CAUTION: Do not overload the filter with dust. High levels of non- fibrous dust particles may obscure fibers on the filter and lower the count or make counting impossible. If more than about 25 to 30% of the field area is obscured with dust, the result may be biased low. Smaller air volumes may be necessary when there is excessive non-asbestos dust in the air.

While sampling, observe the filter with a small flashlight. If there is a visible layer of dust on the filter, stop sampling, remove and seal the cassette, and replace with a new sampling assembly. The total dust loading should not exceed 1 mg.

5.2.9. Blank samples are used to determine if any contamination has occurred during sample handling. Prepare two blanks for the first 1 to 20 samples. For sets containing greater than 20 samples, prepare blanks as 10% of the samples. Handle blank samples in the same manner as air samples with one exception: Do not draw any air through the blank samples. Open the blank cassette in the place where the sample cassettes are mounted on the employee. Hold it open for about 30 seconds. Close and seal the cassette appropriately. Store blanks for shipment with the sample cassettes.

5.2.10. Immediately after sampling, close and seal each cassette with the base and plastic plugs. Do not touch or puncture the filter membrane as this will invalidate the analysis.

5.2.11. Attach and secure a sample seal around each sample cassette in such a way as to assure that the end cap and base plugs cannot be removed without destroying the seal. Tape the ends of the seal together since the seal is not long enough to be wrapped end-to-end. Also wrap tape around the cassette at each joint to keep the seal secure.

5.3. Sample Shipment

5.3.1. Send the samples to the laboratory with paperwork requesting asbestos analysis. List any known fibrous interferences present during sampling on the paperwork. Also, note the workplace operation(s) sampled.

5.3.2. Secure and handle the samples in such that they will not rattle during shipment nor be exposed to static electricity. Do not ship samples in expanded polystyrene peanuts, vermiculite, paper shreds, or excelsior. Tape sample cassettes to sheet bubbles and place in a container that will cushion the samples in such a manner that they will not rattle.

5.3.3. To avoid the possibility of sample contamination, always ship bulk samples in separate mailing containers.

6. Analysis

6.1. Safety Precautions

6.1.1. Acetone is extremely flammable and precautions must be taken not to ignite it. Avoid using large containers or quantities of acetone. Transfer the solvent in a ventilated laboratory hood. Do not use acetone near any open flame. For generation of acetone vapor, use a spark free heat source.

6.1.2. Any asbestos spills should be cleaned up immediately to prevent dispersal of fibers. Prudence should be exercised to avoid contamination of laboratory facilities or exposure of personnel to asbestos. Asbestos spills should be cleaned up with wet methods and/or a High Efficiency Particulate Air (HEPA) filtered vacuum.

CAUTION: Do not use a vacuum without a HEPA filter - It will disperse fine asbestos fibers in the air.

6.2. Equipment

6.2.1. Phase contrast microscope with binocular or trinocular head.

6.2.2. Widefield or Huygenian 10X eyepieces (NOTE: The eyepiece containing the graticule must be a focusing eyepiece. Use a 40X phase objective with a numerical aperture of 0.65 to 0.75).

6.2.3. Kohler illumination (if possible) with green or blue filter.

6.2.4. Walton-Beckett Graticule, type G-22 with 100 plus or minus 2 m m projected diameter.

6.2.5. Mechanical stage. A rotating mechanical stage is convenient for use with polarized light.

6.2.6. Phase telescope.

6.2.7. Stage micrometer with 0.01-mm subdivisions.

6.2.8. Phase-shift test slide, mark II (Available from PTR optics Ltd., and also McCrone).

6.2.9. Precleaned glass slides, 25 mm X 75 mm. One end can be frosted for convenience in writing sample numbers, etc., or paste-on labels can be used.

6.2.10. Cover glass #1 1/2.

6.2.11. Scalpel (#10, curved blade).

6.2.12. Fine tipped forceps.

6.2.13. Aluminum block for clearing filter (see Appendix D).

6.2.14. Automatic adjustable pipette, 100-to 500- m L.

6.2.15. Micropipette, 5 m L.

6.3. Reagents

6.3.1. Acetone (HPLC grade).

6.3.2. Triacetin (glycerol triacetate).

6.3.3. Lacquer or nail polish.

6.4. Standard Preparation

A way to prepare standard asbestos samples of known concentration has not been developed. It is possible to prepare replicate samples of nearly equal concentration. This has been performed through the PAT program. These asbestos samples are distributed by the AIHA to participating laboratories.

Since only about one-fourth of a 25-mm sample membrane is required for an asbestos count, any PAT sample can serve as a "standard" for replicate counting.

6.5. Sample Mounting

Note: See Safety Precautions in Section 6.1. before proceeding. The objective is to produce samples with a smooth (non-grainy) background in a medium with a refractive index of approximately 1.46. The technique below collapses the filter for easier focusing and produces permanent mounts which are useful for quality control and interlaboratory comparison.

An aluminum block or similar device is required for sample preparation.

6.5.1. Heat the aluminum block to about 70 deg.C. The hot block should not be used on any surface that can be damaged by either the heat or from exposure to acetone.

6.5.2. Ensure that the glass slides and cover glasses are free of dust and fibers.

6.5.3. Remove the top plug to prevent a vacuum when the cassette is opened. Clean the outside of the cassette if necessary. Cut the seal and/or tape on the cassette with a razor blade. Very carefully separate the base from the extension cowl, leaving the filter and backup pad in the base.

6.5.4. With a rocking motion cut a triangular wedge from the filter using the scalpel. This wedge should be one-sixth to one-fourth of the filter. Grasp the filter wedge with the forceps on the perimeter of the filter which was clamped between the cassette pieces. DO NOT TOUCH the filter with your finger. Place the filter on the glass slide sample side up. Static electricity will usually keep the filter on the slide until it is cleared.

6.5.5. Place the tip of the micropipette containing about 200 m L acetone into the aluminum block. Insert the glass slide into the receiving slot in the aluminum block. Inject the acetone into the block with slow, steady pressure on the plunger while holding the pipette firmly in place. Wait 3 to 5 seconds for the filter to clear, then remove the pipette and slide from the aluminum block.

6.5.6. Immediately (less than 30 seconds) place 2.5 to 3.5 m L of triacetin on the filter (NOTE: Waiting longer than 30 seconds will result in increased index of refraction and decreased contrast between the fibers and the preparation. This may also lead to separation of the cover slip from the slide).

6.5.7. Lower a cover slip gently onto the filter at a slight angle to reduce the possibility of forming air bubbles. If more than 30 seconds have elapsed between acetone exposure and triacetin application, glue the edges of the cover slip to the slide with lacquer or nail polish.

6.5.8. If clearing is slow, warm the slide for 15 min on a hot plate having a surface temperature of about 50 deg.C to hasten clearing. The top of the hot block can be used if the slide is not heated too long.

6.5.9. Counting may proceed immediately after clearing and mounting are completed.

6.6. Sample Analysis

Completely align the microscope according to the manufacturer's instructions. Then, align the microscope using the following general alignment routine at the beginning of every counting session and more often if necessary.

6.6.1. Alignment

(1) Clean all optical surfaces. Even a small amount of dirt can significantly degrade the .

(2) Rough focus the objective on a sample.

(3) Close down the field iris so that it is visible in the field of view. Focus the of the iris with the condenser focus. Center the of the iris in the field of view.

(4) Install the phase telescope and focus on the phase rings. Critically center the rings. Misalignment of the rings results in astigmatism which will degrade the .

(5) Place the phase-shift test slide on the microscope stage and focus on the lines. The analyst must see line set 3 and should see at least parts of 4 and 5 but, not see line set 6 or 6. A microscope/microscopist combination which does not pass this test may not be used.

6.6.2. Counting Fibers

(1) Place the prepared sample slide on the mechanical stage of the microscope. Position the center of the wedge under the objective lens and focus upon the sample.

(2) Start counting from one end of the wedge and progress along a radial line to the other end (count in either direction from perimeter to wedge tip). Select fields randomly, without looking into the eyepieces, by slightly advancing the slide in one direction with the mechanical stage control.

(3) Continually scan over a range of focal planes (generally the upper 10 to 15 m m of the filter surface) with the fine focus control during each field count. Spend at least 5 to 15 seconds per field.

(4) Most samples will contain asbestos fibers with fiber diameters less than 1 m m. Look carefully for faint fiber s. The small diameter fibers will be very hard to see. However, they are an important contribution to the total count.

(5) Count only fibers equal to or longer than 5 m m. Measure the length of curved fibers along the curve.

(6) Count fibers which have a length to width ratio of 3:1 or greater.

(7) Count all the fibers in at least 20 fields. Continue counting until either 100 fibers are counted or 100 fields have been viewed; whichever occurs first. Count all the fibers in the final field.

(8) Fibers lying entirely within the boundary of the Walton-Beckett graticule field shall receive a count of 1. Fibers crossing the boundary once, having one end within the circle shall receive a count of 1/2. Do not count any fiber that crosses the graticule boundary more than once. Reject and do not count any other fibers even though they may be visible outside the graticule area. If a fiber touches the circle, it is considered to cross the line.

(9) Count bundles of fibers as one fiber unless individual fibers can be clearly identified and each individual fiber is clearly not connected to another counted fiber. See Figure 1 of this appendix for counting conventions.

(10) Record the number of fibers in each field in a consistent way such that filter non-uniformity can be assessed.

(11) Regularly check phase ring alignment.

(12) When an agglomerate (mass of material) covers more than 25% of the field of view, reject the field and select another. Do not include it in the number of fields counted.

(13) Perform a "blind recount" of 1 in every 10 filter wedges (slides). Re-label the slides using a person other than the original counter.

6.7. Fiber Identification

As previously mentioned in Section 1.3., PCM does not provide positive confirmation of asbestos fibers. Alternate differential counting techniques should be used if discrimination is desirable. Differential counting may include primary discrimination based on morphology, polarized light analysis of fibers, or modification of PCM data by Scanning Electron or Transmission Electron Microscopy.

A great deal of experience is required to routinely and correctly perform differential counting. It is discouraged unless it is legally necessary. Then, only if a fiber is obviously not asbestos should it be excluded from the count. Further discussion of this technique can be found in reference 8.10. If there is a question whether a fiber is asbestos or not, follow the rule: "WHEN IN DOUBT, COUNT."

6.8. Analytical Recommendations - Quality Control System

6.8.1. All individuals performing asbestos analysis must have taken the NIOSH course for sampling and evaluating airborne asbestos or an equivalent course.

6.8.2. Each laboratory engaged in asbestos counting shall set up a slide trading arrangement with at least two other laboratories in order to compare performance and eliminate inbreeding of error. The slide exchange occurs at least semiannually. The round robin results shall be posted where all analysts can view individual analyst's results.

6.8.3. Each laboratory engaged in asbestos counting shall participate in the Proficiency Analytical Testing Program, the Asbestos Analyst Registry or equivalent.

6.8.4. Each analyst shall select and count prepared slides from a "slide bank". These are quality assurance counts. The slide bank shall be prepared using uniformly distributed samples taken from the workload. Fiber densities should cover the entire range routinely analyzed by the laboratory. These slides are counted blind by all counters to establish an original standard deviation. This historical distribution is compared with the quality assurance counts. A counter must have 95% of all quality control samples counted within three standard deviations of the historical mean. This count is then integrated into a new historical mean and standard deviation for the slide.

The analyses done by the counters to establish the slide bank may be used for an interim quality control program if the data are treated in a proper statistical fashion.

7. Calculations

7.1. Calculate the estimated airborne asbestos fiber concentration on the filter sample using the following formula:



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where:

AC = Airborne fiber concentration

FB = Total number of fibers greater than 5 m m counted

FL = Total number of fields counted on the filter

BFB = Total number of fibers greater than 5 m m counted in the blank

BFL = Total number of fields counted on the blank

ECA = Effective collecting area of filter (385 mm(2) nominal for a 25 - mm filter.)

FR = Pump flow rate (L/min)

MFA = Microscope count field area (mm(2)). This is 0.00785 mm(2) for a Walton-Beckett Graticule.

T = Sample collection time (min) 1,000 = Conversion of L to cc

Note: The collection area of a filter is seldom equal to 385 mm(2). It is appropriate for laboratories to routinely monitor the exact diameter using an inside micrometer. The collection area is calculated according to the formula:

Area = / (d /2) 2

7.2. Short-Cut Calculation

Since a given analyst always has the same interpupillary distance, the number of fields per filter for a particular analyst will remain constant for a given size filter. The field size for that analyst is constant (i.e. the analyst is using an assigned microscope and is not changing the reticle).

For example, if the exposed area of the filter is always 385 mm(2) and the size of the field is always 0.00785 mm(2) the number of fields per filter will always be 49,000. In addition it is necessary to convert liters of air to cc. These three constants can then be combined such that ECA/(1,000 x MFA)=49. The previous equation simplifies to:



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7.3. Recount Calculations

As mentioned in step 13 of Section 6.6.2., a "blind recount" of 10% of the slides is performed. In all cases, differences will be observed between the first and second counts of the same filter wedge. Most of these differences will be due to chance alone, that is, due to the random variability (precision) of the count method. Statistical recount criteria enables one to decide whether observed differences can be explained due to chance alone or are probably due to systematic differences between analysts, microscopes, or other biasing factors.

The following recount criterion is for a pair of counts that estimate AC in fibers/cc. The criterion is given at the type-I error level. That is, there is 5% maximum risk that we will reject a pair of counts for the reason that one might be biased, when the large observed difference is really due to chance.

Reject a pair of counts if:



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Where:

AC(1) = lower estimated airborne fiber concentration AC(2) = higher estimated airborne fiber concentration AC(avg) = average of the two concentration estimates CV(FB) = CV for the average of the two concentration estimates

If a pair of counts are rejected by this criterion then, recount the rest of the filters in the submitted set. Apply the test and reject any other pairs failing the test. Rejection shall include a memo to the industrial hygienist stating that the sample failed a statistical test for homogeneity and the true air concentration may be significantly different than the reported value.

7.4. Reporting Results

Report results to the industrial hygienist as fibers/cc. Use two significant figures. If multiple analyses are performed on a sample, an average of the results is to be reported unless any of the results can be rejected for cause.

8. References

8.1. Dreesen, W.C., et al., U.S. Public Health Service: A Study of Asbestosis in the Asbestos Textile Industry (Public Health Bulletin No. 241), U.S. Treasury Dept., Washington, DC, 1938.

8.2. Asbestos Research Council: The Measurement of Airborne Asbestos Dust by the Membrane Filter Method (Technical Note), Asbestos Research Council, Rockdale, Lancashire, Great Britain, 1969.

8.3. Bayer, S.G., Zumwalde, R.D., Brown, T.A., Equipment and Procedure for Mounting Millipore Filters and Counting Asbestos Fibers by Phase Contrast Microscopy, Bureau of Occupational Health, U.S. Dept. of Health, Education and Welfare, Cincinnati, OH, 1969.

8.4. NIOSH Manual of Analytical Methods, 2nd ed., Vol. 1 (DHEW/ NIOSH Pub. No. 77-157-A). National Institute for Occupational Safety and Health, Cincinnati, OH, 1977. pp. 239-1 - 239-21.

8.5. Asbestos, Code of Federal Regulations 29 CFR 1910.1001. 1971.

8.6. Occupational Exposure to Asbestos, Tremolite, Anthophyllite, and Actinolite. Final Rule, Federal Register 51:119 (20 June 1986). pp. 22612- 22790.

8.7. Asbestos, Tremolite, Anthophyllite, and Actinolite, Code of Federal Regulations 1910.1001. 1988. pp. 711-752.

8.8. Criteria for a Recommended Standard - Occupational Exposure to Asbestos (DHEW/NIOSH Pub. No. HSM 72-10267), National Institute for Occupational Safety and Health, NIOSH, Cincinnati, OH, 1972. pp. III-1 - III-24.

8.9. Leidel, N.A., Bayer, S.G., Zumwalde, R.D., Busch, K.A., USPHS/NIOSH Membrane Filter Method for Evaluating Airborne Asbestos Fibers (DHEW/NIOSH Pub. No. 79-127). National Institute for Occupational Safety and Health, Cincinnati, OH, 1979.

8.10. Dixon, W.C., Applications of Optical Microscopy in Analysis of Asbestos and Quartz, Analytical Techniques in Occupational Health Chemistry, edited by D.D. Dollberg and A.W. Verstuyft. Wash. D.C.: American Chemical Society, (ACS Symposium Series 120) 1980. pp. 13-41.

Quality Control

The OSHA asbestos regulations require each laboratory to establish a quality control program. The following is presented as an example of how the OSHA-SLTC constructed its internal CV curve as part of meeting this requirement. Data is from 395 samples collected during OSHA compliance inspections and analyzed from October 1980 through April 1986.

Each sample was counted by 2 to 5 different counters independently of one another. The standard deviation and the CV statistic was calculated for each sample. This data was then plotted on a graph of CV vs. fibers/mm ². A least squares regression was performed using the following equation:

CV = antilog ₁₀ [A(log ₁₀ (x)) ² +B(log ₁₀ (x))+C]

where:

x = the number of fibers/mm ²

Application of least squares gave:

A = 0.182205 B = 0.973343 C = 0.327499

Using these values, the equation becomes:

CV = antilog 10 [0.182205(log 10 (x)) 2 + 0.973343(log 10 (x)) + 0.327499]

Sampling Pump Flow Rate Corrections

This correction is used if a difference greater than 5% in ambient temperature and/or pressure is noted between calibration and sampling sites and the pump does not compensate for the differences.

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Where:

Q(act) = actual flow rate Q(cal) = calibrated flow rate (if a rotameter was used, the rotameter value) P(cal) = uncorrected air pressure at calibration P(act) = uncorrected air pressure at sampling site T(act) = temperature at sampling site (K) T(cal) = temperature at calibration (K)

Walton-Beckett Graticule

When ordering the Graticule for asbestos counting, specify the exact disc diameter needed to fit the ocular of the microscope and the diameter (mm) of the circular counting area. Instructions for measuring the dimensions necessary are listed:

(1) Insert any available graticule into the focusing eyepiece and focus so that the graticule lines are sharp and clear.

(2) Align the microscope.

(3) Place a stage micrometer on the microscope object stage and focus the microscope on the graduated lines.

(4) Measure the magnified grid length, PL ( m m), using the stage micrometer.

(5) Remove the graticule from the microscope and measure its actual grid length, AL (mm). This can be accomplished by using a mechanical stage fitted with verniers, or a jeweler's loupe with a direct reading scale.

(6) Let D = 100 m m. Calculate the circle diameter, d(c)(mm), for the Walton-Beckett graticule and specify the diameter when making a purchase:

AL x D

d c = _________

PL

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Example: If PL = 108 m m, AL = 2.93 mm and D = 100 m m, then,

2.93 x 100

d _c = ___________ = 2.71mm

108



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(7) Each eyepiece-objective-reticle combination on the microscope must be calibrated. Should any of the three be changed (by zoom adjustment, disassembly, replacement, etc.), the combination must be recalibrated. Calibration may change if interpupillary distance is changed.

Measure the field diameter, D (acceptable range: 100 plus or minus 2 m m) with a stage micrometer upon receipt of the graticule from the manufacturer. Determine the field area (mm(2)).

Field Area = PI( D /2) 2

If D = 100 MU = 0.1 mm, then

Field Area = PI(0.1 mm /2) 2 = 0.00785mm 2

The Graticule is available from: Graticules Ltd., Morley Road, Tonbridge TN9 IRN, Kent, England (Telephone 011-44-732-359061). Also available from PTR Optics Ltd., 145 Newton Street, Waltham, MA 02154 [telephone (617) 891-6000] or McCrone Accessories and Components, 2506 S. Michigan Ave., Chicago, IL 60616 [phone (312)- 842-7100]. The graticule is custom made for each microscope.

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Figure 1: Walton-Beckett Graticule with some explanatory fibers.
Counts for the Fibers in the Figure

1. New Appendix B to section 1529 filed 2-15-91; operative 2-15-91 pursuant to 
Government Code section 11346.2(d) (Register 91, No. 19).

2. Editorial correction of HISTORY 1 (Register 91, No. 45).

3. Editorial correction of Appendix B (Register 95, No. 41).

4. Repealer and new appendix and Note filed 5-3-96; operative 7-3-96 (Register 96, No. 18).

5. Change without regulatory effect providing more legible Figure 1 filed 1-20- 2008 pursuant to section 100, title 1, California Code of Regulations 
(Register 2009, No. 4).