Example Contributors to Measurement Uncertainty – Direct Air Environmental

Microbiology Analyses (Spore Trap)

(representative list - may not include of all contributors)
(QC sample types in this list are typical of those utilized in AIHA-LAP, LLC laboratories)
See Example Calculations (to the right of the table) and tabbed sheets for additional examples

Representative and
Applicable QC
Contributors to Uncertainty Data Comments to Clarify Contributor Effects
Temperature, Storage, Handling:
shipping time, container & temperature NA No impact on direct air exam samples
lab storage time, conditions & temperature NA No impact on direct air exam samples
contamination in lab storage areas NA No impact on direct air exam samples
Laboratory Subsampling:
sample nonhomogeneity NA Not applicable to direct air exam samples
blending techniques NA Not applicable to direct air exam samples
sample size NA Not applicable to direct air exam samples

Sample Preparation:
With proper care there should be no contamination of daily blanks;
slides & coverslip contamination MB therefore, no impact
With proper care there should be no contamination of daily blanks;
mounting medium MB therefore, no impact
Lab Environmental Conditions:
Samples are not exposed to air for any length of time; therefore
seasonal background spore variances MB there should be no impact
Analysts:
different analysts RS Reference slides analyzed by multiple analysts
analyst training level & experience RS Reference slides analyzed by multiple analysts
data interpretation by analyst RS Reference slides analyzed by multiple analysts
Measuring Instruments:
microscope magnification level used RS Reference slides analyzed with multiple microscopes
eye piece graticule & field of view
calibration RS Reference slides analyzed with multiple microscopes
Test Procedure Variations:
portion and fields of sample analyzed RS Varies by analyst
microbial density RS High concentrations or clumps of spores may impact results
interferences RS Debris level and resolution of spores in field of view
Uncertainty may be concentration dependent. Lab should evaluate
ranges (high, medium, low) RS this as part of method validation.
Data Manipulation:

reading, interpreting and reporting results RS

Accuracy of calculations RS Manual, spreadsheet, LIMS, etc

Typically provided by the customer. This is not part of analytical

uncertainty, but must be considered by labs providing sampling and
area or air volume sampled NA providing combined sampling and analytical uncertainty.
MB = Daily method blank
RS = Daily reference slides

Please note that the original column I (CV of the pair) of the “culturable analyses” tabbed worksheet had a
formula incorrectly entered. The worksheet has been corrected and any affected values have been
highlighted in yellow.
Total Spores/m3 of air by Direct Examination Air (Spore Trap)
Example Using Pooled CV Data from Three Reference Slides
Reference Slide Number

Slide 1 Slide 2 Slide 3

Reading
Number Spores/m3 Spores/m3 Spores/m3
1 813 1640 1213
2 773 1473 1353
3 687 1507 1467
4 707 1420 1460
5 733 1273 1440
6 847 1247 1173
7 760 973 1487
8 793 1747 1373
9 973 1720 1487
10 693 1653 1273

11 960 1940 1453

12 587 1787 1327

13 807 1693 1500

14 1000 1847 1447

15 940 1760 1487
16 853 1320 1053
17 940 1447 1520
18 793 1920 1627
19 793 1807 1160
20 747 1893 1127

21 840 1860 1227

22 820 1667 1427
23 873 2107 1180
24 733 1887 1233
25 833 1853 973

26 853 1407 1133

27 753 1787 1280

28 900 1313 1220

29 647 1900 1480

30 887 1453 1213

Mean 811 1643 1326
std dev 98.9 261.6 162.1
CV 0.122 0.159 0.122
 CV pooled = √[((0.122)2 + (0.159)2 + (0.122)2)/3] = 0.135

Expanded MU @ 95% C.L. (k=2) equals CV pooled X 2 = 0.27 (27%

Bias cannot be determined. No quantitative reference
material available

Example analytical uncertainty for air sample with 890 spores/m3:

Expanded analytical uncertainty = 890 spores/m3 X 0.27 = 240 spores/m3

Example of reporting for air sample with 890 spores/m3:

890 spores/m3 with an analytical uncertainty of +/- 240 spores/m3 at the 95%
confidence level
 RSD)

es/m3:
40 spores/m3

res/m3 at the 95%

 Example Contributors to Measurement Uncertainty – Culturable Environmental
Microbiology Analyses
(representative list - may not include of all contributors)
(QC sample types in this list are typical of those utilized in AIHA-LAP, LLC laboratories)
See Example Calculations (to the
right of the table)

Representative
and Applicable
Contributors to Uncertainty QC Data Comments to Clarify Contributor Effects
Temperature, Storage, Handling:
May affect growth but not under control of lab so no impact on
shipping time, container & temperature NA analytical uncertainty

Affects growth rates of organisms in samples. Study samples

included in some method validation studies may reflect these
lab storage time, conditions & temperature NA contributors.
contamination in lab storage areas NA As long as samples are contained there should be no impact
Laboratory Subsampling:
sample nonhomogeneity DUP Area of bulk samples selected for analysis
blending techniques DUP Extraction/vortexing non-homogeniety of samples
sample size DUP

Sample Preparation:
contamination during preparation BLK Sterility of materials used/autoclave operation

Contributions from this and the rest of Sample Prep contributors

measured only if duplicate samples are prepared; not captured by
sample homogenization/subsampling DUP inter-analyst readings of same sample prep
sample dilution (pipettes, etc) DUP Uncertainty related to method used to make serial dilutions

balance DUP Balance error is often insignificant compared to other MU sources

plating technique DUP How distributed on media
composition of sample DUP Organism competition on media
Lab Environmental Conditions:
Should have minimal impact when proper aseptic techniques are
seasonal background organism variances BLK used
Analysts:

different analysts DUP Must use inter-analyst instead of intra-analyst repeat analyses.

analyst training level & experience DUP Must use inter-analyst instead of intra-analyst repeat analyses.

data interpretation by analyst DUP Must use inter-analyst instead of intra-analyst repeat analyses.
Measuring Instruments:

microscope magnification level used DUP Determines level of detail of organisms that can be easily seen
Proper calibration required to facilitate spore identification based in
eye piece graticule calibration DUP part on size
data logger DUP When used to assist in organism identification, e.g. bacteria
Test Procedure Variations:
media used DUP Different media show preference for different organisms.

Affects rate of growth but cannot be monitored unless using known

incubation conditions DUP concentration control cultures or do time/analysis study
 identification techniques used DUP Especially for bacteria (chemical tests vs. data logger, etc)
microbial density DUP Organism competition (inhibition, overgrowth, etc)
interferences DUP Presence of organisms other than those of interest
Data Manipulation:

reading, interpreting and reporting results DUP

Accuracy of calculations DUP Manual, spreadsheet, LIMS, etc

BLK = Lab Blank prepared when samples are prepared

DUP = Inter-analyst duplicate preparation and analysis
 Total Fungal CFU/swab on MEA media
Example Using Pooled Duplicate CV Data

1st 2nd CV of
number analyst analyst Std Dev of pair
of pair CFU/swab CFU/Swab pair (s/mean) CV2
1 230 140 63.6 0.344 0.118

2 8 6 1.4 0.202 0.041

3 69 54 10.6 0.172 0.030

4 220 200 14.1 0.067 0.005
5 150 155 3.5 0.023 0.001
6 155 130 17.7 0.124 0.015
7 94 125 21.9 0.200 0.040
8 22 16 4.2 0.223 0.050
9 17 17 0.0 0.000 0.000
10 9 7 1.4 0.177 0.031
11 33 41 5.7 0.153 0.023

12 32 30 1.4 0.046 0.002

13 110 120 7.1 0.061 0.004

14 110 100 7.1 0.067 0.005

15 270 290 14.1 0.051 0.003
16 17 29 8.5 0.369 0.136
17 160 170 7.1 0.043 0.002

18 300 330 21.2 0.067 0.005

19 6 8 1.4 0.202 0.041

20 190 260 49.5 0.220 0.048

21 6 9 2.1 0.283 0.080
22 19 11 5.7 0.377 0.142
23 42 40 1.4 0.034 0.001

24 42 49 4.9 0.109 0.012

25 690 590 70.7 0.110 0.012

26 170 240 49.5 0.241 0.058
27 28 38 7.1 0.214 0.046
28 180 160 14.1 0.083 0.007

29 61 68 4.9 0.077 0.006

 30 240 300 42.4 0.157 0.025
∑ CV2 0.988
CV pooled = √ (∑CV2/30) = 0.181

Expanded MU @ 95% C.L. (k=2) equals CV pooled X 2 = 0.36 36%

Example analytical uncertainty for swab sample with
Bias cannot be determined. No quantitative reference material available
440 CFU/swab:
440 CFU/swab
Expanded analytical uncertainty = 440 X 0.36 = 160
Example of reporting for swab sample with 440 CFU/swab:
440 CFU/swab with an analytical uncertainty of +/- 160 CFU/swab
at the 95% confidence level
440 CFU/swab with an analytical uncertainty of +/- 160 CFU/swab at the 95% confidence level
 CFU/swab

b at the 95% confidence level