flat bottles or 150 m2 volumes in Roux flasks. Sterilize by autoclaving at 121 °C for 1 5 min. Cool to 45 °C and place the medical flat bottles on a 1-in-4 sloped surface and the Roux flasks on a flat surface. Allow the agar to solidify.
6.8.2.1.7 Cylinder
A cylinder of length approximately 1 m and of diameter 60 mm to 65 mm, that has a smooth surface and is closed at both ends.
6.8.2.2 Preparation of Bacillus subtilis var, globigii spores
6.8.2.2.1 Prepare about 20 Ml agar slopes (see 6.8.2.1 .6) in medical flat bottles or in Roux flasks, as required, to yield the appropriate amount of spores.
6.8.2.2.2 Use a fresh culture of Bacillus subtilis var. globigii that had been subcultured for three days at 36 °C ± 1 °C.
6.8.2.2.3 Inoculate the agar slopes with the organism and incubate for one week at 36 °C ± 1 °C and then at room temperature until 80 % sporulation has been obtained (usually within about 10 d).

6.8.2.2.4 Make a spore stain of the culture after about eight days to determine the percentage of spores. If the percentage is less than 80 %, leave the cultures until 80 % sporulation has been obtained.
NOTE - Use a 5 % aqueous malachite green stain for determining the percentage of spores. Make a smear on a glass microscope slide and heat fix it. Place the slide over a small beaker of boiling water. (Rest the slide on the rim of the beaker.) Add malachite green fire to the slide. Leave on for five minutes, then wash off with water. Counter stain with safranine for approximately 30 s. Rinse off with water. Check under the microscope.
6.8.2.2.5 Once 80 % sporulation has occurred, very gently wash the culture off the slopes by means of a sterile glass rod and suspend the spores In 10 m? of sterile distilled water. Do not get pieces of agar in the suspension since this will allow the spores to germinate.
6.8.2.2.6 Centrifuge the suspension in sterile tubes with the tops covered with brown paper. Wash the spores three times with sterile distilled water, i.e. decant the supernatant and add fresh sterile water to remove all traces of the medium. Centrifuge each time for 20 min.
6.8.2.2.7 After washing, resuspend the spores in sterile water and heat shock at 60 °C for 30 min or at 70 °C for 20 min for three days in succession.
6.8.2.2.8 Prepare tenfold serial dilutions of spore suspensions and plate out 0,1 m? samples of each dilution on nutrient agar plates (see 6.8.2.1 .5) and incubate the plates at 36 °C ± 1 °C for 24 h to 48 h.
Count the number of colonies on those plates with between 30 colonies and 300 colonies. From this result, calculate the concentration of spores per millilitre of suspension.
6.8.2.2.9 Store the stock culture (2 x 10^ spores to 4 x 10^ spores/mfi) at 4 °C until needed.
6.8.3 Procedure for determining protection factor
6.8.3.1 General
For cabinets of width up to 1 m, carry out five replicate protection tests at the centre of the work-access aperture. For cabinets of width exceeding 1 m but not exceeding 1,9 m, carry out five replicate protection tests at the centre of the aperture, and five each at the centres of the right and left halves of the aperture respectively. In order to avoid confusion from background contamination, carry out the tests in a well-ventilated room, after estimating background contamination.
6.8.3.2 Precede this entire procedure by a control run with the nebulizer switched off.
6.8.3.3 Introduce the cylinder through the work-access aperture of the cabinet to disturb the airflow (to simulate an operator's arm). Centre the cylinder between the side walls of the safety cabinetwork space and, where appropriate, at the centre of the right and left halves of the work access aperture, and normal to the plane of the aperture, extending from the back of the work space to protrude at least 250 mm into the room from the plane of the aperture. Raise the lower surface of the cylinder to
between 65 mm and 75 mm from the cabinet floor.
6.8.3.4 Measurement of the concentration of spores in the spore suspension
6.8.3.4.1 Prepare tenfold serial dilutions of the stock spore suspension (see 6.8.2.2.9) and plate out 0,1 mm samples of each dilution on nutrient agar plates (see 6.8.2.1 .5).
6.8.3.4.2 Transfer a measured volume of the stock spore suspension (5 mUo 10 m?, as appropriate) into the nebulizer (see 6.8.2.1 .4) and weigh the nebulizer with its contents. After spraying, weigh the nebulizer again.
6.8.3.4.3 Prepare tenfold serial dilutions of the spore suspension that remains in the nebulizer and plate out 0,1 mH samples of each dilution on nutrient agar plates.
6.8.3.4.4 At least half of the original volume of the stock spore suspension shall remain in the nebulizer. Determine the volume as follows:
V+(M2-M1) (V/2)
where
M1 is the mass of the nebulizer plus contents before spraying, in grams;
M2 is the mass of the nebulizer plus contents after spraying, in grams; and
V is the volume of the initial spore suspension in the nebulizer, in millilitres;
and assuming the density of the spore suspension to be 1 ,0 g/m8.
6.8.3.4.5 Incubate both sets of inoculated plates (before and after spraying) at 36 °C ± 1 °C for a period of 24 h to 48 h and count the colonies on those plates that have between 30 colonies and 300 colonies on them.
6.8.3.4.6 From these counts, determine the concentration of spores per millilitre in the initial suspension n1 and in the final suspension n2.
6.8.3.5 Place the nebulizer inside the work space, and where appropriate, at the centre of the right and left halves of the work-access aperture, with its outlet or the appropriate extension thereof 100 mm behind the plane of the work-access aperture midway between the side walls of the work space, and directed towards the aperture, with the spray axis parallel to the work surface.
6.8.3.6 For class I cabinets (see figure 6(a)), ensure that the spray axis is below the cylinder and approximately midway between its lower surface and the work surface.
6.8.3.7 For class II cabinets (see figure 6(b)), ensure that the spray axis is level with the upper edge of the aperture.
Dimensions in millimeters
6.8.3.8 Position the slit air samplers outside the cabinet in front of the work-access aperture, with their inlets not more than 200 mm in front of the plane of the aperture. Ensure that the inlets are level with the top of the cylinder, one to the right and one to the left, and each not more than 150 mm from the axis of the cylinder.



6.8.3.9 Switch on the cabinet and allow it to run until normal operating conditions are reached. Adjust each sampler to a sampling rate of not less than 25 d/min and not more than 30 d/min. Place a nutrient agar plate (see 6.8.2.1 .5) in each slit air sampler. Start the samplers. After 30 s, start the nebulizer that contains the stock spore suspension. Run the nebulizer for a period of not less than 4 min to ensure
the dispersal of at least 3x10^ spores. Switch off the nebulizer and continue to run the samplers for a further 5 min.
6.8.3.10 Incubate the culture plates at 36 °C ± 1 °C for 24 h to 48 h.
6.8.3.11 After incubation, count the Bacillus subtilis var. globigii colonies. If there is no growth of Bacillus subtilis var. globigii colonies on the control plates, use the results for the calculation of the protection factor (see 6.8.3.14).
6.8.3.12 If there is growth of Bacillus subtilis var. globigii colonies on the control plates, clean the equipment and repeat the procedure.
6.8.3.13 Determine the challenge dose N, in spores per millilitre, using the equation
N=n2{M1-M2)-(n2-n1)V
where
M1 is the mass of the nebulizer plus contents before spraying, in grams;
M2 is the mass of the nebulizer plus contents after spraying, in grams;
n1 is the concentration of the initial suspension before spraying, in spores per millilitre;
n2 is the concentration of the suspension after spraying, in spores per millilitre; and
V is the volume of the initial spore suspension in the nebulizer, in miliilitres;
and assuming the density of the spore suspension to be 1,0 g/ml
6.8.3.15 Carry out five replicate protection tests. Ensure that no individual value of the protection factor is less than 1,0 x 10^5 (see 4.2.4.3.2).
6.8.4 Potassium iodide method
6.8.4.1 Materials and apparatus
6.8.4.1.1 Potassium iodide, 15g/S solution either in absolute ethanol or industrial methylated spirits with a water content of not more than 5 % (by volume).
6.8.4.1.2 Palladium chloride, 1,0 g/fi solution In 0,1 mol/l hydrochloric acid.
6.8.4.1.3 Aerosol generator assembly that comprises a 3,8 cm diameter spinning disc capable of rotating at 28 000 r/min ± 500 r/min, and a nozzle with a fine hole to deliver the potassium iodide solution (see 6.8.4.1.1) to the spinning disc, the gap between the end of the nozzle and the spinning disc being set to 0,1 mm; also a laboratory stand to hold the aerosol generator above the work surface when necessary.

6.8.4.1 .4 Air samplers, that work on a centripetal principle with a volume flow rate of air of 1 00 ? /min through the front orifice, and a cone that entrains some 3 % of this air, collecting approximately 1 00 % of any potassium iodide particles that enter the sampler; airflow through the samplers being provided by a centrifugal fan coupled to the air samplers by a fixed tube.
NOTE - The particles being heavier than air, follow a straight path through the cone and are deposited on a filter membrane located at the base of the cone, while air is deflected to the outside of the cone (see figure 7),
6.8.4.1 .5 Cylinder of length and diameter approximately 1 m and 60 mm to 65 mm. respectively, that has a smooth surface and is closed at both ends.
6.8.4.1.6 Petri dishes, of diameter 55 mm.
6.8.4.1.7 Filter membranes, of diameter 25 mm and with a pore size of 3 Mm.
6.8.4.2 Procedure
6 8 4 2 1 Set out two Petri dishes (see 6.8.4.1 .6) away from the cabinet being tested, one dish half filled with palladium chloride solution (see 6.8.4.1 .2) and the other one half filled with distilled water.
Replace the lids on each. Set out two filter papers for drying the filter membranes (see 6.8.4.1 .7).
6.8.4.2.2 Introduce the cylinder (see 6.8.4.1 .5) through the work-access aperture of the cabinet to mimic the turbulence produced by an operator's arm in front of the aperture. Centre the cylinder between the side walls of the cabinet work space and normal to the plane of the aperture, extending from the back of the work space to protrude at least 250 mm into the room from the plane of the aperture. Raise the lower surface of the cylinder to between 65 mm and 75 mm from the cabinet floor.
6.8.4.2.3 Place the aerosol generator (see 6.8.4.1 .3). inside the work space, with the leading edge of the disc 100 mm behind the plane of the front aperture.
6.8.4.2.4 For class I cabinets (see figure 6(a)), place the aerosol generator below the cylinder.
6.8.4.2.5 For class II cabinets (see figure 6(b)), ensure that the disc is level with the upper edge of the aperture.
6.8.4.2.6 Position two air samplers (see 6.8.4.1.4) outside the cabinet in front of the work-access aperture, with their inlets 1 50 mm to 160 mm in front of the plane of the aperture. Ensure that the inlets are level with the top of the cylinder, one to the right and one to the left, and each 150 mm from the axis of the cylinder.
6.8.4.2.7 Load each air sampler carefully with a filter membrane using a pair of fine-pointed forceps kept clean and dry solely for this purpose. Adjust the air pressure to 20 cm water gauge (this is consistent with an air sampling rate of 100 fi/min), using a U-tube water manometer one limb of which is attached to a pressure tapping on the rear of the sampler.
6.8.4.2.8 Measure 20 m? of potassium iodide (see 6.8.4.1 .1) into the aerosol generator reservoir with the fluid release valve closed.

6.8.4.2.9 Switch on the cabinet and allow it to run until normal operating conditions are reached. Apply suction to the air samplers and start the spinning disc. Wait 1 5 s and then open the release valve to allow the potassium iodide to feed on to the centre of the disc.
6.8.4.2.10 Turn off the air samplers after the generation of aerosol has stopped. Wait until the suction pump stops completely, and then remove the filter membrane from one sampler using a second pair of fine-pointed forceps designated for this purpose.
6.8.4.2.11 Gently float the filter membrane in the palladium chloride solution contained in the Petridish, with the surface that has been exposed to airflow facing upwards. Within 30 s to 45 s, the membrane will become saturated with palladium chloride and any potassium iodide particles will become visible as brown spots.
6.8.4.2.1 2 Remove the membrane with a third pair of fine-pointed forceps designated for this purpose, immerse the membrane in distilled water for 3 s to 4 s and then place it on a clean filter paper to dry.
Repeat this procedure with the filter membranes from the other air samplers. Replace the lids of the Petri dishes.
NOTE - The solution of potassium iodide used for the tests is flammable and corrosive to untreated steel; consequently the cabinet under test should be wiped clean with a wet cloth and scrupulous care should be taken with the spinning disc equipment.
6.8.4.2.13 Examine each filter with either a low-power binocular microscope or a 15 x magnifier and count the number of brown spots on the filter membrane.
6.8.4.2.14 Calculation of the protection factor
Calculate the number of potassium iodide particles released, N, using the following equation:
N = (3,1 X10^7x M)
where
3,1 X 10^7 is a constant derived from the droplet size, the sampling flow rate and the speed of rotation of the disc; and
M is the volume of potassium iodide solution dispersed by the aerosol generator, in
millilitres.
Then calculate a value for the protection factor, PF (separately for each filter membrane), using the
following equation:
PF = NV/10^4
where
V is the sampling flow rate in cubic metres per minute; and
n is the number of spots on the filter membranes.

NOTES

1 In this case, M is 20 ml (see 6.8.4.2.8) and Vis 100 l/min (see 6.8.4.2.7),
2 Using the above equations and the values of /Wand V given in note 1 , a protection factor of 1,0 x 10^5 would correspond to 62 spots on the filter membrane.
3 When calculating the protection factor, if there was only one spot on the filter membrane, the protection factor would be 2,6 X 1 0^6. If there were no spots on the filter membrane this would indicate that the protection factor was higher than this, and in the above example the protection factor would be recorded as PF > 2,6 x 10^6.
6.8.5 Background tests
Place two air samplers loaded with filter membranes in front of the safety cabinet, 1 50 mm to either side of the aperture centre line and 100 mm from the plane of the aperture. Turn on the sampler suction fan and run it for 10 min without any generation of potassium iodide droplets by the aerosol generator.
Remove the filter membranes and develop and examine them in accordance with 6.8.4.2.12 and 6.8.4.2.13.
NOTES
1 On completion of the background tests in laboratories where no previous tests have taken place within 24 h, the developed membranes should not show any brown spots.
2 In laboratories where protection factor tests have recently taken place (or where protection factor tests have resulted in a considerable leakage of aerosol challenge) it Is particularly advisable to perform background tests before further tests
on the cabinets. A count of more than five spots on one of the two filter membranes following a 1 min test should be regarded as unsatisfactory and further cabinet tests should be postponed until the background is no longer contaminated.
6.9 External contamination test
6.9.1 Principle
The integrity of the air barrier at the work-access aperture is indicated by the measurement of the inward penetration of bacterial spores which are sprayed into the opening with the cabinet operating normally.

6.9.2 Apparatus and materials
As in 6.8.2.1 (the slit air samplers are not required).
6.9.3 Procedure
6.9.3.1 Place the cylinder in the cabinet as described in 6.8.3.3.

6.9.3.2 Distribute at least 12 culture plates (see 6.8.2.1 .2) evenly over the work floor of the cabinet.
6.9.3.3 Load a measured volume of spore suspension into the nebulizer. Position the nebulizer outside the cabinet, with the nebulizer's delivery opening 100 mm in front of the centre of the top edge of the work-access aperture. Ensure that the spray axis is parallel to the work surface and directed into the cabinet.
6.9.3.4 Ensure that the cabinet is operating normally. Uncover the culture plates 1 min before spraying begins. Run the nebulizer for a period of at least 4 min to ensure the dispersal of a challenge dose of at least 3x10^6 spores. Switch off the nebulizer and leave the culture plates uncovered for a further 5 min.
6.9.3.5 In any test, the number of colonies of the test organism counted after incubation at 36 °C ± 1 °C for a period of 24 h to 48 h shall not exceed five. Carry out the test five times, using a new set of culture plates for each test.
6.9.3.6 Carry out a control test with the cabinet motor blower(s) switched off. Ensure that at least 300 colonies are recovered from these plates during the control test. If less than 300 colonies are recovered, repeat the test.
6.10 Cross-contamination test
6.10.1 Principle
Bacterial spores are sprayed across the work space and contamination of the opposite two-thirds of the cabinet is monitored.
6.10.2 Apparatus and materials
As in 6.8.2.1 (the slit air samplers are not required).
6.10.3 Procedure
6.10.3.1 Place the cylinder in the cabinet as described in 6.8.3.3.
6.10.3.2 Distribute at least 12 culture plates (see 6.8.2.1 .2) evenly over the right two-thirds of the work surface of the cabinet and at least 350 mm from the left side.
6.1 0.3.3 Load a measured volume of spore suspension into the nebulizer. Place the nebulizer with its spray axis 1 00 mm above the work surface and 50 mm from the left side of the work surface. Ensure that the spray axis is parallel to the work surface and directed towards the opposite wall.
6.10.3.4 Ensure that the cabinet is operating normally. Uncover the culture plates 1 min before spraying begins and cover them again 5 min after spraying stops. Run the nebulizer for a period of at least 4 min to ensure the dispersal of a challenge dose of at least 1 x 10^5 spores.
6.10.3.5 Count the number of colonies of the test organism after incubation at 36 °C ± 1 °C for a period of 48 h ± 2 h. Carry out the test three times.
6.10.3.6 Carry out the test three more times using reversed positions (i.e. placing the culture plates on the left and the nebulizer on the right).
6.10.3.7 Carry out a control test with the cabinet motor blower(s) switched off. Ensure that 300 colonies are recovered from these plates during the control test. If less than 300 colonies are recovered, repeat the test.

6.11 Test for resistance to corrosion
6.11.1 Test solution
6.11.1.1 Preparation of the sodium chloride solution
Dissolve a sufficient mass of sodium chloride in distilled or deionized water of conductivity not higher than 20 pS/cm at 25 °C ± 2 °C to produce a concentration of the sprayed solution collected of 50 gin ± 5 g/e. The specific gravity range for a 50 g/fi ± 5 g/H solution is 1,025 5 to 1,040 at 25 °C.
Ensure that the sodium chloride contains less than 0,001 % (by mass) of copper and less than 0,001 % (by mass) of nickel as determined by atomic absorption spectrophotometry or another analytical method of similar sensitivity. Ensure that the sodium chloride does not contain more than 0,1 % (by mass) of sodium iodide or more than 0,5 % (by mass) of total impurities calculated for dry salt.
If the pH of the prepared solution, measured at 25 °C ± 2 °C, is outside the range 6,0 to 7,0, investigate for the presence of undesirable impurities in the sodium chloride or in the water (or in both).
6.11.1.2 pH adjustment
So adjust the pH of the solution that the pH of the sprayed solution collected within the spray cabinet (see 6.11.2.1) is between 6,5 and 7,2. Check the pH using electrometric measurement at25 °C ±2 °C, or, in routine checks, with a short-range pH paper which can be read in increments of 0,3 pH units or less. Make any necessary correction by adding hydrochloric acid or sodium hydroxide solution of analytical grade.
Changes in pH can result from the loss of carbon dioxide from the solution when it is sprayed. Such changes can be avoided by reducing the carbon dioxide content of the solution by, for example, heating it to a temperature above 35 °C before it is placed in the apparatus, or by making the solution from freshly boiled water.
6.11.1.3 Filtration
If necessary, filter the solution before placing it in the reservoir of the apparatus (see 6. 11 .2.3), to remove any solid matter which might block the apertures of the spraying device.
6.11.2 Apparatus
Ensure that all components in contact with the spray or the test solution are made of, or lined with, materials resistant to corrosion brought about by the sprayed solution and which, in turn, do not influence the corrosiveness of the sprayed test solutions. The apparatus shall include the components given in 6.11.2.1 to 6.11.2.4.
6.11.2.1 Spray cabinet
The spray cabinet shall be of a capacity not less than 0,2 m^ and preferably not less than 0,4 m^, since, with smaller volumes, difficulties can be experienced in ensuring the even distribution of spray. For large-capacity cabinets, it is necessary to ensure that the conditions of homogeneity and distribution of the spray are met (see 6.1 1 .6). The upper parts of the cabinet shall be so designed that drops of
sprayed solution formed on its surface do not fall on the specimens being tested.

The size and shape of the cabinet shall be such that the collection rate of solution in the cabinet is within the limits specified in 6.1 1.6.3.
6.11.2.2 Heater and temperature control
An appropriate system that maintains the spray cabinet and its contents at the specified temperature (see 6.11 .6.1). The temperature shall be measured at least 100 mm away from the walls.
6.11.2.3 Spraying device
The device for spraying the sodium chloride solution comprises a supply of clean air at a controlled pressure and humidity, a reservoir to contain the solution to be sprayed, and one or more atomizers.
The compressed air supplied to the atomizers is passed through a filter to remove all traces of oil or solid matter and is at an absolute pressure of 70 kPa^1 to 170 kPa.

NOTE - Atomizing nozzles might have a "critical pressure" at which an abnormal increase in the corrosiveness of the salt
spray occurs. If the "critical pressure" of a nozzle has not been established with certainty, control fluctuations in the air pressure within ±0,7 kN/m^2, by installing a suitable pressure regulator valve to minimize the possibility of the nozzle being operated at its "critical pressure".

In order to prevent the evaporation of water from the sprayed droplets, the air is humidified before entering the atomizer, by passing through a saturation tower that contains hot water at a temperature several degrees Celsius higher than that of the cabinet. The appropriate temperature depends on the pressure used and on the type of atomizer nozzle and these shall be so adjusted that the rate of collection of spray in the cabinet and the concentration of the collected spray are kept within the specified limits (see 6.11.6). The level of the water is automatically maintained to ensure adequate humidification.

The atomizers are made of inert material, for example glass or plastics materials. Baffles may be used to prevent direct impact of spray on the test specimens and the use of adjustable baffles is helpful in obtaining uniform distribution of the spray within the cabinet. The level of the sodium chloride solution in the reservoir is automatically maintained to ensure uniform spray delivery throughout the test.
6.11.2.4 Collecting devices
At least two suitable collecting devices, which consist of funnels made of glass or other chemically inert material and with the stems inserted into graduated cylinders or other similar containers, shall be used.
Funnels of diameter 100 mm have a collecting area of approximately 80 cm^. The collecting devices are placed in the zone of the cabinet where the test specimens are placed, one close to an inlet of spray and one remote from an inlet. They are so placed that only spray, and not drops of liquid that fall from specimens or from parts of the cabinet, is collected.
6.11.2.5 Re-use
Ensure that if the equipment has been used for a spray test or for any other purpose and a solution different from the specified sodium chloride solution was used, it is thoroughly clean before use.
Operate the equipment for at least 24 h and measure the pH of the collected solution to ensure that it is correct throughout the entire spraying period, before any specimens are placed in the chamber.
6.11.3 Method of evaluation
To check the reproducibility of the test results for one piece of apparatus or for similar items of apparatus in different laboratories, it is necessary to verify the apparatus at regular intervals, as described in 6.11.3.1 to 6.11.3.4.
6.11.3.1 Reference specimens
To verify apparatus, use four reference specimens of thickness 1 mm ± 0.2 mm. width 50 mm and length 80 mm of CR4 grade steel that complies with SABS ISO 3574:1986, Cold-reduced carbon steel sheet of commercial and drawing qualities, as published by Government Notice No 399 of 1 April 1999 with a practically faultless surface^', and a matt finish (arithmetical mean deviation of the profile Ra = 1 .3 µm ± 0,4 µm). Cut these reference specimens from cold-rolled plate or strip.
Carefully clean the reference specimens immediately before testing. Besides the directions given in 6.11 .4.2 and 6. 11 .4.3, ensure that the cleaning eliminates all traces of dirt, oil and other foreign matter that could influence the test results.
Use one of the following methods:
a) clean the reference specimens by vapour degreasing using a chlorinated hydrocarbon. Use three successive treatments of 1 min each, with an interval of at least 1 min between successive treatments; or
b) thoroughly clean the reference specimens with an appropriate organic solvent (hydrocarbon, that has a boiling point between 60 °C and 120 °C) using a clean soft brush or an ultrasonic cleaning device. Carry out the cleaning in a vessel filled with solvent. After cleaning, rinse the reference specimens with fresh solvent, then dry them; or
c) other cleaning methods may be used after agreement between the interested parties, provided that the results will be comparable.
Determine the mass of the reference specimens to an accuracy within 1 mg. Protect one face of the reference specimens with a removable coating, for example, an adhesive plastic film.
6.11.3.2 Arrangement of the reference specimens

Position the four reference specimens in four different quadrants in the spray cabinet, with the unprotected faces upwards, and at an angle of 20° ± 5° from the vertical.
Use supports made of, or coated with, inert materials such as plastics. Ensure that the upper edges of the reference specimens are level with the top of the salt spray collector. The test duration is 96 h.
6.11.3.3 Determination of mass loss
At the end of the test, remove the protective coating. Remove the corrosion products by immersion in a cleaning solution of hydrochloric acid (^20 = 1,18 g/mO, of recognized analytical grade at 50 % (by volume), in water, inhibited by 3,5 g of hexamethylene tetramine per litre.

After stripping, thoroughly clean the reference specimens with water at ambient temperature, then with acetone, followed by drying.
Determine the mass of the reference specimens to an accuracy within the nearest 1 mg and calculate the mass loss of the exposed surfaces in grams per square metre.

6.11.3.4 Checking of apparatus operation
The operation of the test apparatus is deemed to be satisfactory if the mass loss of each reference specimen is 140 g/m^2 ± 40 g/m^2.
6. 11 .4 Test specimens
6.11.4.1 Two rectangular test specimens of nominal dimensions 100 mm x 15 mm of the metal used to manufacture the cabinet.
6.1 1 .4.2 Thoroughly clean the test specimens before testing (see 6.1 1 .3.1 ). The cleaning method used will depend on the nature of the material, its surface and the contaminants, but abrasives or solvents which might attack the surface of the specimens may not be used.
Ensure that specimens are not recontaminated after cleaning by careless handling.
Specimens intentionally coated with protective organic films should not be cleaned before the lest.
6.11.4.3 If the test specimens are to be cut from a larger coated article, ensure that the coating is not damaged in the area adjacent to the cut. Protect the cut edges by coating them with a material that will be stable under the conditions of the test, such as paint, wax or adhesive tape.
6.1 1 .5 Arrangement of the test specimens
6.1 1 .5.1 So place the test specimens in the spray cabinet that they are not in the direct line of travel of spray from the atomizer.
6.11.5.2 The angle at which the surface of a test specimen is exposed in the spray cabinet is very important. Support the specimen facing upwards in the spray cabinet at an angle as close as possible to 20° to the vertical but within the limits 15° to 30°. In the case of irregular surfaces, for example, entire components, adhere to these limits as closely as possible.

6.11.5.3 So arrange the test specimens that they do not come into contact with the cabinet and that surfaces to be tested are exposed to free circulation of spray. The specimens may be placed at different levels within the cabinet, as long as the solution does not drip from specimens or their supports at one level onto other specimens placed below. However, for a new examination or for tests with a total duration exceeding 96 h, location permutation of specimens is permitted.
6.1 1 .5.4 Ensure that the supports for the test specimens are made of inert non-metallic material such as glass, plastics or suitably coated wood. If it is necessary to suspend specimens, use synthetic fibre, cotton thread or other inert insulating material.
6.11.6 Operating conditions
6.1 1 .6.1 Maintain the temperature inside the spray cabinet at 35 °C ± 2 °C with the minimum possible fluctuation in temperature throughout the duration of the test.
6.1 1 .8.2 Start the test after it has been confirmed that the collection rate (see 6. 1 1 .6.3) and conditions (see 6.1 1 .6.1) are within the specified ranges, and the cabinet is filled with test specimens as planned.
6.11.6.3 Ensure that the solution collected in each of the collecting devices (6.1 1 .2.4) has a sodium chloride concentration of 50 g/l. ± 5 g/l and a pH value in the range 6,5 to 7,2.
Ensure that the average rate of collection of solution in each device, measured over a period of at least 24 h of continuous spraying is 1 mfi/h to 2 m?/h for a horizontal collection area of 80 cm^2.
6.11.6.4 Do not re-use test solution which has been sprayed.
6.11.6.5 During the test, prevent any increase or decrease of cabinet pressure.
6.11.7 Duration of test
6.1 1 .7.1 The duration of the test shall be at least 480 h.
6.11.7.2 Do not interrupt the spraying during the prescribed test period. Do not open the cabinet except for brief visual inspections of the test specimens in position and for replenishing the salt solution in the reservoir, if such replenishment cannot be carried out from outside the cabinet.
6.11.7.3 A periodic visual examination of specimens under test for a predetermined period is allowed, but do not disturb the surfaces under test and only open the cabinet for the minimum period necessary to observe and record any visible changes of the material under test.
6.11.8 Treatment of specimens after test
At the end of the test period, remove the test specimens from the cabinet and allow them to dry for 0,5 h to 1 h before rinsing, in order to reduce the risk of removing corrosion products. Before they are examined, carefully remove the residues of spray solution from the surfaces of the test specimens. A suitable method is to rinse or dip the test specimens gently in clean running water, at a temperature not exceeding 40 "'C and then to dry them immediately in a stream of air, at a pressure not exceeding
200 kPa and at a distance of approximately 300 mm.
6.11.9 Evaluation of results
The material shall be deemed to be corrosion resistant if corrosion of the base metal is not visible to the unaided eye.
6.11.10 Report
6.11.10.1 The report shall indicate the outcome of the test according to the criteria for the evaluation of results. Report the result obtained for each specimen tested and, when appropriate, the average result for a group of replicate test specimens. The report could be accompanied by photographic records of the tested specimens.
6.11.10.2 The following information shall be included in the report:
a) a reference to this compulsory specification;
b) the type and purity of sodium chloride and water used;
c) a description of the material or product tested;
d) the dimensions and shape of the test specimen and the nature and area of the surface tested;
e) the preparation of the test specimen, including any cleaning treatment applied and any protection given to edges or other special areas;
f) known characteristics of any coating with an indication of the surface area;
g) the number of test specimens subjected to the test and a reference to each material or product represented;
h) the method used to clean test specimens after the test, with, where appropriate, an indication of the loss in mass resulting from the cleaning operation;
i) the angle at which the tested surfaces were inclined;
j) the frequency and number of specimen location permutations, if any;
k) the duration of the test and the results of any intermediate inspections;
I) the test temperature;
m) the volume of collected solution;
n) the pH of the test solution and the collected solution;
o) the density of the collected solution;
p) any abnormality or incident that occurred during the entire test procedure; and
q) intervals of inspection.
6.12 Prefilter tests
Use the relevant test methods given in SABS 1424, Filters for use m air-conditioning and general ventilation, as published by Government Notice No. 1 851 of 1 December 1 995, to determine the initial arrestance.
Use a 600 mm x 600 mm sample of the prefilter in its unpleated state.

6.13 Chemical resistance
6.13.1 Reagents
Use the following reagents for the testing of chemical resistance:
hydrochloric acid, 40 g/l^3
sodium hydroxide, 40 g/l
quaternary ammonium compound, 10 g/ l
formaldehyde, 50 g/l.
sodium hypochlorite, 5 g/ l
iodophor, 20 g/ l
phenol, 50 g/ l
ethanol, 70 % (by volume)
6.13.2 Procedure
6.13.2.1 Apply approximately 0,5 m? of each reagent to the surface that has to be tested. Cover each reagent with a watch glass in the centre of the puddle with the concave side down and leave the reagent on the surface for 4 h, ensuring that the test surface is wet throughout the entire period.
6.13.2.2 After the 4 h period, scrub the surface with a stiff brush and hot water at a temperature of 70 °C.
6.13.2.3 Dry the surface and remove any surface stains by washing with alcohol before examination.
6.13.3 Inspection
Inspect the test surfaces for any visible effect other than a slight change of gloss or discoloration when compared with an untreated area.
6.14 Stability test
6.14.1 Principle
The stability test is performed to demonstrate the resistance of a microbiological safety cabinet to overturning under an applied force.
6.14.2 Apparatus
6.14.2.1 Compression force gauge, calibrated in kilograms, accurate to within ± 5 % full scale, or
6.14.2.2 Extension spring balance, calibrated in kilograms, accurate to within ± 5 % full scale.
6.14.3 Procedure
6.14.3.1 Block the cabinet at the front bottom edge to prevent lateral movement. Apply force, as appropriate, at the centre of the rear top edge (see figure 8) to subject the cabinet to a torque of 700 Nm.

6.14.3.2 Measure the cabinet lift at the rear bottom edge.

7 Blower performance test
7.1 Principle
The airflow velocity is increased to determine whether the motor blower has spare capacity.
7.2 Apparatus
7.2.1 Vane anemometer or thermo-anemometer (or both) as appropriate, accurate to within 2 %.
7.2.2 Timer.
7.3 Procedure
7.3.1 Class I
7.3.1.1 With clean HEPA-filters fitted, set the blower speed control so that when the inward airflow velocity is measured in accordance with 6.6.3.1 , the average airflow velocity shall be that specified in
3.6.2.1.
7.3.1.2 After 2 h, measure the average inward airflow velocity in accordance with 6.6.3.1 .
7.3.2 Class II
7.3.2.1 With clean HEPA-filters fitted, set the blower speed control so that when the downward airflow velocity in the work space is measured in accordance with 6.6.3.2.1, the average downward airflow velocity shall be that specified in 3.6.2.2.
7.3.2.2 After 2 h, measure the average downward airflow velocity in accordance with 6.6.3.2.1.
7.3.2.3 When a class II cabinet is fitted with a separate motor blower for the exhaust system, set the blower speed control so that when the inward airflow velocity through the work-access aperture is measured in accordance with 6.6.3.2.2, the average inward airflow velocity shall be that specified in 3.6.2.2.
7.3.2.4 After 2 h, measure the average inward airflow velocity in accordance with 6.6.3.2.2. 

Reference of the measure

Regulations 5.2
Regulations 6.6.1 to 7.3 

Measure also domestic

Yes 

Products affected by the measure.

Description

Microbiological safety cabinets (classes I, II And III) 

Countries/Regions affected by the measure.

Description

All countries