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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
Contributors to Uncertainty
Representative and Applicable QC Data
Comments to Clarify Contributor Effects
Temperature, Storage, Handling:
NA NA NA
No impact on direct air exam samples
Not applicable to direct air exam samples
sample size
NA NA NA
slides & coverslip contamination
MB
With proper care there should be no contamination of daily blanks; therefore, no impact
mounting medium
MB
With proper care there should be no contamination of daily blanks; therefore, no impact
MB
Samples are not exposed to air for any length of time; therefore there should be no impact
shipping time, container & temperature lab storage time, conditions & temperature contamination in lab storage areas
No impact on direct air exam samples No impact on direct air exam samples
Laboratory Subsampling: sample nonhomogeneity blending techniques
Not applicable to direct air exam samples Not applicable to direct air exam samples
Sample Preparation:
Lab Environmental Conditions: seasonal background spore variances Analysts:
RS RS RS
Reference slides analyzed by multiple analysts
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 Varies by analyst
interferences
RS RS RS
ranges (high, medium, low)
RS
Uncertainty may be concentration dependent. Lab should evaluate this as part of method validation.
RS RS
Manual, spreadsheet, LIMS, etc
NA
Typically provided by the customer. This is not part of analytical uncertainty, but must be considered by labs providing sampling and providing combined sampling and analytical uncertainty.
different analysts analyst training level & experience data interpretation by analyst
Reference slides analyzed by multiple analysts Reference slides analyzed by multiple analysts
Measuring Instruments:
Test Procedure Variations: portion and fields of sample analyzed microbial density
High concentrations or clumps of spores may impact results Debris level and resolution of spores in field of view
Data Manipulation: reading, interpreting and reporting results Accuracy of calculations
area or air volume sampled
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 Reading Number 1 2 3 4 5 6 7 8 9 10
Mean std dev CV
Slide 2
Slide 3
Spores/m3 Spores/m3 Spores/m3 813 1640 1213 773 1473 1353 687 1507 1467 707 1420 1460 733 1273 1440 847 1247 1173 760 973 1487 793 1747 1373 973 1720 1487 693 1653 1273
11
960
1940
1453
12 13
587 807
1787 1693
1327 1500
14 15 16 17 18 19 20
1000 940 853 940 793 793 747
1847 1760 1320 1447 1920 1807 1893
1447 1487 1053 1520 1627 1160 1127
21 22 23 24 25
840 820 873 733 833
1860 1667 2107 1887 1853
1227 1427 1180 1233 973
26 27
853 753
1407 1787
1133 1280
28 29
900 647
1313 1900
1220 1480
30
887 811 98.9 0.122
1453 1643 261.6 0.159
1213 1326 162.1 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 Bias cannot be determined. No quantitative reference material available
(27%
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)
Contributors to Uncertainty
Representative and Applicable QC Data
Comments to Clarify Contributor Effects
Temperature, Storage, Handling: shipping time, container & temperature
lab storage time, conditions & temperature contamination in lab storage areas
NA NA NA
May affect growth but not under control of lab so no impact on analytical uncertainty Affects growth rates of organisms in samples. Study samples included in some method validation studies may reflect these contributors. As long as samples are contained there should be no impact
Laboratory Subsampling: Area of bulk samples selected for analysis
sample size
DUP DUP DUP
contamination during preparation
BLK
Sterility of materials used/autoclave operation
sample nonhomogeneity blending techniques
Extraction/vortexing non-homogeniety of samples
Sample Preparation:
sample homogenization/subsampling sample dilution (pipettes, etc) balance plating technique composition of sample
DUP DUP DUP DUP DUP
Contributions from this and the rest of Sample Prep contributors measured only if duplicate samples are prepared; not captured by inter-analyst readings of same sample prep Uncertainty related to method used to make serial dilutions Balance error is often insignificant compared to other MU sources How distributed on media Organism competition on media
Lab Environmental Conditions:
BLK
Should have minimal impact when proper aseptic techniques are used
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.
microscope magnification level used
DUP
Determines level of detail of organisms that can be easily seen
eye piece graticule calibration data logger
DUP DUP
media used
DUP
Different media show preference for different organisms.
incubation conditions
DUP
Affects rate of growth but cannot be monitored unless using known concentration control cultures or do time/analysis study
seasonal background organism variances Analysts:
Measuring Instruments:
Proper calibration required to facilitate spore identification based in part on size When used to assist in organism identification, e.g. bacteria
Test Procedure Variations:
identification techniques used microbial density interferences
DUP DUP DUP
Especially for bacteria (chemical tests vs. data logger, etc) Organism competition (inhibition, overgrowth, etc) Presence of organisms other than those of interest
Data Manipulation: reading, interpreting and reporting results Accuracy of calculations
DUP 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
number of pair
1st 2nd CV of analyst analyst Std Dev 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 4 5 6 7 8 9 10 11
69 220 150 155 94 22 17 9 33
54 200 155 130 125 16 17 7 41
10.6 14.1 3.5 17.7 21.9 4.2 0.0 1.4 5.7
0.172 0.067 0.023 0.124 0.200 0.223 0.000 0.177 0.153
0.030 0.005 0.001 0.015 0.040 0.050 0.000 0.031 0.023
12 13
32 110
30 120
1.4 7.1
0.046 0.061
0.002 0.004
14 15 16 17
110 270 17 160
100 290 29 170
7.1 14.1 8.5 7.1
0.067 0.051 0.369 0.043
0.005 0.003 0.136 0.002
18 19
300 6
330 8
21.2 1.4
0.067 0.202
0.005 0.041
20
190
260
49.5
0.220
0.048
21
6
9
2.1
0.283
0.080
22 23
19 42
11 40
5.7 1.4
0.377 0.034
0.142 0.001
24
42
49
4.9
0.109
0.012
25 26 27 28
690 170 28 180
590 240 38 160
70.7 49.5 7.1 14.1
0.110 0.241 0.214 0.083
0.012 0.058 0.046 0.007
29
61
68
4.9
0.077
0.006
30
240
300
42.4
0.157 ∑ CV2 CV pooled = √ (∑CV2/30) =
0.025 0.988 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