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ODORS GASES SMELLS, DIAGNOSIS & CURES
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How to calculate airborne mold or other airborne particle levels. This document describes proper procedures for evaluating mold and other aerobiological samples for purposes of identification of environmental sample contents and to help in assessing potential exposure of building occupants to levels of indoor particles, mold, allergens, and other materials. Included are procedures for proper particle counting from air samples, lab chemistry for sample preparation, and a directory of field sampling practices and methodology.
References are also given for particle identification. This is Daniel Friedman's general aerobiology & forensic procedure. Other mold test laboratories & forensic laboratories follow similar procedures but details and policy may vary. For clarity, some topics are addressed in separate documents listed here.
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Example % of Trace Calculations for an Olympus CH-2 microscope #5F0837
To form an accurate understanding of the percentage of particle trace that has been examined in the microscope we need to know the physical area of the particle trace and the physical area encompassed by one field of view in the microscope for a given microscope objective (e.g. 40x or 100x objective).
[The actual magnification in the field of view is the product of the microscope objective magnification and the microscope eyepiece magnification. Typically the eyepiece magnification is 10x or 15x.
Typical microscope objective magnifications used for aerobiology, mold particles, pollen, and other small particle identification and counting are 40x, 60x, 100x or 120x.
Indeed while counting is faster and easier at the lower magnifications of 40x or 60x, only by going to 100x oil immersion can we reliably see and count small particles in the 1-5u range, such as Penicillium or Aspergillus spores.
If we are counting less than 100 percent of the particle trace, the dimensions of the trace width and length are required to complete airborne particle concentration calculations. While we give the standardized particle trace dimensions and the percentage represented by one pass across the trace at different magnifications (below), keep in mind that the true particle trace on an air sampler slide varies due to site conditions and other factors - as you can see in our example photo at left.
See LAB & FIELD IAQ EQUIPMENT SOP for a complete table of airborne particle trace dimensions for various air sampling devices.
Air-O-Cell @1000x: .173mm field width/14.4mm x 100 = 1.2% of trace per pass
Air-O-Cell @ 400x:.44mm/14.4mm x 100 = 3.05% of trace per pass
Burkard @ 1000x: .173mm/14.0mm x 100 = 1.24% of trace per pass
Burkard @ 400x: .44mm/14.0mm x 100 = 3.14% of trace per pass
Allergenco @ 1000x: .173mm/.145mm x 100 = 1.193% of trace per pass
Allergenco @ 400x: .44mm/.145mm x 100 = 3.03% of trace per pass (e.g. use .303 as divisor in formula below)
(WARNING: this data is calibrated for a particular microscope in our lab. This number must be calibrated to the microscope, and optics used for the examination of the trace).
A Detailed Example for Airborne Particle Concentration Counting Calculations using the Allergenco Time-Lapse Impaction Sampler
To compute the airborne concentration of any individual particle (or of all particles) in an air test sample we need to know the air sample volume (total liters), for which we need to know the air sampling device flow rate in liters per minute and the number of minutes the sampler was run. We also need to allow for cases in which we count less than 100 percent of the particle trace. The basic formula for calculating the airborne particle concentration in an air sample is
1. Sample Volume in Liters = (Sampler run time in minutes x calibrated sampler flow rate in LPM)One M3 is 1000 cubic liters. So another version of the calculation could use
2. Sample Volume in M3 = [(Flow rate in
LPM)/1000] x [run time in minutes]
To obtain the total volume of air that was processed during an air test for airborne particles we multiply the air flow rate of the sampler (e.g. 15 lpm) by the number of minutes the sampler was operated (e.g. 3 minutes).
E.g. if we ran our Burkard Personal Air Sampler for 7 minutes, knowing that this devices pulls air through its impaction sampler slit at 10 liters per minute, 10 x 7 = 70 liters of air in our sample.
3. Percentage of the total particle trace that was examined. As we explain below, very dense or overloaded samples may have so many particles as to preclude an accurate count of 100 % of the trace sample. If we are counting less than 100% of the sample we need to include that factor in our calculations.
Trace length counted = (Microscope field diam) x (# cross-width traverses)
4. Particles / M3 = (raw particle count / % portion of trace counted) x (1000L/M3 / sample volume in L)
Example 1 - Number of particles represented by a 10 minute air test using an Allergenco impaction sampler
if an Allergenco time-lapse impaction air sampler is run for 10 minutes, 150 L was sampled (10 minutes x 15 LPM)
For the Allergenco, 1 cross trace pass at 1000x = 1.193% of trace
For the Allergenco, 10 cross-trace passes = 11.93% of the trace, or .1193 of the trace.
Example: If Particle "x" was counted at 653 particles in 10 passes of the trace at 1000x.
Example: if 100% of the trace was counted, then the "raw count" will be divided by 1 (100%)
[653 particles / (.1193 of trace/pass )] x [1000 L in 1 M3 of air / 150 L in sample] = 34,490.6 particles/M3 of air in the sample. This number should be documented in the lab as 34,490.6 but in an interpretation may be described as 34,500 particles/M3 of air.
Example 2 - Airborne mold concentration represented by an 8 minute air test using a Burkard Personal Sampler
The Burkard PAS samples air at 10 lpm, so an 8-minute sample represents a test volume of (10 x 8) = 80 liters of air.
We counted 100% of the particle sample trace in this case, identifying the number of occurrences of each particle type found in the sample. Using just one of these particles, Alternaria sp., we found 7 Alternaria spores in the entire trace.
Measure the actual trace length. Divide the trace into equal segments by 4ths
Count 1 segment completely, selecting a representative segment after scanning lengthwise the whole trace. Multiply the actual count by the number of segments (4) to get the "trace total count. ["trace total count"] x [1000/sample volume] = particles/M3
Interferences in Reaching the Objective of 100% Trace Counting - handling overloaded air particle samples
For low density particle traces it is easy and reasonable, as well as most accurate, to count 100% of the particle trace area. Where practical, in our lab we count 100% of the particle trace on the sampling slide or cassette.
Our photo (left) illustrates a reasonable-density air particle sample trace on a slide from one of our Burkard Personal Air Sampler devices. By examination using the naked eye the trace density appears not to be overloaded, though final decision on how to process this sample will be made after the initial examination in the microscope. This particle sample was collected inside the return air plenum of a heating & air conditioning air handler, using passive air sampling methods - we just placed the sampler in the space and ran it for a number of minutes, with minimum disturbance to the environment. And of course the equipment was OFF during the test.
But some airborne particle or dust vacuum particle traces are so dense with particles that an accurate particle count can be difficult, impractical, or even impossible. Our second Burkard PAS particle trace (below left) was collected in the same location as the first particle trace above, but we used our standard "aggressive" sampling method intended to dislodge local particles that might otherwise not appear in the test.
We rapped once with a flashlight on the side of the air handler's air plenum chamber. You can see that this approach made an enormous difference in the number of particles collected even though we ran the air sampling device for the same time interval as in the first test. This sample is probably overloaded.
For example, a very dense particle trace suggests that during the latter portion of the air sampling period, late-arriving particles may impact particles already on the trace medium surface rather than contacting the more sticky trace capture media. As a result, the particle bounce rate increases late in the air sampling period - such particles are lost and thus under-represented in the trace result.
For thick, occluded, dense particle traces too, the absolute number or raw particle count could be in the thousands, making accurate manual counting inaccurate or infeasible. For such dense particle traces our minimum objective is to count 25% of the trace. 20% may be used in the most difficult cases.
We first scan the entire particle trace at lower magnification, perhaps 400x, in order to note the presence or absence of anomalies such as voids in the trace capture media or thick clusters of particles or large particles that can substantially affect the accurate particle count number and that might be missed during a 25% random trace area count approach.
Particle or mold spore cluster or spore chain counting & reporting rules
Similarly, and more important, some mold test labs count a chain of connected mold spores such as Aspergillus spores or Penicillium spores as "one" spore.
Inconsistencies in how labs count clusters and chains of particles can make a very large difference in the airborne particle concentration reported.
We count the individual spores in clusters or chains, and we use that number in our airborne particle concentration calculations.
But because the presence of Pen/Asp spores in chains in an indoor air sample screening for building mold contamination is very important, we also report the presence of spores in chains as a separate technical examination.
Airborne Particle Trace Count Stopping Rules
Airborne Particle Trace Count Stopping Rules may be considered. (when 200 particles/M3 are reached continue that pass across the particle trace to completion, record the total count, calculate the total based on % of trace read, and express it as a minimum with citation of stopping rule.)
See MOLD LAB REPORTS.
Also see ACCURACY vs PRECISION of MEASUREMENTS where we argue that measurements should be reported to include their percentage of error or a +/- figure to give a realistic understanding of the actual reliability of the data.
Also see the following forensic lab & microscopy equipment calibration and adjustment details in these individual lab equipment procedures notebook or policy documents, including the latest microscope calibration dates.
Review microscope setup, Kohler illumination, objective centering, stage micrometer calibration of eyepiece reticule scale, and other preparation procedures in the lab notebook dedicated to each microscope. See LAB PROCEDURES MICROSCOPE TECHNIQUES for a discussion of microscope adjustment, setup, and calibration procedures. For each microscope (or other equipment) the lab maintains a procedures manual that gives these details as well as records of calibration and adjustment dates and results.
Review objective/eyepiece field width measurement data at spread sheet dedicated to each microscope
review list of chemicals, uses, preparations in the Lab Procedures ™ Chemistry & Slide Preparation notebook. Review the MSDS for each lab chemical in the Lab Supplies MSDS notebook. Key files:
Procedures for Qualitative Building Surface Particle Sample Analysis or Vacuum Sample Analysis for Surfaces, Carpets, Soft Goods
Tape or vacuum cassette (tape, Air-o-Cell, or filter cassette) samples are prepared using lab SOP for each sample type. Qualitative analysis and characterization is described in each lab report. Quantitative analysis of surface tape samples is highly questionable since particle density across a building surface cannot be assumed to be uniform.
For Air-o-Cell or MCE filter cassettes, scan the entire trace at low magnification, 40x or 100x, for consistency and for unusual particle clusters to be considered in selecting areas for cross-scans.
For tape samples, the sample may be examined using the low-power stereo microscope to evaluate sample consistency and to select a sample tape segment most-likely to contain significant particles. A 1 cm segment is selected for analysis. If the tape appears to contain a variety of particles by texture, color, etc., multiple 1-cm segments may be required for analysis.
Scan across the trace beginning at one end of the selected area, to and from areas where no particle are visible while crossing the trace. See separate counting rules and stopping rules (if any stopping rules are to be applied), obtaining raw counts of significant or other particles of interest in each pass and totaling for the trace. See Particle Counts.xls for worksheets used for this purpose. The worksheet automates particles/M3 of air when the raw counts and volume of the sample are entered.
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