STANDARD PROCEDURE: NWSP 300.0.R0 (15)

Nonwovens Bacterial Filtration Efficiency

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1. Scope This test method describes the evaluation of bacterial filtration efficiency of nonwoven filter media designed for use as surgical face masks. The method is an in vitro test and therefore in principle will determine the performance of the face mask filter material itself and not the efficiency of a finished mask. It must be recognized that face mask design characteristics have a bearing on ultimate in-use performance. SI values are regarded as the official standard system of measurement for this standard procedure. If other systems of measurement are used in place of SI units (including inch-pound) their values must be reported independently. Systems of measurement must not be combined in any way, but shall be regarded and reported separately.

NOTE 1 SAFETY This standard does not claim to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. It is expected that the person performing this test has been fully trained in all aspects of this procedure.

2. References The following referenced documents are useful for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document applies. 2.1 International standards

a) ISO 5725 -1 Accuracy (trueness and precision) of measurement methods and results – Part 1: General principles and definitions b) ISO 5725 -2 Accuracy (trueness and precision) of measurement methods and results – Part 2: Basic method for the determination of the repeatability and reproducibility of a standard measurement method c) ISO 139 Textiles ― Standard atmospheres for conditioning and testing d) ISO 2859-1 Sampling procedures for inspection by attributes-- Part 1: Sampling

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schemes indexed by acceptance quality limit (AQL) for lot-by-lot inspection e) ISO 3951-1 Sampling procedures for inspection by variables-- Part 1: Specification for single sampling plans indexed by acceptance quality limit (AQL) for lot-by-lot inspection for a single quality characteristic and a single AQL

2.2 NWSP a) NWSP 001.0. Standard Terminology Relating to the Nonwoven Industry, EDANA’s and INDA’s Standard Procedures

2.3 Military Specifications MIL-M-36954C Military Specification, Mask, Surgical, Disposable 3. Terms and Definitions The following referenced terms are of utility for the application of this document: 3.1 Bacterial Filtration Efficiency (BFE) The filtration efficiency of a filter medium is dependent upon the size of the challenge particles, the flow rate of the air stream passing through the filter, and the surface properties of the particles (e.g. either moist or dry). 3.2 Sample For testing purposes a product or a portion of a product taken from a production lot. The sample shall be identifiable and traceable back to the origin. 3.3 Specimen A specific portion of the identified sample upon which a test is performed. Many specimens may be tested from the same sample, using different locations. 4. Principle Essentially the method of determining (BFE) of a surgical face mask comprises the five following stages: a) Aerosol generation

A system which delivers a constant controlled amount of aerosolized bacteria of a known and precisely controlled average particle size and particle size range.

b) Aerosol chamber The nebulizer may be contained in this chamber or be separate and the chamber

may have the facility to vent in additional filtered air.

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c) Filter housing Sited at the exit of the aerosol chamber and in which is placed the filter medium to

be tested. d) Bacterial collection device This assays the number of viable droplets or particles in the aerosol which

challenge the test medium with no specimen in place and which pass through the test medium with the specimen in place.

e) Vacuum pump and flow meter A post filter vacuum created by the vacuum pump induces airflow at a controlled rate.

Providing the method utilized is reproducible with little variability and incorporates all the above stages it is acceptable. Annex A describes a commonly used protocol and is cited to give guidance for the interpretation of the principles of the test procedure. Prospective users of this protocol are advised of the limitations of the method as detailed in clause A 1 in so far as the method is applicable to the filter medium itself and not the finished article. 5. Conditioning not applicable 6. Sampling Please refer to NWSP 005.0, if applicable 7. Apparatus see Annex A 8. Procedure see Annex A 9. Calculation see report, o)

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10. Report In addition to the precise test results, the report shall include the following information:

a) Reference the test method used b) Complete identification of all materials tested and method of sampling c) Name and address of testing institution d) Make and model of testing equipment e) The particle size range of the challenge aerosol f) The bacteria challenge employed g) The airflow rate at which the test was conducted h) Efficiencies quoted should be the average of at least five individual readings

obtained with adequate controls i) Laboratory testing conditions; Barometric pressure, Temperature, Relative

humidity j) Number of specimens tested k) For computer processed data, identify the software used and the version l) Deviation from the standard test procedure, if any m) Anything unusual noted during the testing n) Where an average BFE is given the number of tests performed should be

indicated. o) Calculation of efficiency:

%100)(

)(% xrthoutFilteColoniesWi

thFilterColoniesWirthoutFilteColoniesWiBFE

11. Precision to be determined

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ANNEX A (informative)

Bacterial Filtration Efficiency

This annex describes a commonly used protocol and is cited merely to assist the prospective test laboratory to interpret the principles embodied in this method. A 1. Principle

The following method is used for determining the bacterial filtration efficiency of surgical face mask material and other filter materials. The method was developed by Dr Paul S. Nicholes of the University of Utah. N.B. It should be noted that the method assesses the filtration efficiency of a material and not the efficiency of face mask per se.

A 2. References

a) Military specification - MIL-M-36954C, 1975, p. 21 b) Andersen 2000 Inc. “Instructions and Care of the Andersen Viable Sampler”.

5.1.71 c) Andersen 2000 Inc. Catalog

A 3. Apparatus (see figures 2 and 3) a) Glass cloud chamber - custom made. b) Andersen air sampler c) Andersen air sampler - adapted for plastic plates d) Flow meter e) Air pressure source f) Pressure gauge g) Filter apparatus h) Condenser i) Incubator j) Agar sterilizer k) Autoclave l) Colony counter m) Syringe pump n) Automatic petri plate filler o) Chicago nebulizer p) Vacuum pump q) Spectrophotometer

A 4. Material and Reagents a) Tryptic soy agar - Gibco b) Difco bacto-peptone

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c) Distilled water d) Nutrient broth e) Sodium chloride (reagent grade) This methodology can also be modified to accommodate other sources of nebulized bacteriological challenges e.g. the Collison nebulizer can accommodate other particle size challenges. In all references to performance the choice of nebulizer and particle size distribution of the challenge medium should be quoted.

NOTE A1 SAFETY WARNING

All normal safety precautions essential for safe handling of potentially hazardous organisms and essential for a properly run microbiological laboratory should be enforced. Routine checks should be made on the test apparatus to ensure good seals and connections are maintained and that there are no leaks. It will be noted that the Andersen sampler assembly is provided with a back-up filter holder which will accommodate a high efficiency filter medium such as a glass fiber filter which has a high collection efficiency for sub-micron particles. Additionally, a high efficiency bacterial filter covers the pressure equalizing part in the aerosol chamber to protect the environment from aerosolized bacteria.

A 5. Procedure A 5.1 Preparation of media and reagents

a) Tryptic soy agar plates Tryptic soy agar is mixed with distilled water at a concentration of 40 grams per

liter and autoclaved at 121°C for 20-30 minutes. 27 ml ± 1 ml are dispensed per glass Andersen plate.

b) Nutrient broth 8 grams of powdered nutrient broth is dissolved per liter of distilled water. 30

ml volumes are dispensed into 125 ml flasks and autoclaved at 121°C per 30 minutes. The broth is kept at 10°C ± 1°C until it is used.

c) Peptone dilution medium 15 grams of Difco-peptone and 9 grams of sodium chloride (Reagent Grade) in

1 liter of distilled water. 75-100 ml volumes are dispensed into 125 ml flasks. The flasks are autoclaved at 121°C for 30 minutes and stored at room temperature.

d) Tryptic soy agar slants The agar is prepared as in 1 above and 6 ml are placed in a 10 ml test tube

and autoclaved at 121°C for 20 minutes. The tubes are placed on a slant and allowed to solidify.

A 5.2 Preparation of test culture

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This methodology cites the use of Staphylococcus aureus as the challenge organism, however alternative organisms such as Staphylococcus epidermidis which may be potentially less hazardous may be employed. a) New slants New slants are inoculated with isolated colonies from previously run culture

plates of Staphylococcus aureus UT 15. The slant cultures are incubated at 37°C ± 1°C for 24-48 hours with the screw caps loosened. At the end of the incubation period the caps are tightened and the slant cultures are stored at room temperature. Slant cultures may be used until a loss in viability is noted. New slants are prepared every two weeks. Slants can be identified by a number which corresponds to month and day of inoculation, for example, if the slant is started on August 6th, the identifying number is 806.

b) Test broth Staphylococcus aureus UT 15 is aseptically transferred from a mature slant to

30 ml of nutrient broth using a sterile inoculating loop. The broth is agitated for about 30 seconds and then incubated for 24 hours without agitation. The culture is again agitated for about 30 seconds just prior to use.

c) Culture dilution The culture is diluted to an appropriate concentration with peptone to deliver a

total count of 2200 ± 500 viable particles on the control plates when the aerosol is collected in the Andersen sampler.

A 5.2.1 The dilution procedure is as follows:

a) The spectrophotometer (B & L Spectronic 20) is set at 625 nm and zeroed at 0 % transmittance. b) Peptone dilution medium Peptone dilution medium is added to a clean sterile cuvette. The cuvette is

then placed in the spectrophotometer which is then adjusted to read 100 % transmittance.

c) The cuvette is then emptied and allowed to drain The undiluted culture is then added to the cuvette. The culture turbidity is

measured on the spectrophotometer and recorded in percent transmittance, T.

d) Peptone dilution medium is added Peptone dilution medium is added to the culture until the turbidity measures

72 % T. With each addition of peptone the culture is swirled with a vortex mixer.

e) The 72 % T culture must then be diluted The 72 % T culture must then be diluted to yield 2200 ± 500 viable particles

per 0,2 ml test broth aerosolized. A serial dilution is used. For example, if a 1/3000 dilution factor is required the dilution procedure would be as follows in figure 1:

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Figure A1 Only clean, sterile glassware is used with culture handling

1 ml 1 ml 1 1 ml

2 ml Peptone

9 ml Peptone

9 ml Peptone

9 ml Peptone

1/3 1/3 1/30 1/300 1/3000 Curvette 72%

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A 5.3 Apparatus operation a) Once the test culture has been prepared

Once the test culture has been prepared it is drawn into a sterile 5 cc Hamilton gas tight syringe. The syringe is then connected to the Chicago syringe pump.

b) The tryptic soy agar plates The tryptic soy agar plates are then placed into the Andersen sampler. All fittings and hoses are inspected to insure that all connections are made properly.

c) The syringe pump The syringe pump is then used to prime the nebulizer. The timer activating the syringe pump is shut off when the test solutions come within approximately 6 mm of the capillary tube tip.

d) The apparatus is now ready to operate Using the first set of plates as a waste set, the flows are set and the apparatus is used as follows The vacuum to the Andersen sampler is turned on and adjusted to pull 28 lpm airflow. The flow is measured by a Gilmont flow meter which has been calibrated by a dry gas meter. The air to the nebulizer is turned on and the pressure is adjusted to 15 psi. The timer activating the syringe pump is set for one minute. After 1 minute has elapsed, the syringe pump is switched off. After 1 minute 45 seconds total elapsed time, the air to the nebulizer is switched off. After 2 minutes total elapsed time, the vacuum from the Andersen sampler is switched off. The Andersen plates are then removed from the sampler and replaced with fresh plates. After the waste set of plates a control is run without any test material in place. Filter samples are then run using a fresh set of plates for each sample. It is suggested that one control run be made for every 6 samples tested. The average control value used for each batch of 6 test samples is the average of the two control runs prior to and subsequent to those six tests.

A 5.4 Incubation, counting and calculations

a) Incubate The inoculated plates are then inverted and incubated at 35-37°C for 24-48 hours.

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b) After incubation The colonies are counted using an automatic colony counter. All colonies are

counted on plates 1 and 2. On plates 3-6 only those colonies deposited in line (deposit areas) with the air stream holes are counted. These numbers are then converted using the positive hole conversion table supplied by Andersen. Ref: Andersen A.A. (1958). New Sampler for the collection, sizing and enumeration of viable airborne particles. Journal of Bacteriology 76 471-484.

This table converts the numbers of holes filled with viable particles to the probable number of actual particles in the aerosol. This table takes into account the probability that as the number of holes filled with viable particles increases, then the chances of a second or third viable particle also entering the same hole also increases.

c) The counts The counts are totaled and the filtration efficiencies are calculated as follows:

%100)(

)(% XrageControlAve

MaskDataIndividualrageControlAveBEF

A 5.5 Particle size of challenge aerosol

Average droplet or particle size is calculated based on the distribution of particle collection in the Andersen sampler and using a weighted average calculation. Andersen particle fractionating samplers are also available with eight multi-orifice impactor stages which separate the particles into 8 fractions from 11 microns and above down to 0,4 micron diameter.

Typical particle size distribution (microns)

Stage 0 1 2 3 4 5 6 7

Stage 6

- >7 4.4 - 7 3.3 – 4.7 2.1 – 3.3 1.1 – 2.1 0.65 – 1.1 -

Stage 8

>11 7-11 4.7 - 7 3.3 – 4.7 2.1 – 3.3 1.1 – 2.1 0.65 – 1.1 0.48 - 0.65

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Figure A2 Schematic diagram of a six-stage Andersen sampler

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Figure A3 Bacterial filtration efficiency

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A 5.6 Additional data reporting All of the following data is also recorded at the time of the test: a) Room temperature b) Relative humidity c) Barometric pressure d) Culture turbidity e) Dilution factor f) Slant number

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ANNEX B

Modification Track Sheet

History starting with WSP 2005 or later edition

Present document version number (name)

Previous version (s) Changes made

NWSP 300.0.R0 (15) (Nonwovens Bacterial Filtration Efficiency)

Originally published as WSP 300.0 (2005)

NWSP numbering Modification track

sheet