SEPA
EPA/600/R-14/239 | September 2014 | www.epa.gov/ord
United States
Environmental Protection
Agency
Decontamination Process
Indicators: Biological Indicators
Assessment and Lessons
Learned in the Development of
Biological Indicators for
Chlorine Dioxide Fumigation
Office of Research and Development
National Homeland Security Research Center
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EPA 600-R-14-239
Decontamination Process Indicators: Biological Indicators
Assessment and Lessons Learned in the Development of Biological Indicators for Chlorine
Dioxide Fumigation
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
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Disclaimer
The United States Environmental Protection Agency, through its Office of Research and Development's
National Homeland Security Research Center, funded and managed this investigation through EP-C-09-027
WA 0-51, 1-51, 2-51, 3-51 and 4-51 with ARCADIS U.S., Inc. This report has been peer and
administratively reviewed and has been approved for publication as an Environmental Protection Agency
document. It does not necessarily reflect the views of the Environmental Protection Agency. No official
endorsement should be inferred. This report includes photographs of commercially available products. The
photographs are included for purposes of illustration only and are not intended to imply that EPA approves
or endorses the product or its manufacturer. Environmental Protection Agency does not endorse the
purchase or sale of any commercial products or services.
Questions concerning this document or its application should be addressed to:
M. Worth Calfee, Ph.D.
Decontamination and Consequence Management Division
National Homeland Security Research Center
U.S. Environmental Protection Agency (MD-E343-06)
Office of Research and Development
109 T.W. Alexander Drive
Research Triangle Park, NC 27711
Phone:919-541-7600
Fax: 919-541-0496
E-mail: Calfee.Worth@epamail.epa.gov
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Acknowledgments
This effort was managed by the principal investigator from the Office of Research and Development's
National Homeland Security Research Center (NHSRC).
The authors wish to thank Joe Dalmasso for his input.
Additionally, the authors would like to thank the peer reviewers for their significant contributions.
Specifically, the efforts of Lawrence Kaelin (EPA OEM), Timothy Dean (EPA ORD), and Sanjiv Shah
ORD) are recognized. Quality Assurance review by Joan Bursey (SEE, EPA ORD) is also greatly
appreciated.
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Table of Contents
Disclaimer ii
Acknowledgments iii
Table of Contents iv
List of Tables ix
List of Appendices x
List of Acronyms and Abbreviations xi
Executive Summary xiii
1 Introduction 1
1.1 Process 1
1.2 Project Objectives 3
1.3 Experimental Approach Overview 3
1.4 Data Treatment 4
1.4.1 Stumbo-Murphy-Cochran (SMC) Method 6
1.4.2 Quantitative Method 6
2 Materials and Methods 7
2.1 Experimental Approach and Test Matrices 7
2.1.1 Task 1: Testing of Candidate Burden Materials 7
2.1.2 Task 2: Testing of Bl Carrier Materials 12
2.2 Test Materials and Deposition 13
2.2.1 Bl Preparation 13
2.2.2 Spore Preparation 15
2.2.3 Bl Carrier Inoculation 17
2.2.4 Control Bis 17
2.2.5 Well-Plate Inoculation 18
2.2.6 Off-The-Shelf Bis 18
2.2.7 Building Material Coupons 20
2.3 Fumigation Methods 21
2.4 Sampling and Analytical Procedures 22
2.4.1 Qualitative Bl Analysis 23
2.4.2 Qualitative Well-plate Analysis 23
2.4.3 Quantitative Bl Analysis 24
2.4.4 Chlorine Dioxide Monitoring 24
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2.5 Sampling Strategy 25
2.5.1 Sampling/Monitoring Points 25
2.6 Sampling Handling and Custody 26
2.6.1 Preventing Cross-Contamination 26
2.6.2 Sample Containers 28
2.6.3 Sample Identification 28
2.6.4 Information Recorded by DTRL Personnel 28
2.6.5 Sample Preservation 28
2.6.6 Sample Holding Times 28
2.6.7 Sample Custody 29
2.6.8 Sample Archiving 29
3 Results and Discussion 30
3.1 Fumigations 30
3.2 Test A - Burden Scoping Test 30
3.3 Test B - Material Scoping Test 34
3.4 Barrier Investigation 36
3.5 COTS Bl Comparisons 36
3.5.1 COTS Yakibou B. atrophaeus Bis 36
3.5.2 Mesa EtO Bl 37
3.5.3 RCT B. atrophaeus Bis 37
3.5.4 MesaStrip and Releasat® Bis 37
3.5.5 ProLine PCD Bis 38
3.6 Proximity Investigation 39
3.7 CT Investigation 39
3.8 Burden Investigation 44
3.8.1 Cellobiose 44
3.8.2 Dithiothreitol 46
3.8.3 Carrageenan 47
3.8.4 Glutathione 49
3.8.5 Gelatin 49
3.8.6 Casein 53
3.9 Variability Investigation 59
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3.9.1 Spore Preparations 59
3.9.2 Effect of Vortex Mixing Bis during Analysis Procedures 63
3.9.3 Effect of RH 64
3.9.4 Quantitative Analysis 67
3.9.5 Fumigation Repeatability (Tests S, T, U and V) 70
3.9.6 Effects of Spore Population Density 72
3.9.7 CT Investigation of Low Inoculum 76
3.9.8 Age of Bl 78
4 Quality Assurance 81
4.1 Sampling, Monitoring, and Analysis Equipment Calibration 81
4.2 Data Quality 82
4.3 QA/QC Checks 82
4.4 Acceptance Criteria for Critical Measurements 83
4.5 Data Quality Audits 89
4.6 QA/QC Reporting 89
4.7 Amendments to Original QAPP 89
5 Summary 90
References 92
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List of Figures
Figure 1-1. Conceptual Diagram of the Fumigation System 3
Figure 1-2. Theoretical Survival Curve Types of Bis 5
Figure 2-1. Stainless Steel Carrier with Burden and Inoculum 13
Figure 2-2. Commercial Off-the-Shelf Bis 19
Figure 2-3. 18 mm Coupons 21
Figure 2-4. Glovebox Fumigation Chamber 22
Figure 2-5. Well Plates with Alamar Blue Showing Growth (pink) and No-Growth (purple) Results 23
Figure 2-6. Springs used to Organize Bis 27
Figure 3-1. Survival (1000 ppm CI02) of B. atrophaeus Bis (n=5) with Low Concentration
Burdens 32
Figure 3-2. Survival (1000 ppm CI02) of B. atrophaeus Bis (n=5) with High Concentration
Burdens 33
Figure 3-3. Survival (1000 ppm CI02) of G. stearothermophilus Bis (n=5) with Low Concentration
Burdens 34
Figure 3-4. Survival (1000 ppm CI02) of G. stearothermophilus Bis (n=5) with High
Concentration Burdens 34
Figure 3-5. Survival (1000 ppm CI02) for B. atrophaeus Protected by NuFab Barrier (Test K)
(n=8) 36
Figure 3-6. Survival (1000 ppm CI02) of Two COTS Bis (Test U) (n=30) 37
Figure 3-7. Survival (1000 ppm CI02) of 3 Commercially-Available Bl types (Test V) (n=30) 38
Figure 3-8. Survival (1000 ppm CI02) of Proline Bis (n=10) with Various Lumen Lengths 39
Figure 3-9. Survival (1000 ppm CI02) of Apex Bl (n=30) Based on Proximity to Other Bis (Test L) 41
Figure 3-10. D-Value (1000 ppm CI02) for B. atrophaeus Bis from Proximity Investigation (Test L) 41
Figure 3-11. Survival of Bl (n=30) Types at Various CT Values (Tests M and N) 44
Figure 3-12. Survival (1000 ppm CI02) of G. stearothermophilus Bis with CLB Burden Test D
(n=30) and TestE(n=10) 45
Figure 3-13. Survival (1000 ppm CI02) of B. atrophaeus Bis with DTT Burden (Test C (n=30), Test
E (n=10), and Test F (n=10)) 46
Figure 3-14. Survival (1000 ppm CI02) of G. stearothermophilus Bis (n=10) with CAR Burden
(TestE) 48
Figure 3-15. Survival (1000 ppm CI02) for B. atrophaeus Bis with CAR Burden from Test C
(n=30), Test E (n=10) and Test F (n=10) 48
Figure 3-16. Reinterpretation of Figure 3-15 49
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Figure 3-17. Survival (1000 ppm CI02) of G. stearothermophilus Bis (n=30) with GEL Burden
(Test D) 51
Figure 3-18. Survival (1000 ppm CI02) of B. atrophaeus Bis (n=10) with GEL Burdens (Test E) 52
Figure 3-19. Survival (1000 ppm CI02) of 1.0% GEL B. atrophaeus Bis from Test D (n=30), Test E
(n=10), Test G (n=5), Test H (n varies between 5 and 30), and Test I (n varies
between 5 and 30) 53
Figure 3-20. Survival (1000 ppm CI02) of B. atrophaeus Bis (n=10) with CSN Burden (Test E) 54
Figure 3-21. Survival (1000 ppm CI02) of B. atrophaeus Bis (n=10) with CSN Burden (Test F) 55
Figure 3-22. Variability in Survival Rates of B. atrophaeus Bis with 1.0% Casein 57
Figure 3-23. Survival (5000 ppm*hours, 1000 ppm CI02) of Two Spore Preparations (n=10) as a
Function of CSN Concentration (Test O) 59
Figure 3-24. Survival (1000 ppm CI02) of Yakibou Spore Bis (n=20) with CSN Burden (Test P) 60
Figure 3-25. Survival (1000 ppm CI02) of Raven Spore Bis (n=20) with CSN Burden (Test P) 60
Figure 3-26. Calculated Average D-Values for Test P and Test Q Bis 61
Figure 3-27. Calculated D-Values of Yakibou Bis per Exposure Time 62
Figure 3-28. Survival (1000 ppm CI02) of Test U and Test V Bis (n=30) 63
Figure 3-29. Survival (1000 ppm CI02) of B. atrophaeus Bis (n =30) with GEL Burden at 75% RH*
(Test I) 65
* All Bis survived fumigation at 60 % RH 65
Figure 3-30. Survival (1000 ppm CI02) of B. atrophaeus Bis (n=30) with CSN Burden at 75% RH*
(Test I) 65
Figure 3-31. Quantitative Analysis of CFU (n-5) following 1000 ppm CI02 fumigation (Test R) 68
Figure 3-32. Qualitative Interpretation of Figure 3-31 Quantitative Results 68
Figure 3-33. Test R Qualitative Results from Qualitative Bis (n=5) 69
Figure 3-34. D-Values (1000 ppm CI02) of B. atrophaeus Bis versus Fumigation Time (Test R) 69
Figure 3-35. Survival (1000 ppm CI02) from Test S and Test T Bis (n=20) 70
Figure 3-36. Two D-Value Methods of 1 % CSN B. atrophaeus Bis from Two Fumigations 71
Figure 3-37. Survival (1000 ppm CI02) of 1% Casein Bis (Qualitative Analysis) 72
Figure 3-38. Survival (1000 ppm CI02) of 103CFU Inoculum B. atrophaeus Bis (n=10) 73
Figure 3-39. Survival (1000 ppm CI02) for Low inoculum Bis (n=20) in Test AA 74
Figure 3-40. Survival (1000 ppm CI02) of 105CFU Inoculum B. atrophaeus Bis (n=10) 75
Figure 3-41. Survival of 2 % CSN B. atrophaeus Bl (n=20) at Three CI02 Concentrations (Test Z,
AA, and AB) 77
Figure 3-42. Effect of Age of CSN Bl on Survival Rates (Test J) 78
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List of Tables
Table 2-1. Numbers of Replicates per Experimental and Control Bl Type for Task 1 8
Table 2-2. Burden Additives for Custom Bis 8
Table 2-3. Summary of Test Variables during Task 1 Burden Tests 9
Table 2-4. Ancillary Tests for Characterization of Bis 10
Table 2-5. Number of Replicates per Experimental and Control Bl Type for Task 2 12
Table 2-6. Material Test 12
Table 2-7. Carrier Materials for Custom Biological Indicators 14
Table 2-8. Source and Lot of Inoculant used on Bis 16
Table 2-9. Characteristics of Control Bis 18
Table2-10. Monitoring Methods 26
Table 2-11. Critical and Non-Critical Measurements 26
Table 3-1. Average Conditions during Fumigations 31
Table 3-2. Survivability of B. atrophaeus Bis with Burdens 32
Table 3-3. Survivability of G. stearothermophilus Bis with Burdens 33
Table 3-4. Survival rates of Bis based on Carrier Material 35
Table 3-5. Survival (1000 ppm CI02) of Apex and Raven Bis (n=30) Placed Close (compact)
and Widely Dispersed 40
Table 3-6. Concentration*Time Values and Survival Rates for Test M (1000 ppmvCI02) 42
Table 3-7. Concentration*Time Values and Survival Rates for Test N (250 ppmv CI02) 43
Table 3-8. Survival Rates of B. atrophaeus Bis with Less Than 0.25% CLB burden 46
Table 3-9. Survival Rates of 10 mM DTT on B. atrophaeus Bis (n=10) 47
Table 3-10. Survival Rates of Test C B. atrophaeus and G. stearothermophilus Bis with 5 mM
GLU Burden 50
Table 3-11. Tested Concentrations of GEL as a Burden on B. atrophaeus and G.
stearothermophilus Bis 50
Table 3-12. Survival Rates GEL B. atrophaeus Bis (Test D) 51
Table 3-13. Survival Rates of Gelatin B. atrophaeus Bis for Some Concentrations 52
Table 3-14. Survival Rates of B. atrophaeus Bis with CSN Burdens 56
Table 3-15. Fumigation Conditions for the 9-hour Exposure of Multiple Tests 58
Table 3-16. Effect of Vortex Mixing on Survival Rate Determination 64
Table 3-18. Target and Actual CT Exposure for Tests Z, AA, and AB 76
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Table 3-19. Recovery (CFU) from Test AA and Test AB Coupons 78
Table 3-20. Fumigation Conditions for Low Inoculum Tests 79
Table 3-21. D-Values (hours) for Bl Types during Low Inoculum Tests 79
Table 3-22. Correlation between D-Values of Selected Bis and Fumigation Conditions 80
Table 4-1. Sampling and Monitoring Equipment Calibration Frequency 81
Table 4-2. QA/QC Sample Acceptance Criteria 83
Table 4-3. Accuracy and Completeness DQIs for Critical Measurements 84
Table 4-4. Completeness of DQIs 85
Table 4-5. Precision Acceptance Criteria for Critical Measurements 86
Table 4-6. Observed Precision of Critical Measurements 87
List of Appendices
Appendix A Miscellaneous Operating Procedures (MOPs)
Appendix B DTRL - QC Checklist for Data Reviewers
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List of Acronyms and Abbreviations
jjm
Micrometer(s)
AAC
Amino Acid cocktail
ALG
Alginate (sodium salt)
ALU
6061 Aluminum
APPCD
Air Pollution Prevention and Control Division
ATCC
American Type Culture Collection
B.
Bacillus
Bl
Biological Indicator
BSC
Biological Safety Cabinet
CAR
Carrageenan
CBD
Chipboard Discs
CEM
Cellulose Ester Membranes
CER
Ceramic Tile
CFU
Colony Forming Unit(s)
CLB
Cellobiose
CI02
Chlorine Dioxide
COC
Chain of custody
COTS
Commercial off-the-shelf
CRK
(Adhesive) Cork Dots
CSN
Casein
CT
Concentration-Time
CUP
C14500 Copper disc
Dl
De-ionized (Water)
DAS
Data Acquisition System
DCMD
Decontamination and Consequence Management Division
DMS
Dimethyl Sulfoxide
DQI
Data Quality Indicator
DQO
Data Quality Objective
DTRL
Decontamination Technologies Research Laboratory
DTT
Dithiothreitol (Test E onward)
ECBC
Edgewood Chemical Biological Center
EMS
Environmental Monitoring System
EPA
U. S. Environmental Protection Agency
EtO
Ethylene oxide
FCL
Ferrous Chloride
FLT
(Adhesive) Felt Dots
g
Gram(s)
G.
Geobacillus
GEL
Gelatin
GLU
Glutathione
GS
Geobacillus(G.) stearothermophilus
HBB
High Bay Building
HCI
Hydrochloric Acid
HMA
Humic Acid (sodium salt)
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HSRP Homeland Security Research Program
ISO International Organization for Standardization
kDa KiloDalton
Kl Potassium Iodide
KIPB Phosphate Buffer Solution Containing Kl
L Liter(s)
Ipm Liter(s) per minute
LR Log reductions
mg Milligram(s)
mm Millimeter(s)
mM Millimolar
mol Mole
MOP Miscellaneous Operating Procedure
mSM Modified Standard Method
NA Not applicable
NHSRC National Homeland Security Research Center
NIST National Institute of Standards and Technology
ORD Office of Research and Development
pAB pH-Adjusted bleach
PBST Phosphate Buffered Saline with 0.05 % TWEEN®20
PCD Process Challenge Device
PPE Personal Protective Equipment
ppmv Part(s) per million volume
QA Quality Assurance
QAPP Quality Assurance Project Plan
QC Quality Control
RCT Mesa Labs culture test kits
RH Relative Humidity
RTP Research Triangle Park
RUB (Adhesive) Rubber Dots
SD Standard Deviation
SM Standard Method
SMC Stumbo-Murphy-Cochran
SST Stainless Steel
TSA Tryptic Soy Agar
TSB Tryptic Soy Broth
UV Ultraviolet (light)
WACOR Work Assignment Contracting Officer Representative
WOD Wooden Discs
XYZ Porous Polypropyl
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Executive Summary
The objective of the research presented in this report was to develop a "custom Bl(s)" that could be used to
indicate the efficacy of fumigation more accurately, when CI02 fumigation is used to decontaminate building
interiors following a B. anthracis contamination incident. The custom Bl(s) would be engineered to yield
complete kill after exposure to 9000 ppm*hours of CI02 gas, while at the same time providing Growth
results at fumigation conditions unlikely to deactivate B. anthracis spores. The resulting custom Bl(s) would
therefore have significant utility in 1) modeling decontamination and kill kinetics of building material-bound
spores more accurately, 2) providing an easily deployed method to assess decontaminant effectiveness
with laboratory and field applications, and 3) offering pertinent information to a "multiple lines of evidence"
approach to building clearance, thereby potentially reducing the number of surface and air samples needed
to be collected to build confidence in clearance decisions.
To achieve the objective, custom Bis were prepared by numerous approaches, resulting in an increased
resistance of the indicator spores to CI02 gas. These approaches included using carrier materials other than
stainless steel or paper as is used in commercial off the shelf (COTS) Bis; or combining the spore
suspension with a protective chemical additive (i.e., burden material) prior to pipetting spores onto the
carrier material. The Bis were then exposed to CI02 for between 1000 ppm*hours and 20000 ppm*hours,
typically at 1000 ppm CI02. After exposure, the Bis were placed in growth media and incubated for seven
days to test for viable spores.
While no Bl modification tested achieved a precise Bl deactivation point of 9000 ppm*hours exposure to
CI02, results from this study suggest sources of Bl kill point variability may be an important focus of future
research in this area.
Burdens can have the effect of increasing survival rates of Bis. Burdens that seemed to increase Bl survival
to 7000 ppm*hours and yet not provide protection so that all Bis survived 9000 ppm*hours included
cellobiose, dithiothreitol, carrageenan, gelatin, and casein, all of which could be evaluated further. Most
promising was 1% casein as a burden on low inoculum (103 CFU) B. atrophaeus Bis. Results were variable,
however, with large variations between batches and fumigations due to unidentified factors apparently
related to production.
While coupon materials did affect the survival rates of Bis, none of the carriers showed promise, providing
either too much or too little protection. Unlike burdens, carriers cannot be tested in different concentrations.
Fumigated wooden carriers would not support growth of the target organism, and were therefore not a
suitable carrier material. When rubber was used as the carrier material, 100% of Bis demonstrated growth
following exposures, suggesting that rubber surfaces may be difficult to decontaminate with CI02.
Either semi-permeable barriers or lumens (open tubes) can be used as physical barriers, and types of both
were demonstrated to extend the survival rates of spores. More research should be conducted on semi-
permeable membranes, as incorporation of membranes into the Bl manufacturing process would be easy to
implement. Bis incorporating tortuous paths such as lumens should also be further investigated.
Various COTS Bis were investigated, including B. atrophaeus Bis from Apex Laboratories., Raven
Laboratories, and three variations from Mesa Laboratories. Some were more hardy, and some were less
hardy, than the target 9000 ppm*hour full kill. Moreover, the spore preparation was found to have an impact
on spore survival rates. Because spore preparations exhibit this variability, the behavior of Bis can fluctuate
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from batch to batch, though there is also variability between batches using the same spore preparation,
which suggests some other production factor may be causing the variation in survival rates. Future research
should focus on removing variation from spore preparations, or focus on species that are more easily
destroyed, thus removing the significance of spore variation. Regardless, the apparent variability in kill
points amongst COTS Bis suggests that Bl variability may be unavoidable, and therefore to some degree
acceptable.
This study reaffirmed previous reports that Bl D-values are non-linear over the duration of the fumigation.
Many Bis resilient enough to survive 7000 ppm*hours would also tend to survive 9000 ppm*hours due to a
subpopulation of spores with higher resistance than the main population, due either to a protective location
or inherent hardiness. While a Bl with very hardy spores may predict the behavior of bacterial spores in an
actual event, it may represent too high a benchmark. One possible explanation of this tailing effect was the
protective bio-burden of clumping in high-inoculum Bis. To reduce the bio-burden, lower inoculum Bis were
tested, and showed a lower tendency towards the long-surviving tail and an increasing resistance with
increasing casein burden. These techniques may be used to tune a Bl to better model the inactivation of
any target organism. To produce a good model of Bacillus anthracis with CI02 fumigations, the authors
would recommend side-by-side comparisons of Bacillus anthracis to Bis with low inoculum and 1 % and 2%
casein burden.
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1 Introduction
This project supports the mission of the U.S. Environmental Protection Agency's (EPA) Office of
Research and Development (ORD), Homeland Security Research Program (HSRP). The EPA's National
Homeland Security Research Center (NHSRC), conducts high-quality research to support the HSRP, by
providing information pertinent to the decontamination of contaminated areas, such as those resulting
from an act of terrorism. Previously, biological indicators (Bis) from Apex Laboratories (Sanford, NC,
USA), consisting of Bacillus (B.) atrophaeus spores on stainless steel coupons, were used in laboratory
decontamination studies of building materials as the standard surrogates for spores of B. anthracis.
However; recent systematic decontamination studies conducted jointly by EPA and Edgewood Chemical
Biological Center (ECBC) within the US Army, using chlorine dioxide (CI02) as the fumigant of choice,
showed that B. anthracis spores on certain building materials (such as bare pine wood, painted wall
wallboards, painted I-beam steel, and concrete cinder blocks) are more resilient to decontamination than
B. atrophaeus Bis and required a considerably higher Concentration-Time (CT) to achieve zero viable B.
anthracis spores in samples recovered from building material [1], Subsequently, EPA's HSRP initiated a
study to develop and evaluate a Bl designed specifically for Homeland Security decontamination
applications.
The objective of the research presented in this report was to develop a "custom Bl(s)" that could be used
to indicate the efficacy of CI02 fumigation more accurately, when CI02 fumigation is used to
decontaminate building interiors following a B. anthracis contamination incident. The developed Bl would
be engineered to yield complete kill after exposure to 9000 ppm*hour of CI02 gas. This target kill point
was selected based upon previous laboratory data and target exposure criteria for fumigations following
the 2001 anthrax incidents [2] [3], The resulting custom Bl(s) would therefore have significant utility in 1)
modeling decontamination and kill kinetics of building material-bound spores more accurately, 2)
providing an easily deployed method to assess decontaminant effectiveness with laboratory and field
applications, and 3) offering pertinent information to a "multiple lines of evidence" approach to building
clearance (http://www.epa.aov/osweroe1/docs/misc/cdc-epa-interim-clearance-strateav.pdf). thereby
potentially reducing the number of surface and air samples needing to be collected to build confidence in
clearance decisions.
To achieve the objective, custom Bis were prepared by numerous approaches, resulting in an increased
resistance of the indicator spores to CI02gas. These approaches included using carrier materials to
construct the Bis, rather than using stainless steel or paper as is used in commercial off the shelf (COTS)
Bis; or combining the spore suspension with a protective chemical additive (i.e., burden material) prior to
pipetting spores onto the carrier material. As used here, burden material refers to any chemical added to
a Bl that will act to partially shield (chemically or physically) the biological portion of the Bl (i.e., B.
atrophaeus or Geobacillus (G.) stearothermophilus spores). This latter approach proved more promising,
as the volumes and concentrations of the burden materials could be varied to achieve a target
inactivation point, once a dose-dependent relationship of the burden and spore survival was established.
The procedures and data in this report document the recent efforts to develop the above-described
custom Bl.
1.1 Process
Numerous custom Bis were designed, procured, and subsequently subjected to bench-scale fumigations
with CI02, under highly-controlled environmental conditions. Bis were evaluated for their viability following
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the fumigation. An opaque chamber (830 series glove box, Plas-Labs, Inc., Lansing, Ml, USA) was used
to maintain and control a leak-free fumigation atmosphere and allow for the periodic addition and removal
of Bis during fumigation. CI02 was generated by a ClorDiSys-GMP (ClorDiSys, Inc., Lebanon, NJ, USA),
which passes 2 % chlorine in nitrogen through sodium chlorite cartridges. The generator includes real-
time feedback control of CI02 concentration in the chamber atmosphere via an internal photometric
monitor. A second photometric monitor, was controlled by an Environmental Monitoring System (EMS)
(ClorDiSys, Inc., Lebanon, NJ, USA), and used to assess the accuracy of the primary monitor.
Modified Standard Method (mSM)-4500 samples (see Section 2.4.4.2) were collected a minimum of every
60 minutes to confirm the concentration of CI02 in the test chamber. A fan inside the chamber provided
internal mixing. Pressure relief valves and check valves prevented over-pressurization of the chamber.
Humidity within the chamber was controlled by a custom-built data acquisition system (DAS). A relative
humidity (RH)/temperature sensor (Vaisala, Vantaa, Finland) was used in a feedback loop to control RH.
When the Vaisala RH sensor read lower than the RH setpoint, solenoid valves were opened to inject
humid air from a gas humidity bottle into the chamber. The gas humidity bottle (Fuel Cell Technologies,
Albuquerque, NM, USA), heated to 60 °C, passes compressed air through Nafion® tubes surrounded by
deionized water, creating a warm air stream saturated with water vapor. Temperature was controlled by
circulation of cooling water through radiators. Figure 1-1 shows the schematic of the configuration used
for the tests. HOBO RH sensor/loggers (Onset Computer Corporation, Bourne, MA, USA) were placed
throughout the chamber to assess RH spatial variability within the chamber.
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Isolation Chamber
Airlock
Digital signal line
DAS
Radiators
Cooling
Water
RH/Temp Sensor
mSM-4500
Gas
Humidity
Bottle
EMS CI02
concentration
CI02 Generator
Digital control line
Heated tubing for gas flow
Cooling water line
Figure 1-1. Conceptual Diagram of the Fumigation System
1.2 Project Objectives
The objective of this project was to develop a custom Bl that would provide reliable results of No Growth
after fumigation with 9000 ppm*hours CI02, while at the same time providing Growth results at fumigation
conditions unlikely to deactivate B. anthracis spores.
1.3 Experimental Approach Overview
The experimental approaches that were used to meet the objectives of this project are:
• Fumigation using a glovebox chamber.
• Use of burdens (chemical additives) in Bl spore inoculums. Burdens were added to alter spore
survival on the Bl carrier, either through physical or chemical mechanisms.
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• Use of Bl carrier materials that are more difficult to decontaminate. Such materials may alter Bl
spore survival by providing physical protection, or by catalytically reducing fumigant
concentrations within close proximity of the material-bound spores.
• Use of material barriers or lumens to physically delay or lessen spore exposure to the fumigant.
• Use of multiple fumigation time-points to evaluate spore kill as a function of time and exposure
(concentration xtime, CT).
• All fumigation testing was conducted at EPA's Research Triangle Park (RTP), NC campus, within
the High Bay Building (HBB). The general test method for the fumigation tests was as follows:
1. Design and order (from Bl vendor) custom Bis.
2. Receive Bis. Label and group by type and exposure duration (time).
3. Establish the target temperature and RH for the trial in the fumigation chamber.
4. Charge the chamber with CI02 to achieve the target concentration.
5. Through the chamber airlock, place the appropriate Bis for the trial in the chamber. The Bis were
present for CI02 ramp-up for some tests (Step 4).
6. Maintain the target concentration, temperature, and RH for the specified time (Note: Time zero was
defined as the time at which the target concentration was achieved in the chamber).
7. Use the airlock to remove Bis at desired time points during fumigation.
8. After the final exposure time, aerate the chamber for a defined length of time and until a safe CI02
concentration was achieved in the chamber.
9. Process and analyze Bis in HBB Room H130A or in the NHSRC-RTP Microbiology Laboratory,
(E390).
This report presents the developmental tests conducted toward the creation of a custom Bl and evolved
from two primary tasks: Task 1, Testing of Candidate Burden Materials, and Task 2, Testing of Coupon
Materials. This work spanned five years and was performed under an approved Quality Assurance
Project Plans (QAPP). The methods approved and reported within the QAPP are summarized in sufficient
detail in this report. During the project period, while many tests evolved from these two primary tasks, a
number of others were conducted to characterize Bl stability, reproducibility and comparability to
commercial off-the-shelf (COTS) Bis. The following sections describe the sequence and reasoning behind
each of these tests.
1.4 Data Treatment
Survivability was calculated by a simple percentage of Bl replicates showing growth out of the total
number of replicates. The perfect Bl formulation would have high survivability at 7000 ppm*hours and 0 %
survivability at 9000 (± 500) ppm*hours. The Bl formulations in Task 1 and Task 2 closest to this perfect
survivability rate were chosen for additional tests. Maximum Bl survival rates were propagated to all
earlier time points; i.e., if 20 % of Bis survive at 9000 ppm*hours, then the Bl survival rate at 7000
ppm*hours must be at least 20 % theoretically. This information is listed in Tables as "Maximum Survival
Rate".
4
-------
Figure 1-2 shows the classifications of Bis identified and discussed in this study.
Hypothetical Survival Rates of Bl Categories
120
~ Target Bl
Overprotected Bl
A Underprotected Bl
)( Sigmoidal Bl
)i( False-Positive Bl
0
5000
10000
15000
CT (ppm*hours)
Figure 1-2. Theoretical Survival Curve Types of Bis
The optimal Bl for this effort would have a time lag before survival rates are affected, giving the target Bl
a high probability of surviving 7000 ppm*hour CI02 fumigations, but would be completely inactivated at
9000 ppm*hours. Three of the survival curve categories show this time lag or shoulder. An overprotected
Bl would survive at exposures equal to or greater than 9000 ppm*hours, and would falsely indicate an
inadequate fumigation. The under-protected Bl is inactivated much faster than B. anthracis, and so is not
indicative of adequate fumigation (typical of current COTS Bis). The sigmoidal Bl has a tailing as well as
a shoulder. The sigmoidal Bl may show survival rates similar to the target Bl at exposures less than 9000
ppm*hours, but then may prove very difficult to achieve complete deactivation. This type of Bl would be
more likely to falsely indicate an inadequate fumigation for the 9000 ppm*hours target CT required for full
kill of B. anthracis spores. Finally, the false positive Bl is linear and has a deactivation at the target 9000
ppm*hours, but has a higher likelihood of falsely indicating a successful fumigation at exposures less than
9000 ppm*hours.
D-value (or decimal reduction time) is a measure of the time a deactivation technique requires to effect a
90% (1 Log10 reduction) reduction in population. This measurement assumes a first order reaction, so that
if it takes one hour to reduce the population of a Bl from 1 x 105 colony forming units (CFU) to 1 x 104
CFU, then after five hours there would be less than one CFU present. Put another way, the time it takes
to reduce the population from 100,000 to 10,000 CFU is the same it would take to reduce the population
from <10 CFU to <1 CFU. D-values were calculated according to the two methods described in Section
1.4.1 and 1.4.2. D-values were used even in cases where there was an obvious time lag. However, it is
common practice to characterize Bis by D-values, even though their kill kinetics are often known to be
other than first order.
5
-------
1.4.1 Stumbo-Murphy-Cochran (SMC) Method
The SMC method can be used for qualitative results under any conditions that produce a fractal survival
set; i.e., for all time points that had both some deactivated Bis and some Bis showing growth. The D-
value equation is shown in Equation 1.
U,
A =7 \ (Eqn. 1)
' (logJV„-logtfJ
where
Dt = D-value at time point /',
Uj = fumigation time,
N0 = the original population (CFU) of the Bl before fumigation, and
Nuj = the Most Probable Number, calculated by ln(n/ri), where
nj = total number of replicate Bis at time point i, and
H = number of Bis negative for growth at time point i.
When multiple time points produce fractal values, the D-value for each time point was averaged to
provide a D-value for the Bl under that fumigation condition.
1.4.2 Quantitative Method
When quantitative populations of Bis are available, then the D-value may be calculated directly per
Equation 1, but where:
N0 = the original population (CFU) of the Bl before fumigation, and
Nui = the population quantitatively determined at time point /'.
6
-------
2 Materials and Methods
2.1 Experimental Approach and Test Matrices
2.1.1 Task 1: Testing of Candidate Burden Materials
In Task 1, several burden materials were experimentally tested with one carrier material (stainless steel)
to determine both compatibility with the surrogate organisms and the ability, if any, to increase the CT
required for Bl inactivation.
The growth and production of spores, preparation of burden-amended inocula, and inoculation of Bl
carriers with (1 x 106 spores) B. atrophaeus or G. stearothermophilus spores was performed by Yakibou
(formerly Apex Laboratories of Sanford, NC, now Yakibou, Inc., Holly Springs, NC,
http://ivdesiqnhouse.com/vak/). Apex laboratories was acquired by Mesa Laboratories (Lakewood, CO)
during the timeframe of this testing. Ten candidate burden materials at two concentrations (and one "no
burden" control) were identified and tested under this task. Burden materials were chosen based upon
their water solubility, shelf stability, and having a chemically reduced oxidation state (able to be oxidized).
The resulting custom Bis were challenged by fumigation with 1,000 parts per million volume (ppmv)
gaseous CI02 at 75% RH and 24 °C in the Decontamination Technologies Research Laboratory (DTRL)
located in HBB Room H224. The test samples were collected at 5, 7, and 9 hour exposure times (5,000,
7,000, and 9,000 ppm*hours).
Each fumigation test included positive and negative control Bis. The negative control Bis were the same
stainless steel discs used by Yakibou for Bis, not inoculated, yet packaged in Tyvek® envelopes in the
same manner as test Bis. Since Bis are produced to be used in sterilization environments and not
produced for scientific study, the Bl vendor did not guarantee the sterility of non-inoculated Bis. An
additional set of laboratory blank Bis were generated by autoclaving negative control Bis upon arrival.
These laboratory blank Bis were used to assess the aseptic technique of the handling laboratories. The
positive control Bis consisted of standard Bis of each organism that were not fumigated. The spores were
inoculated onto stainless steel discs and packaged in Tyvek® envelopes by Yakibou. Performance control
Bis were also included for the longest exposure time in each test. These Bis included the burden but no
spores on the Bl carrier. After fumigation, the Bis were placed in a bacterial growth medium, which was
then spiked with the test organism (~ 1 x 103 CFU), as a control to demonstrate that the fumigated
coupon would not inhibit growth of the test organism. The number of replicates per test and control Bl
type is shown in Table 2-1.
Table 2-2 is a summary of the burdens used during testing, and Table 2-3 summarizes the tests
conducted; each one of the burden tests was developed based upon the results of previous tests. For
ease in presentation, each test is given an alphabetical designation in the order that they were conducted.
B. atrophaeus and G. stearothermophilus are referred to as BG and GS, respectively.
7
-------
Table 2-1. Numbers of Replicates per Experimental and Control Bl Type for Task 1
Sample
Time
Point
(hours)
Replicates
Total
Test/Control
Bl
Inoculated
Burden
Spiked
Media
Spiked
Fumigated
Performance
Baseline
0
3
3
C
No
Yes
Yes
No
Positive
Control
0
3
3
C
Yes
Yes
No
No
Performance
Control
5,7,9
3
9
c
No
Yes
Yes
Yes
Experimental
5,7,9
5
15
T
Yes
Yes
No
Yes
30
Note: 30 Bis * 11 burden materials (including no burden control) * 2 burden concentrations * 2 organisms =
1,320 Custom Bis, + 10 positive controls and 3 negative controls per fumigation.
Table 2-2. Burden Additives for Custom Bis
#
Chemical Name
Burden
Code
Sigma
Catalog #
Formula Wt.
Low
Concentration
High
Concentration
1
Humic Acid (sodium salt)
HMA
H16752
2-500 kDa
1.25%
5%
2
Amino Acid cocktail
AAC
Cysteine
W326305
121.16 g/mol
15 mM
59 mM
Methionine
M9625
149.21 g/mol
Glutamine
G3126
146.14 g/mol
3
Ferrous Chloride
FCL
44939
198.81 g/mol
63 mM
250 mM
4
Glutathione
GLU
G6529
307.32 g/mol
25 mM
100 mM
5
Dithiothreitol
DTT
43816
154.25 g/mol
63 mM
250 mM
6
Gelatin
GEL
G7765
-60 kDa
2.5 %
10 %
7
Alginate (sodium salt)
ALG
180947
10-600 kDa
0.5 %
2 %
8
Carrageenan
CAR
C1013
Variable
0.25 %
1 %
9
Dimethyl Sulfoxide
DMS
D8418
78.13 g/mol
10 %
40 %
10
Cellobiose
CLB
22150
342.3 g/mol
83 mM
333 mM
kDa = kiloDalton
g/mol = grams per mole
mM = millimolar
8
-------
Table 2-3. Summary of Test Variables during Task 1 Burden Tests
Test
Date
Burdens*
Burden
Concentrations
Spores
Time Points
Purpose
A
1/20/2010
10 kinds
(see Table 2-2)
(see Table 2-2)
BG, GS
5, 7, and 9 hours
Primary
identification of
effective burdens
C
4/14/2010
CAR
0.063% (GS) and
0.125% (BG)
BG, GS
4, 8, 8.5, 9, 9.5,
10, and 10.5 hours
Follow-up
investigation of
promising burdens
GLU
5 mM
DTT
5 mM
D
5/5/2010
GEL
0.1%, 1.0%
BG, GS
4, 8, 8.5, 9, 9.5,
10, and 10.5 hours
Follow-up
investigation of
promising burdens
CLB
0.17%
E
7/21/2010
GEL ,
0.25%, 0.50%,
0.75%, 1.00%,
1.25%, 1.50%,
2.00%
BG
1, 5, 7 and 9 hours
Follow-up
investigation of
promising burdens
CAR
0.01%, 0.03%,
0.05%, 0.1%,
0.25%
CLB
0.10%, 0.25%,
0.50%, 0.75%,
1.00%, 1.50%,
2.00%, 4.00%
DTT
10 mM, 20 mM, 40
mM, 50 mM
new burden
CSN
0.1%, 1.0%
CAR
0.05%, 0.075%,
0.100%, 0.125%,
0.250%
GS
1, 5, 7 and 9 hours
Follow-up
investigation of
promising burdens
CLB
0.10%, 0.25%,
0.50%, 0.75%,
1.00%, 1.50%,
2.00%, 4.00%
F
9/15/2010
GEL
1.6%, 1.7%, 1.8%,
1.9%, 2.0%
BG
1, 5, 7 and 9 hours
Follow-up
investigation of
promising burdens
CLB
0.005%, 0.010%,
0.050%
DTT
10 mM, 12 mM, 14
mM, 16 mM
CSN
0.10%, 0.25%,
0.50%, 0.75%,
1.00%, 1.20%,
G
11/10/2010
GEL
1.0%. 1.5%, 1.6%,
1.7%
BG
1,5, 7, 8 and 9
hours
Follow-up
investigation of
promising burdens
CLB
0.050%, 0.060%,
0.070%, 0.10%
9
-------
Test
Date
Burdens*
Burden
Concentrations
Spores
Time Points
Purpose
CSN
0.8%, 0.9%, 1.0%,
1.1%
GEL
0.8%, 0.9%, 1.0%
1,5, 7, 8 and 9
hours
Follow-up
investigation of
promising burdens
H
2/22/2011
CSN
0.90%, 1.00%,
1.05%, 1.10%,
1.15%, 1.20%
BG
GEL
0.8%, 0.9%, 1.0%
1,5, 7, 8 and 9
hours
Follow-up
investigation of
promising burdens
I
3/8/2011
CSN
0.90%, 1.00%,
1.05%, 1.10%,
1.15%, 1.20%
BG
O
2/28/2012
CSN
0.85%, 0.90%,
0.95%, 1.00%,
1.05%, 1.10%,
Yakibou
prepared
and Apex
purchased
spore
inocula
5 hours only;
replication 1
Investigated the
differences in spore
preparations
(vendors) on
burden Bis
P
3/12/2012
CSN
0.85%, 0.90%,
0.95%, 1.00%,
1.05%, 1.10%,
Same as
TestO
5, 7, and 9 hours;
replication 2
Q
3/20/2012
CSN
0.85%, 0.90%,
0.95%, 1.00%,
1.05%, 1.10%,
Same as
TestO
5, 7, and 9 hours;
replication 3
R
7/24/2012
CSN
0.5%, 1.0%
0, 1, 2, 3, 4, 5, 6,
7, 8, and 9 hours
Investigated D-
value with
quantitative and
qualitative analysis
* Burden abbreviations are defined in Table 2-2.
Additional tests conducted to characterize stability, reproducibility comparability of custom Bis to COTS
Bis are listed in Table 2-4. During Test K, physical barriers instead of burdens were tested to determine if
they could be used to provide protection to spores. Three barriers were used: one layer of Breathe Easy
(Breath Easy membranes, Diversified Biotech, USA Scientific Part# 9123-6100), two layers of the Breath
Easy membrane, and 1 layer of NuFab (DuPont, Wilmington, DE, USA discontinued product). For the
physical barrier tests, spore suspensions were evaporated to dryness in the wells of micro-titer plates;
barrier membranes were then affixed to the tops of the plates to seal the wells.
Table 2-4. Ancillary Tests for Characterization of Bis
Test
Fumigation
Date
Purpose
Details
J
4/26/2011
Tested age of Bl (stability over time)
and vortexing vs. not vortexing
The burden on some Bis resulted in
encapsulated growth (in a bubble) during
analysis, resulting in a false-positive result
due to the lack of turbid media. This test
attempted methods to liberate surviving
spores from the Bl carrier prior to
incubation to circumvent this issue
10
-------
Test
Fumigation
Date
Purpose
Details
K
5/24/2011
Tested D-value
Tested at 0, 1.5, 2.5, 3, 3.5, 4, 4.5 and 5
hours
Tested Breath Easy film on well
plates
Tested at 2, 6, and 9 hours with no filter, 1
layer of filter, and 2 layers of filter
Tested Nufab on well plates
Tested at 2, 6, and 9 hours
L
8/1/2011
Tested Apex vs. Raven Bis
Investigated effect of proximity with
compact (dense) and loose packing of Bis
during exposure.
M
9/21/2011
Tested Apex vs. Raven Bis
1000 ppmv CIO2 fumigation
N
11/1/2011
Tested Apex vs. Raven Bis and
RCT culture test kits (see Section
2.2.6.6)
250 ppmv CIO2 fumigation
S
10/9/2012
0% and 1.0% CSN concentrations
to measure D-value and kill point.
Triplicate identical Bis/test conditions.
Tested at 0,1,4,8,12,16, and 20 hours
T
10/30/2012
Repeat of Test S
Triplicate identical Bis/test conditions.
Tested at 0,1,4,8,12,16, and 20 hours
U
1/22/13
Repeat of Test S, adding Mesa
COTS ethylene oxide (EtO) Bl
Triplicate identical Bis/test conditions.
Tested at 0,1,4,8,12,16, and 20 hours
Included COTS Bis
V
3/13/13
Repeat of Test S, adding three
Mesa COTS Bis
Tested at 0,1,4,8,12,16, and 20 hours
Included three COTS Bis:
• Mesa Laboratories Releasat® for
Chlorine Dioxide Sterilization, 106S.
atrophaeus (reorder no. RCD/50)
• Mesa Laboratories MesaStrip for Low
Temperature Steam Formaldehyde
Sterilization, 10aG. stearothermophilus
(reorder no. SGMLF/6).
• Mesa Laboratories MesaStrip for
Steam Sterilization, 106S. atrophaeus
(reorder no. SGMG/6)
w
8/20/13
Low Inoculum Test on unburdened
vs. 1.0%, 2.0%, and 5.0% CSN
burdened Bis. Tested at 0,1,2,4,6,
and 9 hours
Included ProLine Process Challenge
Device (PCD) COTS Bis (see Section
2.2.6.7) with and without lumens
X
8/27/13
Low Inoculum Test on unburdened
vs 1.0%, 2.0%, and 5.0% CSN
burdened Bis. Tested at 0,2,4,6, 9,
and 12 hours
Included ProLine PCD COTS Bis without
lumens, and modified to have
foreshortened inlet (not reported)
Y
9/9/13
Low Inoculum Test on unburdened
vs 1.0%, 2.0%, and 5.0% CSN
burdened Bis. Tested at 0,6, 9, and
12 hours
z
01/28/14
Low Inoculum Test on unburdened
vs 1.0% and 2.0% CSN burdened
Bis. Tested at 0, 6000, 9000, and
12000 ppm* hours.
Included inoculated coupons of
carpet, wood, and aluminum
Fumigated with 2000 ppm CIO2
AA
03/05/14
Fumigated with 1000 ppm CIO2
AB
03/20/14
Fumigated with 500 ppm CIO2
11
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2.1.2 Task 2: Testing of Bl Carrier Materials
In this task, several carrier materials, unrelated to those used in Task 1, were evaluated for their
compatibility with surrogate organisms and their ability to increase the CT required for Bl inactivation. The
number of replicates per material type and indicator organism is shown in Table 2-5. Under this task,
carriers of 11 material types were used for the custom Bis (Table 2-6). Criteria for choosing carrier
materials included being commercially available with relatively uniform size, shape, porosity; being of a
size amendable to packaging within a Tyvek® envelope; and being of a material type that is easily
sterilized.
Carrier materials were inoculated with 1 x 106 spores (either B. atrophaeus or G. stearothermophilus),
and placed into Tyvek® envelopes. The resulting custom Bl test samples were challenged by fumigation
with 1,000 ppmv gaseous CI02 at 75% RH and 24 °C in the DTRL. The test samples and controls
(negative, positive, and performance controls, discussed further in Section 2.1.3) were collected at 5, 7,
and 9 hour exposure times.
Table 2-5. Number of Replicates per Experimental and Control Bl Type for Task 2
Sample
Time
Point
(hours)
Replicates
Total
Test/Control
Bl
Inoculated
Media
Spiked
Fumigated
Performance
Baseline
0
3
3
C
No
Yes
No
Positive Control
0
3
3
C
Yes
No
No
Performance Control
5,7,9
3
9
c
No
Yes
Yes
Experimental
5,7,9
5
15
T
Yes
No
Yes
Note: 30 Bis * 10 carrier materials (including stainless steel) * 2 organisms = 600 Custom Bis, + 3
negative control Bis.
Table 2-6. Material Test
Test
Date
Materials
Spores
Time Points
B
2/3/2010
10 types
(see Table 2-7)
BG, GS
5, 7, and 9 hours
12
-------
2.2 Test Materials and Deposition
2.2.1 Bl Preparation
The prepared Bis were stored and transported to EPA as individually packaged Bis in a Tyvek® envelope.
Table 2-2 shows the burden additives and concentrations initially prepared for Task 1, Test A. Additional
burdens at several concentrations were investigated for successive tests as discussed in Section 3.
Casein, burden code CSN, was sourced from Sigma (P/N C7078 Sigma-Aldrich Corporation, St. Louis,
MO, USA) and added starting at Test E. Table 2-7 shows the materials tested for Task 2. Figure 2-1
shows the mixture of burden and inoculum dried on the stainless steel carrier.
Figure 2-1. Stainless Steel Carrier with Burden and Inoculum
13
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Table 2-7. Carrier Materials for Custom Biological Indicators
#
Material
Material
Code
Supplier and
Location
Cat#
Surface
dimensions
Thickness
Inoculum
location
1
Stainless
Steel
SST
Yakibou* (Sanford, NC)
NA
10 mm
diameter
0.19 mm
Surface
2
Wooden
Discs
WOD
American Woodcrafters
Supply Company
(Riceville, IA, USA)
DIS-050
12.7 mm
diameter
3.18 mm
Surface
3
Adhesive
Felt Dots
FLT
Aetna Foot Care
Products (Allentown, PA,
USA)
1/2" Dots
12.7 mm
diameter
1.6 mm
Surface
4
Adhesive
Rubber Dots
RUB
Aetna Foot Care
Products (Allentown, PA,
USA)
016205
12.7 mm
diameter
2.4 mm
Surface
5
Adhesive
Cork Dots
CRK
Aetna Foot Care
Products (Allentown, PA,
USA)
004377
12.7 mm
diameter
1.6 mm"
Surface
6
Ceramic Tile
CER
Mosaic Basics (Atlanta,
GA, USA)
NA
9.5 mm x 9.5
mm
3.18 mm
un-glazed
side
7
6061
Aluminum
ALU
Mc Master Carr (Atlanta,
GA, USA)
89015K
86
12.7 mm
diameter
2 mm
Surface
8
Chipboard
Discs
CBD
Wolter Pyro Tools
(Montello, Wl, USA)
CBD-
58-16
15.9 mm"
diameter
1.6 mm
Surface
9
0.2 |jm pore-
size
Cellulose
Ester
CEM
Whatman, GE
Healthcare Bio-Sciences,
(Pittsburgh, PA, USA)
104017
12
47 mm
diameter
135 |jm
Top
Surface
10
C14500
Copper
discs
CUP
Storm Copper
Components (Decatur,
TN, USA)
NA
12.7 mm
diameter
2.5 mm
Surface
11
Porous
Polypropyl
XYZ
Permaplas Corp.
(Fayetteville, GA, USA)
20201
13.1 mm
diameter
2.6 mm
Surface
* Then known as Apex Laboratories.
NA = Not applicable.
14
-------
2.2.2 Spore Preparation
The different types of spore preparations used for this study are described below.
2.2.2.1 Performance Control Spore Preparation
An inoculum containing approximately 1 x 104 CFU mL"1 was used for all performance control spikes. This
inoculum was a dilution of an original preparation of ATCC 9372 B. atrophaeus from Apex Laboratories,
Lot 712691.
2.2.2.2 Burden and Material Bl Spore Preparations
In general, custom Bis used an inoculum prepared and dispensed onto carriers by Yakibou. Table 2-8
shows the details of the inoculant used on burden and material Bis.
15
-------
Table 2-8. Source and Lot of Inoculant used on Bis
Test
Bl Batch
Date
B. atrophaeus
ATCC 9372
G. Stearothermophilus
ATCC 12980
Provider
Lot
CFU Recovered
Provider
Lot
CFU
Recovered
A
12/17/09
Yakibou*
2598GL
7.9 x 10s
Yakibou*
0598ST
6.2 x 10s
B
12/21/09
Yakibou*
2598GL
8.1 x 10s
Yakibou*
0598ST
7.0 x 10s
C
3/31/10
Yakibou*
2598GL
6.0 x 10s
Yakibou*
0598ST
5.1 x 10s
D
3/31/10
Yakibou*
2598GL
6.0 x 10s
Yakibou*
0598ST
5.1 x 10s
E
7/14/10
Yakibou*
2598GL
7.6 x 106t
Yakibou*
0598ST
4.6 x 10s
F
8/30/10
Yakibou*
See note
1.0 x 107
G
10/21/10
Yakibou*
See note
3.1 x 10s
H
12/30/10
Yakibou*
See note
4.6 x 10s
I
12/30/10
Yakibou*
See note
4.6 x 10s
J
Varies. See Section 3.9.7 for details
O, P and Q
1/17/12
Yakibou*
2566GL
1.2 x 10s
Mesa Labs
1073081
1.4 x 10s
R
6/6/12
Yakibou
2566GL
3.8 x 10s
S, Tand U
9/25/12
Yakibou
2566GL
6.0 x 10s
V
2/21/13
Yakibou
2566GL
6.8 x 10s
(1% CSN Bl)
W, X, and Y
8/14/13
Yakibou
2566GL
1.2 x 103
(1% CSN Bl);
2.3 x102
(unburdened Bl)
or
1.1 x 105
(1% CSN Bl);
1.3 x 104
(unburdened Bl)
(see Section 3.9.6)
Z, AA, and AB
1/6/14
Yakibou
2566GL
9.9 x102
(unburdened Bl),
1.4 x 103
(1% CSN Bl);
1.3 x 103
(2% CSN Bl)
*Then known as Apex Laboratories.
+ The dithiothreitol (DTT) samples alone were only 2 x 104 concentration following heat shock, suggesting the
heat shock with the DTT either suppresses germination/growth or is lethal to the spores. With the heat shock
eliminated, the concentration for the DTT samples was 3.5 x 10s.
Note: The lot number was unavailable from the manufacturer, but the manufacturer did confirm that the source
was American Type Culture Collection (ATCC) 9372.
16
-------
The recovery (CFU) values listed are from Bis without burden unless otherwise noted. For Tests W, X,
and Y, the recovery from Bis with burden was an order of magnitude higher than the recovery from Bis
without burden, presumably due to more efficient spore dislodgement from the carrier during extraction.
2.2.2.3 Well Plate Spore Preparation
The spores for Test Kwere prepared from a serial dilution of ATCC 9372 B. atrophaeus spore stock
solution obtained from Raven Laboratories, batch 304GB. Dilutions were made with 40% ethanol in sterile
deionized water. Suspension recovery was 1.8 x 108 CFU/mL.
2.2.3 Bl Carrier Inoculation
Custom Bis were typically inoculated by Yakibou with 20 |jl_ of spore suspension. Carriers with burden
were inoculated with 40 |jl_ of a 50:50 mixture of burden and spore suspension. For performance control
Bis, 20 |jl_ aliquots of the most concentrated burden were pipetted onto the carriers. Carriers were
typically dried 2.5 hours in a flowing air oven at 37 - 38 °C before packaging into uniquely labeled Tyvek®
envelopes.
2.2.4 Control Bis
Each test included control Bis (Table 2-9) used to evaluate data quality. The negative control Bis were
the same stainless steel discs used by Yakibou for Bis, not inoculated, and packaged in Tyvek®
envelopes. These negative Bis were generally autoclaved before use and were not fumigated (laboratory
blanks). Some negative Bis were simply Bis that were not inoculated, but not autoclaved and not
guaranteed sterile from the Bl vendor (field blanks). The positive control Bis were standard stainless steel
Bis of each organism prepared by Yakibou. The spores were inoculated onto stainless steel discs and
packaged in Tyvek® envelopes. The positive control Bis were not exposed to the fumigant. Burden tests
included five replicate positive control Bis; material tests used three replicates as baseline controls, which
were included in each fumigation. Turbidity control Bis included burden but no inoculum, and were
incubated along with test Bis to determine if the presence of burden material could lead to a turbid result
that could be misinterpreted as growth.
Performance control Bis (non-spore-inoculated carriers) were subjected to the longest fumigation duration
for each test. The Bis were then aseptically placed into tryptic soy broth (TSB) and the medium was
spiked (inoculated) with 0.1 mL of a ~5 x 103 CFU mL"1solution of the target surrogate spores used during
the test (either B. atrophaeus ATCC 9372 or G. stearothermophilus ATCC 19280). The inoculated tubes
were then incubated for 7-9 days at the temperature most favorable for growth (35 °C ± 2 °C for B.
atrophaeus and 55 °C ± 2° C for G. stearothermophilus) and afterwards visually inspected to confirm
compatibility of the fumigated burden with viable spores (presence of turbid (cloudy) culture media,
indicative of bacterial planktonic growth).
17
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Table 2-9. Characteristics of Control Bis
Control Bl
Information Provided
Inoculated
Fumigated
Burden
Negative Control Bl
(coupon or Bl without
biological agent)
Controls for sterility of
materials and methods
used in the procedure.
No
No
Yes
Positive control
(Bl or inoculated
material not fumigated)
Shows incubation
tubes ability to support
and show growth
Yes
No
Yes
Turbidity Control
(Bl with material or
burden, not inoculated
but fumigated)
Provides information
about the tendency
towards false positives
of candidate Bl
No
Yes
Yes
Performance Controls
Confirms compatibility
of the fumigated
burden with growth of
viable spores
After fumigation
Yes
Yes
2.2.5 Well-Plate Inoculation
The well plates used for Test Kwere sterile, polystyrene cell culture plates (Corning Incorporated P/N
3548, Corning, NY, USA). Each well was inoculated with B. atrophaeus ranging from 102to 106 for the
test samples or with 40% ethanol in Dl water for the negative control samples and allowed to dry
overnight. After fumigation, each well plate was charged with 725 |jl_ of 3% Alamar Blue solution (P/N
BUF012A, AbD Serotec, Oxford, UK).
2.2.6 Off-The-Shelf Bis
COTS Bis were used throughout the test sequence as comparisons to the custom Bis. These COTS Bis
are discussed below.
2.2.6.1 Mesa Ethylene Oxide (EtO) Bis
The EtO Bis used for Test U were COTS Bis recommended for ethylene oxide gas sterilization. This Bl
was an 8 mm x 12 mm stainless steel oblate disc inoculated with B. atrophaeus ATCC 9372 spores.
Batch 301GB, used for Test U, had a nominal population of 2.8 x 106 spores. Batch 301GBN was
identical to the Raven B. atrophaeus (P/N 1-6100-ST) Bl.
2.2.6.2 Mesa Strips
Mesa Laboratories MesaStrip Bis consisted of a 6.4 mm x 38.1 mm strip of Schleicher & Schuell filter
paper (#470) inoculated with bacterial spores and sealed in a glassine envelope. For this project, two
varieties of MesaStrip were used: P/N SGMG/6 were inoculated with approximately 1 x 106B. atrophaeus
spores., and P/N SGMLF/6 were inoculated with approximately 1 x 106 G. stearothermophilus spores.
2.2.6.3 Mesa Laboratories Releasat® for Chlorine Dioxide Sterilization
The Releasat® Bl (P/N RCD/50) used for Test V was a 19 mm x6.3 mm paper carrier inoculated with
approximately 1 x 106 B. atrophaeus spores, sealed in a glassine envelope. The kits included culture
18
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tubes of specially formulated soybean casein digest culture medium containing a color indicator that turns
yellow in the presence of bacterial growth.
¦
IjJjw
e ¦
Mesa strip Proline PCD Releasat®
f"*».
V
Apex B. atrophaeus Bl Mesa EtO Bl/Raven B. atrophaeus Bl
Figure 2-2. Commercial Off-the-Shelf Bis
2.2.6.4 Apex B. atrophaeus Bl
The Apex B. atrophaeus Bl was used in tests L, M, and N, and was identical to the stainless steel
Yakibou Bl with no burden. The Apex B. atrophaeus Bl was an COTS product made by Apex before the
sale of Apex to Raven/Mesa Laboratories, it was a stainless steel disc inoculated with approximately 1 x
106 spores of B. atrophaeus, and sealed in a Tyvek® envelope.
2.2.6.5 Raven B. atrophaeus Bl
The Raven B, atrophaeus (P/N 1-6100-ST) Bl used for Tests L, M, and N was a stainless steel carrier
inoculated with approximately 1 x 10° spores of B. atrophaeus, and sealed in a Tyvek® envelope.
MESAstrip
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-J
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19
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2.2.6.6 Mesa Laboratories RCT
RCT kits were manufactured by the Raven Laboratories division of Mesa Laboratories. The kits included
a 19 mm x 6.3 mm strip of paper inoculated with B. atrophaeus strain ATCC 9372, with a mean strip
recovery of 2.1 x 106 CFU/strip. Batch 298GB (Lot number 1S62983) was used for Test N. The kits are
designed specifically for use with CI02 sterilization. Each kit contained 25 spore strips (as previously
described) individually wrapped and 25 culture media tubes. Following testing, each strip was aseptically
placed into culture media tube and incubated for seven days, then evaluated based on color/ turbidity of
tube. The tubes that retained the purple color and were not turbid were considered negative for growth,
and tubes that exhibited a yellow color change and were turbid were considered positive for growth.
2.2.6.7 Mesa Laboratories ProLine PCD
Mesa Laboratories ProLine Process Challenge Device (PCD) Bis consisted of a 9 mm paper disc
inoculated with B. atrophaeus spores inside a glassine envelope. These Bis are designed to go inside a
lumen or other sterilizable tubing 1.6 mm to 14.3 mm ID. These Bis were tested with various lumen
lengths from 0 cm to 122 cm of 1.6 mm ID tubing. The nozzle was completely removed from the Bl for
Test X.
2.2.7 Building Material Coupons
For tests Z, AA, and AB, wooden, carpet, and aluminum coupons were used in conjunction with the Bis
(Figure 2-3). Decontamination kinetics of these coupons (surrogates for materials inside a building) were
compared to the developed custom Bis. Coupons were made of wood or carpet affixed to an aluminum
stub (P/N 16119 http://www.tedpella.com/SEM html/SEMpinmount.htm. Ted Pella, Inc. Redding, CA,
USA) using a carbon based adhesive, the third material being the aluminum stub itself. Wooden coupons
were prepared from commercially available 19 mm oak stair plugs (http://www.craftparts.com/oak-stair-
pluqs-p-3943.html?cat id=257. Woodworks. Ltd., Haltom City, TX, USA). The original planed but
unfinished surface of the wood was used as the inoculation surface. The carpet coupons were made by
punching an 18 mm core from a commercial carpet square (Ultimate Temptation model, color: allurement,
carpet model number 85128/695, pile height 0.64cm, Sherwin-Williams, Cleveland, OH, USA), which was
glued onto the aluminum stub. The adhesive was allowed to dry for 48 hours before undergoing
sterilization by a steam autoclave on a gravity cycle following NHSRC RTP Microbiology Laboratory
internal Miscellaneous Operating Procedure (MOP) 6570 (Appendix B). Each sterilization batch included
all coupons for a single test. Three sample coupons were analyzed for growth/no growth from each
sterilization batch as an indication of sterilization efficacy.
The liquid inoculum was obtained from Yakibou, Inc. (same inoculum as used for Bis), and the inoculation
of the coupons was performed by the NHSRC RTP Microbiology Laboratory. Each coupon was
inoculated with 0.1 mL of the spore suspension with mean population of 5.3 x 106 CFU/mL distributed
across the coupon and allowed to dry overnight in a biological safety cabinet (BSC) before use.
20
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Figure 2-3. 18 mm Coupons
2.3 Fumigation Methods
Fumigation conditions were established in a glovebox (P/N 830-ABC Glovebox, PlasLabs, Lansing, Ml,
USA) (Figure 2-4) that permitted removal of Bis during exposure. The atmosphere in the glovebox was
brought to 75% RH by injection of hot, moist air generated by a Gas Humidity Bottle (P/N HF-HBA, Fuel
Cell Technologies, Inc., Albuquerque, NM, USA). Injection of the hot moist air was regulated by a
feedback loop from an RH sensor (P/N HMD40Y or P/N HMD53W, Vaisala, Vandaa, Finland). Once the
RH conditions were met, a CI02 generator (P/N GMP or Minidox, ClorDiSys Solutions Inc., Lebanon, NJ,
USA) supplied fumigant until the set-point was reached, as determined by an internal photometer. This
phase is called the conditioning phase. Once the target concentration is reached, the exposure phase
begins.
21
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Figure 2-4. Glovebox Fumigation Chamber
For the initial tests, Bis were present in the glovebox for the conditioning phase as well as the exposure
phase. For a two-hour exposure, the Bis would have been present for the ramp-up from ambient to target
concentration, as well as the two-hour exposure at target concentration. Beginning with Test H and for all
subsequent tests, Bis were placed in the glovebox once the target concentration was reached. This
change in procedure was made to reduce the variability in exposures (exposure time, and total CT)
between tests, and to achieve more precise exposures (CTs).
2.4 Sampling and Analytical Procedures
All materials needed to process the samples were prepared in the NHSRC RTF Microbiology Laboratory.
These materials included, but were not limited to, growth media, culture broth, agar plates, and/or sterile
liquids. Quality assurance (QA) checks (listed in Table 4-2) conducted on these materials in the NHSRC
RTF Microbiology Laboratory verified that the materials were not contaminated with any organism and
supported the growth of target organisms. All quality control (QC) records pertaining to these materials
are retained in the NHSRC RTF Microbiology Laboratory. All personnel in the NHSRC RTF Microbiology
Laboratory operate under an approved Facility Manual specific for the Lab, which contains MOPs that are
relevant to this project.
22
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2.4.1 Qualitative Bl Analysis
MOPs 6560 and 6566 describe the qualitative Bi analyses (all associated MOPs can be found in
Appendix B). To analyze, the Bis were aseptically transferred in a Class II BSC, to 15 mL polypropylene
culture tubes (P/N 169897, USA Scientific Inc., Oca la FL, USA) containing 10 mL of tryptic soy broth
(TSB). The Bis were either allowed to drop aseptically from the packaging into the TSB tubes or
disposable sterile thumb forceps were used to transfer them. The tubes were incubated at the appropriate
temperature for the target organism specified by manufacturer's instructions (e.g., all B, atrophaeus were
incubated at 35 ± 2 °C, and all G. stearothermophilus were incubated at 55 ± 2 °C). The medium within
the tubes was inspected visually for turbidity seven days later. To confirm the qualitative results and to
verify that the turbidity was caused by the target organism, 10 % of the samples that were turbid were
plated to confirm that the growth was from the target organism (by colony morphology). Additionally, all
(100%) samples found to have No Growth were plated (0.1 mL) (to confirm that there indeed was no
growth).
For tests A-l, the qualitative confirmation was completed using a sterile disposable 10 pL loop to remove
a 10 pL aliquot from the TSB tube containing the Bl and aseptically spread the aliquot onto a tryptic soy
agar (TSA) medium plate. For Tests J-Y, the qualitative confirmation was completed by plating a 100 pL
aliquot from the TSB tube containing the Bl sample, directly onto TSA. All TSA plates were incubated at
the proper temperature for the target surrogate, per manufacturer's instructions. Prior to removing either
the 10 pL or 100 pL aliquot from the qualitative samples, some of the TSB tubes containing the Bl
samples (Tests J-Y), were homogenized by vortex mixing for a quick 2-5 second burst. Note that the
vortexing of the samples did not have any effect on the growth of the Bl samples based on data from Test
J (see section 3.9,2). TSA plates were analyzed and results documented following18-24 hours of
incubation at the appropriate temperature for the target organism.
2.4.2 Qualitative Well-plate Analysis
Well plates used in Test K were charged with 725 pL of TSB amended with 3 % Alamar blue solution and
incubated for seven days at 35 °C ± 2 °C. The 3 % Alamar blue is a colorimetric indicator of bacterial
growth, which turns pink when growth occurs or remains purple/blue in the absence of growth (Figure 2-
5), The color change makes identification of growth-positive samples easier and more reliable. Alamar
blue was used for the well-plate tests to aid in detecting growth, since well-plates could not be held in
front of a light and inspected individually as done with culture tubes.
Figure 2-5. Well Plates with Alamar Blue Showing Growth (pink) and No-Growth (purple)
Results
23
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2.4.3 Quantitative Bl Analysis
For quantitative analysis, Bis were placed in sterile 18 mm borosilicate glass tubes (Fisherbrand P/N 14-
961-32, ThermoFisher Scientific, LLC. Waltham, MA, USA), containing 10 mL of Phosphate Buffered
Saline with 0.05% TWEEN®20 (PBST) made according to MOP 6562 (Appendix B). Bis soaked in the
PBST for a minimum of 15 minutes, as an initial procedural step. The tubes containing the Bis were then
placed in an ultrasonic cleaner for three seven-minute intervals at 44 kHz ± 6 % (Bransonic Ultrasonic
Cleaner, P/N 8510R-MT, Danbury, CT). The location of the tubes was changed between each seven-
minute interval to increase uniform exposure of tubes. After sonication, 10 mL of PBST was removed and
transferred to a 50 mL conical tube. The Bl was discarded and the PBST extraction was treated with a
heat shock per MOP 6576 (80 ± 2 °C for ten minutes). Following heat shock, the samples were re-
homogenized by vortex. The liquid extracts were then tenfold serially diluted and spread plated according
to MOP 6535a. Plates were incubated at 35 ± 2 °C for 18-24 hours.
2.4.4 Chlorine Dioxide Monitoring
CI02 measurements were conducted using two techniques; a ClorDiSys Solutions, Inc. photometric
monitor and Standard Methods for the Examination of Water & Wastewater Method 4500-CI02 (mSM-
4500). The first technique was used continuously for real-time control of the chamber fumigant
concentration at 1,000 ppmv. The mSM-4500-CI02 was used periodically as a confirmation of the
photometer. Because wavelengths of light in the visible and UV regions cause spontaneous breakdown of
CI02, all sampling methods used opaque sample lines as a precaution.
2.4.4.1 Photometric Monitoring
The ClorDiSys CI02 monitor is a photometric system operating in absorbance mode with a fixed path cell.
A pump provides flow of the test gas from the test point to the analytical cell. The maxima and minima of
an unspecified, proprietary CI02-specific absorbance band are continuously monitored and used to
calculate the absorbance. Calibration was performed by the manufacturer with National Institute of
Standards and Technology (NIST)-traceable transmission band-pass optical filters and was performed in-
house every six months with manufacturer reference filters. The monitor includes a photometer zero
function to correct for detector aging and accumulated dirt on the lenses. Daily operation of the
photometers included moments when clean, CI02-free air was being cycled through it. If the photometer
read > 0.1 mg/L during these zero air purges, then the photometer was re-zeroed. The photometer was
cleaned if the concentration measurements were not within 10% of the mSM-4500 values.
2.4.4.2 mSM-4500-CI02
Two variations of the 1992 18th edition Standard Method (SM)-4500-CI02 titration were used during this
testing: (1) amperometric titration (SM-4500-CI02-E) and (2) iodometric titration (SM-4500-CI02-B). The
SM-4500-CI02 collection method has been modified (mSM) to include gas-phase sampling based upon a
buffered potassium iodide bubbler sample collection, and restricting the official method to a single titration
based upon analyzing the combined chlorine, CI02, and chlorite as a single value. This method can only
be applied only where chlorine and chlorite are not present. Since the modified method described below
is applied to gas-phase samples, the presumption of the absence of chlorite and chlorate is valid. The
presence of chlorine would be indicated by a difference in CI02 concentration as measured by the
photometer and titration.
24
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The modified method was performed as follows:
a. Add 20 ml_ of phosphate buffer solution, pH 7.2, containing potassium iodide (Kl) (KIPB solution,
25 g Kl/ 500 mL of phosphate buffer) to two impingers. (P/N PRG-5795, Prism Research Glass,
Inc., Raleigh, NC, USA)
b. Set CI02 gas flow from the chamber into the impingers containing KIPB solution in series at a
flow rate of 1 L/min for two minutes.
c. Combine the 20 mL of KIPB solution from each impinger into a 200 mL volumetric flask and rinse
the impingers thoroughly with deionized water.
d. Add 5 mL of 6 N hydrochloric acid (HCI) to the solution.
e. Place solution in dark for five minutes.
f. Titrate the solution with 0.1 N sodium thiosulfate. The end point is determined visually (yellow to
clear for mSM-4500-CI02-B) or amperometrically (mSM-4500-CI02-E).
g. Record the volume of sodium thiosulfate titrated. Conversion calculations from titrant volume to
CI02 concentration are based on SM 4500-CI02:
CIO? (mq/L) = Volume of Sodium Thiosulfate (mL) x N x 13.490 (Eqn 2)
Volume of Gas impinged (L)
2.5 Sampling Strategy
2.5.1 Sampling/Monitoring Points
Photometer and mSM-4500-CI02 samples were taken from ports in the isolation chamber. Each port from
the well-mixed chamber was expected to be representative of the bulk concentration.
The RH and temperature sensors were co-located on the meter. The meter was placed far enough from
the walls of the chamber to be unaffected by any difference between wall temperature and the bulk
atmosphere within the chamber. A HOBO sensor was placed in the center beside the Bis. Table 2-10
details the parameters for the monitoring methods.
25
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Table 2-10. Monitoring Methods
Monitoring Method
Sampling Flow Rate
Measurement Range
Measurement Frequency and
Duration
Photometer
5 Lpm nominal
50 -10,000 ppmv CIO2
Real-time; six per minute
mSM-4500-CI02
0.5 Lpm
36 -10,000 ppmv CIO2
Every 30 - 60 minutes;
four minutes each
Vaisala RH/ Temperature
Meter
NA
0 -100% RH,
-40 °C to 60 °C
Real-time; six per minute
K-type thermocouple
(Omega Engineering,
Stamford, CT, USA) for
chamber and coolant
temperature
NA
-200 °C to 1350 °C
Real-time; six per minute
HOBO RH/
Temperature Sensor
NA
5 - 95% RH,
0-100 °C
Real-time, three per minute
Bis
NA
0 to >1 x 10s spores
Growth/No Growth determinations
Viable population evaluated at end
of experimentation as compared
with time 0
Table 2-11 lists the critical and non-critical measurements for each sample.
Table 2-11. Critical and Non-Critical Measurements
Sample Type
Critical Measurements
Non-critical Measurement
mSM-4500-CI02
Collected gas volume, titrant volume
Temperature, collection time
Fumigation Conditions
RH, temperature, photometric CIO2 reading
Bis
Exposure time, proximity*, lumen length*
vortexed or not vortexed*
* Measurements critical for specific tests only.
2.6 Sampling Handling and Custody
2.6.1 Preventing Cross-Contamination
Cross-contamination of Bis during fumigation was prevented by the Tyvek® or glassine (manufacturer
dependent) envelopes that enclosed each Bl. Samples were also separated and organized by attachment
to 12.7 mm diameter stainless steel springs.
Each extractive CI02 sample was placed in its own sample jar. Glassware was triple-rinsed with deionized
water before reuse.
26
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Figure 2-6. Springs used to Organize Bis
All work involving the growth/culturing and analysis of the samples was performed while using the proper
personal protective equipment (PPE). Processing and analysis was completed within the confines of a
BSC. During transfer of the samples from the Tyvek envelopes to sterile tubes containing the culturing
broth, aseptic technique was used to prevent contamination or cross-contamination of samples. To
prevent any form of cross-contamination, all B. atrophaeus samples were manipulated separately from G.
stearothermophilus samples. Prior to any analysis or processing, the workspaces were cleaned and
made free of debris. The BSC was thoroughly cleaned by wiping surfaces in the following order: pH-
adjusted bleach (pAB), deionized water, and a 70-90 % solution of denatured ethanol. The BSC was
cleaned in this manner before work began, after each use, between sample sets involving different
species of bacteria, and any other time in which a contamination event was suspected to have occurred.
An ultraviolet (UV) light was used in the BSC for decontamination only after all work was completed and
no personnel were working in the immediate area to prevent exposure to UV light. All biological waste
material that was accumulated from the processing and analysis was properly disposed of in order to
prevent possible contamination.
Samples were manipulated in the following order to prevent any form of cross-contamination: negative
controls (lowest concentration of bacteria), sample sets (all unknown and variable concentrations of
bacteria), positive controls (highest concentration of bacteria). Samples that were visually identified as No
27
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Growth were streak-plated to confirm the absence of growth prior to the streak-plating of any samples
positive for growth. Every Bl that was incubated for qualitative analysis was placed in a new sterile,
disposable tube. In addition, all glass borosilicate tubes used for quantitative analysis and extraction were
used only once and then disposed of to ensure no cross-contamination occurred between samples.
2.6.2 Sample Containers
Tyvek®-wrapped Bis provided by the vendors were delivered by the manufacturer in plastic bags
containing silica desiccants. These bags served as the sample containers until analysis. Bis were
segregated by organism and stored in a stable indoor humidity-, light-, and temperature-controlled
secondary containment. CI02 extractive samples were typically processed immediately and hence were
not stored in a container. Coupons were aseptically placed in a 50 mL conical tube and transported to the
NHSRC RTP Microbiology Laboratory for analysis.
2.6.3 Sample Identification
Each Bl, coupon or sample was identified by a unique sample ID that was documented in an explicit
laboratory log that included records of its associated test number, inoculum level, sampling method, and
the date sampled. Each Bl was marked with the material descriptor and unique code number. Sample IDs
included descriptors, for project number (WA 51), test ID, inoculum type, burden type, burden
concentration, material type, sample purpose (test, control, field blank, etc.) and replicate number as
applicable. Once samples were transferred to the NHSRC RTP Microbiology Laboratory for
microbiological analysis, each plate was additionally identified by replicate number and dilution. The
NHSRC RTP Microbiology Laboratory also included on each plate the date it was placed in the incubator.
2.6.4 Information Recorded by DTRL Personnel
DTRL personnel were responsible for recording data collected during the fumigation such as sample
volumes, titration volumes, and other data used to characterize the fumigation conditions. Field personnel
also recorded the times that Bis were exposed and removed from the fumigation chamber.
2.6.5 Sample Preservation
Bis were placed inside a permeable envelope that allows penetration of the fumigant but prevents
movement of microorganisms from the outside to the inside, and vice versa, thereby preserving the Bl
from contamination. Before use, Bis were stored in packaging containing desiccant, preventing hydration
of the spores. After exposure or use, Bis were stored under ambient laboratory conditions before
analysis.
2.6.6 Sample Holding Times
After sample collection for a single test was complete, all biological samples were transported to the
NHSRC RTP Microbiology Laboratory immediately, with appropriate chain of custody (COC) form(s).
Samples of other matrices were stored no longer than five days before the primary analysis. Typical hold
times, prior to analysis, for most biological samples was < two days. CI02 extractive samples were
typically processed immediately and hence were not stored.
28
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2.6.7 Sample Custody
Careful coordination with the NHSRC RTP Microbiology Laboratory was required to achieve successful
transfer of uncompromised samples in a timely manner for analysis. Test schedules were confirmed with
the NHSRC RTP Microbiology Laboratory prior to the start of each test. Accurate records were
maintained whenever samples were created, transferred, stored, analyzed, or destroyed. The primary
objective of these procedures was to create a written record used to trace the possession of the sample
from the moment of its creation through the reporting of the results. Details of the chain of custody
procedures were documented in the approved QAPP.
2.6.8 Sample Archiving
All coupons were archived for a minimum of two weeks following completion of analysis. This time
allowed for review of the data to determine if any re-plating of selected samples was required. Samples
were archived by maintaining the primary extract at 4 ± 2 °C in a sealed extraction vessel. Incubated Bis
were not typically archived after the seven-day plating.
29
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3 Results and Discussion
Tests results are arranged below for the sake of clarity rather than in chronological order. Test A and Test
B were scoping tests designed to identify burdens or carrier materials that might modify the behavior
(resistance to CI02) of a Bl towards the target Bl. Subsequent investigations consisting of a test or a
series of tests provided more in-depth knowledge of the behaviors of Bis and their constituent parts.
3.1 Fumigations
All fumigations with the exception of Tests N, Z, and AB were intended to have fumigation conditions at
2.77 mg/L (1000 ppm) CI02 and 75% RH at 23.8 °C. The range of exposure times varied depending on
the purpose of the test. Fumigations are complex operations, and are difficult to replicate. Fumigation
conditions for all tests are summarized in Table 3-1.
3.2 Test A - Burden Scoping Test
The effect of both low and high concentrations (see Table 2.1) often different burdens on the survivability
of B. atrophaeus Bis is shown in Table 3-2 and Figures 3-1 and 3-2.
These tests were conducted to identify burden materials that 1) increased the survival of the test
organism, and 2) demonstrated a dose-dependent increase in Bl survival. Burdens demonstrating dose-
dependent effects on Bl survival were more desirable as protection from the fumigant could be increased
or decreased by altering the burden concentration. The data demonstrate that many burdens provided too
much protection, resulting in 100% survival rates even at 9000 ppm*hours. Increasing concentration
increased protection for three burdens: carrageenan (CAR), glutathione (GLU), and humic acid (HMA).
The custom Bis that had a low concentration of CAR looked promising, and were evaluated further (Tests
C and E). HMA also showed great promise, with increased protection with the increase in burden
concentration. However, HMA also showed a tendency towards false positives as indicated by turbidity
control Bis. The turbidity control Bis included burden but no inoculum, and were incubated along with test
Bis. HMA, as well as FCL, produced an effect that was interpreted as growth, even though the Bis did not
include inoculum. These materials thus demonstrated a tendency towards false positives and were
rejected. The HMA Bis also seemed to interfere with the growth of low inoculum spikes of fumigated Bis,
with only 19 of 24 of the spiked control samples showing growth. For these reasons, HMA was not
studied further as a burden.
Similarly, the results from G. stearothermophilus Bis are shown in Table 3-3 and Figures 3-3 and 3-4.
Overall, G. stearothermophilus Bis showed similar survival rates to B. atrophaeus Bis, with CLB, DTT,
FCL, GEL, and GLU significantly increasing both organisms resistance to CI02. The low concentration
amino acid coctail (AAC) provided protection to the G. stearothermophilus Bl, but the reliability at lower
exposure times was too low to be considered for further investigation. Similar to the B. atrophaeus Bis,
carrageenan (CAR) provided partial protection for the G. stearothermophilus Bis, as did HMA.
30
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Table 3-1. Average Conditions during Fumigations
CI02 (mg/L)
Titration Data
CIO2 (mg/L)
Photometer Data
RH
(%)
Temperature
(°C)
Test
ID
Test Date
Average
SD*
Average
SD
Average
SD
Average
SD
A
1/20/2010
2.6
0.04
2.5
0.1
75.1
0.1
23.7
0.1
B
2/3/2010
2.6
0.3
2.5
0.3
74.6
0.0
23.2
0.2
C
4/14/2010
2.8
0.4
2.8
0.2
75.1
0.8
25.8
0.3
D
5/5/2010
2.7
0.1
2.7
0.0
75.3
0.2
25.6
0.1
E
7/21/2010
2.9
0.1
NA
NA
75.4
0.3
23.9
0.1
F
9/15/2010
3.0
0.1
2.8
0.1
75.2
0.2
24.0
0.1
G
11/10/2010
2.8
0.1
2.7
0.2
75.1
0.1
22.6
0.1
H
2/22/2011
2.7
0.1
2.6
0.1
58.0
0.3
23.8
0.1
I
3/8/2011
2.7
0.3
2.5
0.3
75.5
1.0
23.5
0.5
J
4/26/2011
2.7
0.0
2.8
0.1
75.6
1.8
23.8
0.1
K
5/24/2011
2.8
0.1
2.8
0.1
75.1
0.1
23.9
0.1
L
8/1/2011
2.9
0.1
2.9
0.1
75.3
0.3
24.8
0.2
M
9/21/2011
2.8
0.1
2.9
0.1
75.1
0.1
24.1
0.04
N
11/1/2011
0.7
0.1
0.6
0.0
73.8
0.4
24.1
0.1
O
2/27/2012
3.2
0.5
3.0
0.3
74.2
0.2
23.8
0.2
P
3/12/2012
2.8
0.1
2.5
0.1
75.5
0.7
23.8
0.1
Q
3/20/2012
2.9
0.1
2.8
0.5
75.2
0.4
23.8
0.1
R
7/24/2012
3.0
0.1
3.1
0.1
75.3
0.0
24.0
0.0
S
10/9/2012
2.9
2.9
2.9
0.1
75.1
0.0
23.8
0.1
T
10/30/2012
2.9
2.9
2.9
0.1
75.1
0.1
23.2
0.3
U
1/22/2013
2.9
0.1
2.7
0.1
75.0
0.0
23.7
0.2
V
3/13/2013
2.7
0.1
2.8
0.1
75.0
0.1
23.7
0.3
w
8/20/2013
2.5
0.1
2.4
0.1
75.1
0.0
22.9
0.2
X
8/27/2013
2.8
0.2
2.8
0.2
75.4
0.5
23.5
0.4
Y
9/9/2013
2.9
0.1
2.8
0.1
75.0
0.2
23.7
0.1
z
1/28/2014
5.7
0.5
5.5
0.2
73.9
2.4
23.6
0.3
AA
3/5/2014
2.8
0.1
2.8
0.1
75.4
0.5
20.5
0.7
AB
3/19/2014
1.5
0.3
2.1
0.4
74.1
2.8
23.8
0.0
*Standard deviation.
31
-------
Table 3-2. Survivability of B. atrophaeus Bis with Burdens
Low Concentration Burden
High Concentration
Burden
CIO2 pprrfhours (nominal)
5000
7000
9000
5000
7000
9000
Amino Acid cocktail (AAC)
20%
0%
20%
0%
0%
0%
Alginate (sodium salt) (ALG)
0%
0%
0%
0%
40%
0%
Carrageenan (CAR)
100%
80%
20%
100%
100%
80%
Cellobiose (CLB)
100%
100%
100%
100%
100%
100%
Dimethyl Sulfoxide (DMS)
0%
0%
0%
0%
0%
0%
Dithiothreitol (DTT)
100%
100%
100%
100%
100%
100%
Ferrous Chloride (FCL)
100%
100%
100%
100%
100%
100%
Gelatin (GEL)
100%
100%
100%
100%
100%
100%
Glutathione (GLU)
100%
100%
80%
100%
100%
100%
Humic Acid (sodium salt) (HMA)
20%
0%
0%
100%
100%
100%
NO
"ni
>
to
w)
3
-------
High Concentration Burden
Burden
120
"ni
>
100
>
3
80
CO
(/)
3
-------
Low Concentration Burden
Burden
"ro
">
3
l/l
Q.
O
E
ro
ai
15
120
100
80
60
40
20
0
4000
6000 8000
CT (ppm*hours)
10000
AAC
ALG
CAR
CLB
DMS
DTT
FCL
GEL
GLU
HMA
Figure 3-3. Survival (1000 ppm CI02) of G. stearothermophilus Bis (n=5) with Low
Concentration Burdens
Low Concentration Burden
Burden
ro
>
Q.
O
E
ro
ai
15
120
100
80
60
40
20
0
4000
6000 8000
CT (ppm*hours)
10000
AAC
ALG
CAR
CLB
DMS
DTT
FCL
GEL
GLU
HMA
Figure 3-4. Survival (1000 ppm CI02) of G. stearothermophilus Bis (n=5) with High
Concentration Burdens
3.3 Test B - Material Scoping Test
Table 3-4 shows the survival rates of B. atrophaeus and G. stearothermophilus on carrier materials.
34
-------
Table 3-4. Survival rates of Bis based on Carrier Material
B. atrophaeus Survival
Rate (%)
G. stearothermophilus
Survival Rate (%)
CI02 ppm*hours
(nominal)
5000
7000
9000
5000
7000
9000
CBD- Chipboard
0
0
0
0
20
0
CEM - CE Membranes
0
0
0
0
0
0
CER- Ceramic Tile
0
0
0
0
0
0
CRK - Adhesive Cork
0
0
0
40
20
20
CUP- C14500 Copper
0
0
0
0
0
0
FLT - Adhesive Felt
20
0
0
0
0
0
RUB- Adhesive Rubber
100
100
100
100
100
100
SST- Stainless Steel
0
0
0
0
0
0
WOD- Wooden Discs
0
0
0
20
0
0
XYZ - Porous
Polypropyl
0
0
0
40
40
0
Rubber provided complete protection to both spore types, and was thus unsuitable for further study.
Porous polypropyl (XYZ) provided partial protection to G. stearothermophilus Bis, but survival rates at
5000 ppm*hours were considered too low for follow-up. Subsequent testing was performed on stainless
steel carriers only.
All tests included control Bis (see Section 1.3.1) to validate the test methods. The performance controls, a
type of control Bl, were fumigated for the entire duration of the test, placed in extraction fluid, and then the
extraction fluid was spiked with spores of the target organism. Fumigated wooden Bis of this type would
not support growth of spiked B. atrophaeus or G. stearothermophilus spores, thereby indicating a
tendency towards false negative results. Wooden Bis would thus require different laboratory methods.
Because wood is a natural material with natural variation, and is often treated prior to consumer usage,
different laboratory methods might be necessary for each batch to avoid false negative results. Based on
these data, wood would not be a viable carrier.
Overall, the data suggest that finding a suitable carrier material to develop the target Bl successfully was
not likely, and the quest for a suitable carrier material using this approach should be discontinued. Lower
protection factors of the materials (as compared to the burden approach) and the inability to adjust the
protection were the two leading reasons for abandoning this approach. The results do suggest that
decontamination of rubber materials may be extremely difficult, as 100 % of the Bis survived all exposure
points, for both organisms tested.
35
-------
3.4 Barrier Investigation
During Test K, physical barriers instead of burdens were tested to determine if physical barriers could be
used to provide protection to spores dried in micro-titer plate wells. Five levels of inoculum, from 100
CFU/micro-titer plate to 1 x 106 CFU/micro-titer well plate were tested. Three barriers were used: one
layer of Breathe-Easy (Diversified Biotech, Dedham, MA, USA), two layers of the Breathe-Easy
membrane, and 1 layer of NuFab (DuPont, Wilmington, DE, USA, discontinued product). The negative
control samples on the Breath-Easy well plates showed growth, so results from the Breathe-Easy are not
reported due to data quality concerns. Figure 3-5 shows the survival rates for micro-titer plates protected
by the 1 layer of NuFab.
NuFab Barrier
120
100
_ 80
£
.1 60
'>
00 40
20
0
CT (ppm*hours)
Figure 3-5. Survival (1000 ppm CI02) for B. atrophaeus Protected by NuFab Barrier (Test
K) (n=8)
While lower inocula did not survive even short fumigation exposure, the higher two inocula showed some
protection by the NuFab barrier. The 1 x 106 inoculum showed the desired response curve, but complete
kill was achieved at a time point earlier than the target nine hours. Follow-up tests could not be performed
because the product had been discontinued.
3.5 COTS Bl Comparisons
None of the COTS Bis tested showed promise as an ideal Bl candidate for the purposes of this study
(i.e., inactivation following exposure to 9000 ppm*hours CI02). The sections below give detailed results.
3.5.1 COTS Yakibou B. atrophaeus Bis
The D-value evaluation of COTS Yakibou Bis (Test K) was inconclusive, with only two time points
showing fractional survival rates. All Bis survived the longest fumigation exposure time (five hours), in
contrast to previous tests and studies that showed kill points in the first few hours of exposure.
Inoculum
(CFU)
EMPTY
0.00E+00
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
2000
4000
6000
8000
10000
36
-------
3.5.2 Mesa EtO Bl
Test U included an investigation of the Mesa EtO Bl as compared to the COTS Yakibou B. atrophaeus Bl.
Survival rates are shown in Figure 3-6.
Survival Rates of B. atrophaeus Bis during
Test U
-Yakibou COTS
MESA EtO Bl
5000 10000 15000
CT (ppm*hours)
20000
25000
Figure 3-6. Survival (1000 ppm CI02) of Two COTS Bis (Test U) (n=30)
Test U suggested the Yakibou Bl was hardier than the Mesa EtO Bl. The Yakibou COTS Bl was so hardy
that it showed a 40% survival rate after 20 hours at 1000 ppmv, or 20,000 ppm*hours.
3.5.3 RCT B. atrophaeus Bis
RCT Bis performed very similarly to the stainless steel Raven Bl. The results are discussed in Section 3.7
where the CT investigation results are discussed.
3.5.4 Mesa Strip and Releasat® Bis
Three COTS Bis were fumigated during Test V. The survival rates are shown in Figure 3-7.
37
-------
Survival Rates of Various Bis during Test V
120
100
80
MesaStrip Ba Bl
Releasat ® Bl
g 60
>
*_
3
l/l
40
MesaStrip Gs Bl
20
0
1000
2000
3000
4000
5000
6000
CT (ppm*hours)
Figure 3-7. Survival (1000 ppm CI02) of 3 Commercially-Available Bl types (Test V)
(n=30)
None of the COTS Bis used in Test V are representative of the ideal Bl. Survival rates are very low at four
hours (4000 ppm*hours), whereas the ideal Bl would have a kill point close to the 9000 ppm*hour mark.
There are large differences in the ability of the Bl to survive a one-hour fumigation, with the two paper
strip Bis showing more hardiness.
3.5.5 ProLine PCD Bis
ProLine PCD Bis, fumigated in Test W, were very resistant to CI02 fumigation and may not show promise
as a surrogate Bl. Survival rates are shown in Figure 3-8.
Based on the limited data from the 6000 and 8000 ppm*hour marks, the lumens did provide some
protection to the Bl. The approach of physical barriers using lumens could be further investigated with a
less hardy organism. Interestingly, this Bl, though similar to MesaStrip BG Bis, was much hardier,
possibly due to the spore preparation. There was no difference in the behavior of the removed nozzle Bl
(see Table 2-4) and the original Bl as evaluated in Test X.
38
-------
Survival Rates of Proline Bis with various
lumen lengths
120
lumen
Length
~ Test W 0 inch
Test W 6 inch
A Test W 12 inch
20
X Test W 48 inch
0
)K Test X 0 inch
0 2000 4000 6000 8000 10000 12000 14000
CT (ppm*hours)
Figure 3-8. Survival (1000 ppm CI02) of Proline Bis (n=10) with Various Lumen Lengths
3.6 Proximity Investigation
Test L was conducted to determine if placing Bis very close together biased survivability. While the
distance between the closest Bis (0.5 mm) was much larger than the mean free path of the gas
molecules, there was some concern that a demand for the fumigant by the Bl itself or the Bl packaging
could create a localized minimum in gas concentration. Thirty (30) COTS Bis of two types (Apex BG and
Raven BG) were placed in two configurations: one that had all Bis packed closely together (0.5 mm apart)
and one that had one cm between Bis. The results are shown in Table 3-5. Figure 3-9 shows the results
of the Apex Bl.
Most Raven Bis were deactivated, even after one hour of exposure, leaving no basis for determining the
effect of the proximity of other Bis. The Apex Bis resulted in fractional kill data, which are more suited for
D-value evaluation. Survival rates at one- and two-hour exposures were very similar for both Bl
configurations, suggesting that the close proximity of other Bis did not provide any protection. D-values,
shown in Figure 3-10, also suggest no difference between the two configurations.
3.7 CT Investigation
The concentration-time (CT) investigation tested the survival rate of two types of Bis (Apex B. atrophaeus
and Raven B. atrophaeus) after exposure to CI02 at common CTs but at two fumigant concentrations,
1000 ppmv and 250 ppmv CI02. Mesa Laboratories RCT culture test kits were also tested at 250 ppmv
CI02. The exposure times and resulting CT for the two fumigations are shown in Tables 3-7 and 3-8.
39
-------
Table 3-5. Survival (1000 ppm CI02) of Apex and Raven Bis (n=30) Placed Close
(compact) and Widely Dispersed
Bl Type
Distance
Between
Bis
Hours
Exposed
No.
Surviving
Survival (%)
Maximum
Survival Rate
(%)
Paired t-test p
value
1
17
57
57
2
4
13
13
0.5 mm
3
5
17
17
4
2
7
7
5
0
0
3
Apex
6.5
1
3
3
0.14
1
16
53
53
2
4
13
13
10 mm
3
0
0
7
4
0
0
7
5
2
7
7
6.5
0
0
0
1
1
3
3
2
0
0
3
0.5 mm
3
0
0
3
4
1
3
3
5
0
0
3
Raven
6.5
1
3
3
0.50
1
1
3
3
2
1
3
3
10 mm
3
0
0
3
4
1
3
3
5
0
0
0
6.5
0
0
0
40
-------
>
'>
Survival Rates of Apex Bis Based on
Proximity
60
50
^ 40
20
10
0
2000 4000 6000
CT (ppm*hours)
8000
¦Compact - Survival
Distant - Survival
Compact - Maximum
Survival Rate
¦Distant- Maximum
Survival Rate
Figure 3-9. Survival (1000 ppm CI02) of Apex Bl (n=30) Based on Proximity to Other Bis
(Test L)
l
_ 0.8
l/>
2 0.6
0.2
D-Value for Compact and Full
Exposure Apex Bis
A
0)
= 0.4 - • Compact - Survival
Distant - Survival
2000 4000 6000 8000
CT (ppm*hours)
Figure 3-10. D-Value (1000 ppm CI02) for B. atrophaeus Bis from Proximity Investigation
(Test L)
41
-------
Table 3-6. Concentration*Time Values and Survival Rates for Test M (1000 ppmv CI02)
Bl Type
Minutes
Exposed
Nominal
ppm*hours
No.
Surviving
Survival
(%)
Maximum
Survival Rate
(%)
0
0
10
100
100
15
250
30
100
100
30
500
29
97
97
45
750
26
87
87
x
60
1000
22
73
73
0)
Q.
<
90
1500
8
27
27
120
2000
3
10
13
180
3000
3
10
13
240
4000
0
0
13
300
5000
1
3
13
360
6000
4
13
13
0
0
10
100
100
10
167
16
53
53
20
333
2
7
7
40
667
0
0
3
£
60
1000
1
3
3
0)
>
re
90
1500
0
0
3
0£
120
2000
0
0
3
180
3000
0
0
3
240
4000
0
0
3
300
5000
0
0
3
360
6000
1
3
3
42
-------
Table 3-7. Concentration*Time Values and Survival Rates for Test N (250 ppmv CI02)
Bl Type
Minutes
Exposed
Nominal
ppm* hours
No.
Surviving
Survival
(%)
Maximum
Survival Rate
(%)
60
250
30
100
100
120
500
30
100
100
Apex
180
750
22
73.3
73.3
240
1000
23
76.7
76.7
300
1250
12
40
40
360
1500
9
30
30
480
2000
5
16.7
16.7
10
42
30
100
100
20
83
28
93.3
93.3
30
125
20
66.7
66.7
Raven
60
250
6
20
20
120
500
2
6.7
13.3
180
750
1
3.3
13.3
360
1500
4
13.3
13.3
480
2000
1
3.3
3.3
10
42
30
100
100
20
83
30
100
100
30
125
30
100
100
RCT
40
167
25
83
83
60
250
10
33
33
120
500
2
6.7
6.7
240
1000
0
0
0
480
2000
0
0
0
The results of these two fumigations are shown in Figure 3-11.
43
-------
120
100
- 80
(D
>
'>
s_
3, 60
40
20
0
CT (ppm*hours)
Figure 3-11. Survival of Bl (n=30) Types at Various CT Values (Tests M and N)
These two tests showed similar response of Bl survival rates based on CT exposure for both Bl types,
tested at the two concentrations rather than exposure time. The results suggest that kill kinetics are
exposure-dependent, not concentration or time (alone) dependent. Figure 3-11 also demonstrates the
difference between different manufacturers of Bis, with Apex B. atrophaeus Bis being significantly hardier
than Bis from Raven or Mesa Laboratories (RCT). The cause of the hardiness of the Apex B. atrophaeus
Bis is unknown.
3.8 Burden Investigation
Many burdens were evaluated on B. atrophaeus and G. stearothermophilus Bis. The following sections
describe the fumigation process and the results of each burden.
3.8.1 Cellobiose
The effect of cellobiose (CLB) burden was tested on both B. atrophaeus (Tests A, D, E, F, G) and G.
stearothermophilus (Test A, D, E) Bis. Burden concentrations ranged from 0.10 % to 11.3 %.
Survival rates of the G. stearothermophilus Bis with CLB concentrations lower than 0.5 % are
shown in Figure 3-12. All higher concentrations exhibited 100 % survival rates. Not all time
points were tested at all concentrations.
Survival of COTS Bis as a function of CT at two
fumigation conditions (1000 ppm and 250 ppm
CIO2)
I v^\
\ \
"" H ^ ^ "i x "ft
Bl Type and CI02
concentration
Apex (250 ppm)
Apex (1000 ppm)
Raven (250 ppm)
Raven (1000 ppm)
RCT (250 ppm)
500
1000
1500
2000
44
-------
Survival of Cellobiose Bl
120
Cellobiosose
concentration
100 —£
80
~ 0.10% Test E
>
40
0.17% Test D
20
0.25% Test E
0
0 2000 4000 6000 8000 10000 12000
CT (ppm*hours)
Figure 3-12. Survival (1000 ppm CI02) of G. stearothermophilus Bis with CLB Burden Test
The ideal Bl would show high survival rates at five- and seven-hour exposures but poor survival rates at
nine hours. A Bl with the ideal Bl characteristics would indicate the likelihood that B. anthracis spores
survived a decontamination attempt. While CLB protected the Bl at the seven- or eight-hour mark, as
shown by the high survival rates at those time points, use of CLB did not culminate in the eventual kill by
the fumigant by the nine-hour time point. CLB burden at 0.17 % provided too much protection to Bis of
both types, with survival rates over 80 % even after 10,500 ppm*hours. G. stearothermophilus Bis with a
CLB burden could be further investigated at concentrations less than 0.10 % but did not look promising
based on the Test D results.
The survival rates of the B. atrophaeus Bis with concentrations of CLB lower than 0.25 % are shown in
Table 3-8. All B. atrophaeus Bis with CLB burdens of 0.25 % and higher showed 100 % survival rates at
all time-points.
Concentrations of CLB below 0.05% did not provide enough protection for the B. atrophaeus Bl to survive
even one hour of fumigation during Test G. The results from fumigation G indicate that CLB must be more
than 0.05 % to survive seven hours of fumigation. Test E demonstrated that 0.1 % CLB provided too
much protection, even with the inadvertent spike in CI02 concentration experienced during this test.
However, CLB Bl data from Test G are not consistent with Test E or Test F.
CLB is not recommended as a candidate burden due to the inconsistencies seen in Test G and the lack
of sensitivity to fumigation time.
D (n=30) and Test E(n=10)
45
-------
Table 3-8. Survival Rates of B. atrophaeus Bis with Less Than 0.25% CLB burden
0.005%
CLB
0.01%
CLB
0.05%
CLB
0.06%
CLB
0.07%
CLB
0.10%
CLB
0.17%
CLB
Hours
Exposure
Test F
Test F
Test G
Test F
Test G
Test G
Test G
Test E
Test D
1
0%
0%
0%
50%
20 %
40 %
20 %
100%
NA
4
NA
NA
NA
NA
NA
NA
NA
NA
100%
5
0%
0%
0%
20%
0%
0%
20 %
100%
NA
7
0%
0%
0%
30%
10%
0%
0 %
100%
NA
8
NA
NA
0%
NA
5%
0%
0 %
NA
97%
9
0%
0%
5%
10%
5%
5%
5 %
90%
100%
3.8.2 Dithiothreitol
Concentrations of dithiothreitol (DTT) used as a burden on both B. atrophaeus and G. stearothermophilus
Bis ranged from 5 mM to 250 mM.
Figure 3-13 shows the survival rates for B. atrophaeus Bis with DTT burden. The 10 mM DTT burden
exhibited nearly perfect behavior during Test E, with modest survival rates at 5000 ppm*hours and no
survival at 9000 ppm*hours. However, 20 mM of DTT provided full protection, with 100 % survival at 9000
ppm*hours.
NO
"ni
>
'>
3
CO
B. atrophaeus Bis with DTT Burden
10 mM Test E
DTT concentration
—5 mM Test C
~ 10 mM Test E
10 mM Test F
12 mM Test F
14 mM Test F
16 mM Test F
A 20 mM Test E
)( 40 mM Test E
50 mM Test E
4000
6000 8000
CT (ppm*hours)
10000
12000
Figure 3-13. Survival (1000 ppm CI02) of B. atrophaeus Bis with DTT Burden (Test C
(n=30), Test E (n=10), and Test F (n=10))
46
-------
In Test F, B. atrophaeus Bis with DTT burden showed very high survival rates (90-100 %) at all
fumigation time points, in contrast to results from Test E, which did experience a high-concentration spike
of CI02. Survival rates from 10 mM DTT B. atrophaeus Bis from the two tests are shown in Table 3-9.
Because the results of Test E were not repeated during Test F, the effect of the burden was masked by
some other unknown stronger variable, possibly the variations in fumigation conditions. DTT was not
further evaluated as a burden on B. atrophaeus Bis, but may be of interest, especially at higher CI02
concentrations.
Table 3-9. Survival Rates of 10 mM DTT on B. atrophaeus Bis (n=10)
Hours
Test E
Test F
1
100%
90%
5
30%
90%
7
0%
100%
9
0%
90%
Little protection of G. stearothermophilus Bis was offered by 5 mM DTT in Test C. All other concentrations
of DTT showed 100 % growth. Based on these results, concentrations of DTT between 5 mM and 63 mM
could be of interest as burdens on G. stearothermophilus Bis.
3.8.3 Carrageenan
Carrageenan (CAR) was used as a burden on both B. atrophaeus and G. stearothermophilus Bis at
concentrations ranging from 0.01 %-0.25 % and 0.05 % and 1 % respectively.
The survival rates from G. stearothermophilus Bis with CAR burden are shown in Figure 3-14. The seven-
hour time point seems to be an outlier. However, poor survival rates after one hour of fumigation and non-
linear response to fumigation time indicate this Bl should not be included for further study.
Figure 3-15 shows the survival rates for CAR B. atrophaeus Bis. As discussed earlier, the Test A results
showed promise. However, Test C was not consistent with Test A, and the Test E results are counter-
intuitive, with survival rates of nine-hour exposure higher than survival rates of one-hour exposure.
Nonetheless, survival rates did generally vary as a function of burden concentration. These results could
be indicative of the effects of post-exposure handling, unusual sensitivity to variations in fumigation
conditions, or protection of the Bl by a high-concentration burst of CI02 (as experienced early in the Test
E fumigation).
Indeed, the Test E results are so counterintuitive, that they are presented in Figure 3-16, assuming an
inadvertent switch of one-hour and nine-hour samples during laboratory evaluation. Evaluation of these
results indicates that CAR may be a very promising burden, with a concentration between 0.1 % and 0.25
%. Due to the lack of confidence in the CAR data, this Bl was not further evaluated. Future studies may
consider further investigation of the 0.1 % and 0.25 % CAR Bl.
47
-------
G. stearothermophilus Bis with CAR Burden
CAR burden
concentration
0.050 %
0.075 %
0.100%
0.125%
0.250 %
2000
4000 6000
CT (ppm*hours)
10000
Figure 3-14.Survival (1000 ppm CI02) of G. stearothermophilus Bis (n=10) with CAR
Burden (Test E)
NO
"ni
>
">
3
CO
120
100
B. atrophaeus Bis with CAR Burden
CAR Concentration
and Test
—~—0.01 % Test E
-A—0.03 % Test E
)( 0.05 % Test E
0.08 % Test E
0.10% Test E
0.125% TestC
0.25% Test E
0.25% Test A
1.0 % Test A
2000 4000 6000 8000
CT (ppm*hours)
10000
12000
Figure 3-15. Survival (1000 ppm CI02) for B. atrophaeus Bis with CAR Burden from Test C
(n=30), Test E (n=10) and Test F (n=10)
48
-------
NO
~ro
>
'>
3
CO
120
100
80
60
40
20
B. atrophaeus Bis with CAR Burden - Modified
CAR Concentration
and Test
—~—0.01 % Test E
-A—0.03 % Test E
X 0.05 % Test E
0.08 % Test E
0.10% Test E
0.125% TestC
0.25% Test E
0.25% Test A
1.0 % Test A
2000 4000 6000 8000
CT (ppm*hours)
10000
12000
Figure 3-16. Reinterpretation of Figure 3-15
3.8.4 Glutathione
Test A demonstrated that glutathione (GLU) could provide protection to both B. atrophaeus and G.
stearothermophilus Bis. The lower concentration (25 mM) provided full protection at seven hours and 80
% protection at nine hours for Test A. As a follow-up, Test C at a lower concentration of 5 mM on both Bis
was tested, with the supposition that the lower concentration would provide less protection. However,
GLU Bis of both species had 100 % survival rates after nine hours of exposure.
GLU Bis also showed high variability and insensitivity to fumigation time (see Table 3-10). For these
reasons, GLU was not considered a candidate burden for subsequent tests.
3.8.5 Gelatin
Table 3-11 shows the concentrations of gelatin (GEL) that were tested as burdens on both B. atrophaeus
and G. stearothermophilus Bis. Replicate concentrations have been shaded.
49
-------
Table 3-10. Survival Rates of Test C B. atrophaeus and G. stearothermophilus Bis with
5 mM GLU Burden
Exposure
Time
(Hours)
B. atrophaeus Bl
G. stearothermophilus
Bl
% Surviving
% Surviving
4.0
93
100
8.0
87
83
8.5
77
93
9.0
97
93
9.5
97
100
10.0
97
100
10.5
100
100
Table 3-11. Tested Concentrations of GEL as a Burden on B. atrophaeus and G.
stearothermophilus Bis
Test A
Test D
Test E
Test F
Test G
Test H
Test I
Both Bis
B. atrophaeus Bis only
2.5 %
0.1 %
0.25 %
1.6 %
1.0 %
0.8 %
0.8 %
10.0%
1.0 %
0.50 %
1.7 %
1.5 %
0.9 %
0.9 %
0.75 %
1.8 %
1.6 %
1.0 %
1.0 %
1.00 %
1.9 %
1.7 %
1.25 %
2.0 %
1.50 %
2.00 %
As discussed in Section 3.2 gelatin provided full protection during Test A, allowing nearly full
survival at 2.5 %. Successive tests were conducted with lower concentrations of GEL.
Figure 3-17 shows the results of G. stearothermophilus Bis with GEL burden. Increasing the
GEL concentration from 0.1 % to 1 % did not increase the survival of the G. stearothermophilus
Bl, though during Test A these Bis with 2.5 % GEL showed complete protection (100 % growth).
The GEL G. stearothermophilus Bl is indicative of a sigmoidal Bl and is not recommended for
further study. Complete kill was never achieved for G. stearothermophilus GEL Bis, even after
10,000 ppm*hours.
50
-------
G. stearothermophilus Bis with GEL burden
120
100
80
GEL Concentration
60
0.1% GEL
40
¦ —*—1.0 % GEL
0
0 2000 4000 6000 8000 10000 12000
CT (ppm*hours)
Figure 3-17. Survival (1000 ppm CI02) of G. stearothermophilus Bis (n=30) with GEL
Burden (Test D)
As seen in Table 3-12, both 0.1 % and 1.0 % GEL provided too little protection to B. atrophaeus
Bis.
Table 3-12. Survival Rates GEL B. atrophaeus Bis (Test D)
Hours
0.1%
GEL
1.0%
GEL
4.0
0 %
20 %
8.0
0 %
7 %
8.5
0 %
3 %
9.0
0 %
0 %
9.5
0 %
10 %
10.0
0 %
0 %
10.5
0 %
0 %
51
-------
Figure 3-18 shows the promise of GEL as a burden on B. atrophaeus Bis. From Test A, 2.5 % GEL
provided too much protection (100 % survivability), while Test D suggested 1.0 % was too low (20 %
survival after just four hours). Taken within the context of this test alone, GEL is very promising: a nice
correlation of survivability to concentration at one hour and strong protection up to seven-hour exposure.
However, combining the results from Test E and Test D suggests variability between the two data sets.
Two sources of variability are changes in fumigation conditions and changes in manufacturer. The
survival rates of some GEL B. atrophaeus Bis are shown in Table 3-13. The GEL Bl results from Test G
were consistent with Test F, but not consistent with Test E.
"S
>
¦>
120
100
80
60
40
20
0
B. atrophaeus Bis with GEL Burden
2000
GEL
concentration
—~—0.25 %
—A—0.50 %
—*—0.75 %
1.00 %
1.25 %
1.50 %
2.00 %
4000 6000
CT (ppm*hours)
8000
10000
Figure 3-18. Survival (1000 ppm CI02) of B. atrophaeus Bis (n=10) with GEL Burdens (Test
E)
Table 3-13. Survival Rates of Gelatin B. atrophaeus Bis for Some Concentrations
1.0% GEL
1.5% GEL
1.6% GEL
1.7% GEL
Test
Hours
Exposure
D
E
G
H
I
G
E
G
F
G
F
1
NA
10 %
100%
100%
100%
100%
20%
100%
100%
100%
100%
5
20%
(four hour
exposure)
0%
80%
100%
100%
100%
0%
100%
100%
100 %
100%
7
NA
0%
100%
100%
100%
100%
0%
100%
100%
100%
100%
8
7%
NA
85%
100%
97%
100%
NA
100%
NA
100%
NA
9
0%
0%
35%
100%
100%
100%
0%
100%
100%
100%
100%
52
-------
Figure 3-19 shows a graphical representation of the survival rates of a single Bl (1.0 % gelatin on B.
atrophaeus Bis) over a range of fumigations. As indicated in Table 3-13, there was a wide range of
responses for this Bl. As discussed earlier, there was a spike in CI02 concentration during Test E, which
could explain the lower survival rates, but the remaining fumigations had no known significant anomalies.
Each manufactured batch of 1.0 % GEL Bis behaved differently (Test H and Test I were manufactured on
the same date, from the same spore lot).
Survival Rates for 1% GEL B. atrophaeus
Bis
4 6 8
Hours Exposure to 1000 ppmv CI02
Test ID
¦~—Test D
Test E
¦A—Test G
*-Test H
Test I
Figure 3-19. Survival (1000 ppm CI02) of 1.0% GEL B. atrophaeus Bis from Test D (n=30),
Test E (n=10), Test G (n=5), Test H (n varies between 5 and 30), and Test I (n
varies between 5 and 30)
Test H and Test I B. atrophaeus Bis with GEL burden showed high survival rates (90% or higher) at all
fumigation conditions. GEL Bis did not produce repeatable results, though the cause of variability is
unclear.
3.8.6 Casein
Casein (CSN) was used as a burden on B. atrophaeus Bis for 19 tests starting with Test E.
Concentrations tested ranged from 0.1 % to 10 %.
Figure 3-20 shows survival rates for B. atrophaeus Bis with CSN burden from Test E. This new burden
showed promise, providing full protection at 10 % and partial protection at lower concentrations.
Interestingly, the presence of the CSN on the Bl resulted in encapsulated bacterial growth during the first
days of growth, preventing the broth from becoming cloudy but creating a visible bubble on the surface of
the Bl. Nonetheless, CSN was chosen for follow-up testing in Test F, which was designed to pinpoint a
concentration of CSN that might better approximate the 9,000 ppm*hours kill point.
53
-------
120
B. atrophaeus Bis with CSN Burden
ra
*_
3
l/l
100
80
£ 60
40
20
2000 4000 6000
CT (ppm*hours)
8000
CSN
concentration
—~—0.1%
-*—10%
10000
Figure 3-20. Survival (1000 ppm CI02) of B. atrophaeus Bis (n=10) with CSN Burden (Test
E)
Figure 3-21 displays the survival rates of the B. atrophaeus Bis burdened with CSN from Test F. As in
Test E, CSN clearly demonstrates an ability to protect the spores, allowing growth at conditions that
inactivate the unburdened Bl. In general, there was a dose-dependent response to increased
concentration of CSN. While the 1.0 % shows a nearly perfect response, the 100 % growth at seven
hours seems an outlier taken in the context of the 40 % growth rate of the Bl with 1.2 % CSN. Also visible
in Figure 3-21 is the fact that increased protection of the Bl reduces the ability to reach complete kill
conditions after 9000 ppm*hours.
Table 3-14 shows the survival rates of B. atrophaeus with selected concentrations of CSN burden across
several fumigations, demonstrating the variability encountered between different batches of Bis and
between different fumigations. Figure 3-22 shows the survival rates of 1.0 % casein Bis for five different
fumigations. Tests H and I were of the same batch of Bis.
54
-------
Test F B. atrophaeus Bis with CSN burden
"to
">
1-
3
l/l
120
100
4000
6000 8000
CT (ppm*hours)
10000
CSN
concentration
0.10%
0.25 %
0.50 %
0.75 %
1.00%
1.20 %
12000
Figure 3-21. Survival (1000 ppm CI02) of B. atrophaeus Bis (n=10) with CSN Burden (Test
F)
55
-------
Table 3-14. Survival Rates of B. atrophaeus Bis with CSN Burdens
0.8 %
CSN
0.9 % CSN
1.0% CSN
1.1% CSN
Hours
Exposu
re
%
%
%
%
%
%
%
%
%
%
%
%
Survivi
Survivi
Survivi
Survivi
Survivi
Survivi
Survivi
Survivi
Survivi
Survivi
Survivi
Surviv
ng Test
ng
ng Test
ng Test
ng
ng
ng
ng
ng
ng
ng
ing
G
Test G
H
I
Test E
Test F
Test G
Test H
Test I
Test G
Test H
Test I
1
100
100
100
100
100
100
100
100
100%
100%
100%
100%
5
40
0
100
60
60
90
80
100
100%
80%
100%
100%
7
10
50
100
50
0
100
40
100
100%
70%
100%
100%
8
0
0
100
17
NA
NA
0
100
100%
0%
100%
100%
9
0
0
100
17
20
10
0
100
100%
0%
100%
97%
56
-------
120
100
80
"ro
60
*_
3
40
l/l
20
0
Variability in Survival Rates of
B. atrophaeus Bis with 1% CSN
2000 4000 6000 8000
CT (ppm*hours)
10000
12000
—*—1.0%
CSN
1.0%
CSN
SO
0s-
o
t-H
t
CSN
1.0%
CSN
-©- 1.0%
CSN
Figure 3-22. Variability in Survival Rates of B. atrophaeus Bis with 1.0% Casein
Table 3-15 shows the fumigation conditions associated with the Bl survival curves, shown in Figure 3-22,
as well as some additional fumigations to be discussed in Section 3.9. Table 3-15 also shows the
correlation between the survival rate of the 1% Casein B. atrophaeus Bl and some fumigation conditions.
The strongest correlation is with CI02 concentration, even though the difference between the highest and
lowest average concentration is less than 10%. Furthermore, the highest survival rates occur at the
lowest concentrations, which suggest coincidence rather than correlation. The correlation in the
ppm*hours belies the true source of variation. For instance, it would seem that the lower CT at the 9 hour
mark of Test H and I could have contributed to the high survival rate, but the 8-hour exposure during Test
G, for instance, had a CT of 8136 ppm*hours, and still had a 0% survival rate. Variations in fumigation
conditions do not seem to be the source of variations in survival rates.
57
-------
Table 3-15. Fumigation Conditions for the 9-hour Exposure of Multiple Tests
9 Hour Exposure
Test E
Test F
Test G
Test H
Test 1
Test P
Test Q
Test R
Test S
Test T
Survival Rate
Correlation
(Pearson
correlation
coefficient - r)
Average CI02
(mg/L)
2.9
3.0
2.8
2.7
2.7
2.8
2.9
3.0
2.9
2.9
-0.78
ppm*hours
9345
9566
9113
8968
8665
94823
9805
9746
9285
93234
-0.70
Max CI02 (mg/L)
10.8
3.1
3.1
2.9
3.5
2.9
8.0
3.1
3.1
3.2
-0.14
Average RH (%)
75.4
75.2
75.1
58.0
75.5
75.5
75.2
75.0
75.1
75.1
-0.64
Max RH (%)
77.8
76.2
76.1
59.0
81.1
77.7
76.9
75.3
75.6
75.7
-0.44
Average
Temperature (°C)
23.9
24.0
22.6
23.8
23.5
23.8
23.8
23.7
23.8
23.2
0.18
Max Temp (°C)
24.0
24.4
23.0
23.9
24.2
24.0
24.0
24.0
24.0
24.2
0.19
Survival Rate of
1.0% CSN
B. atrophaeus Bl
20 %
10 %
0 %
100 %
100 %
15 %
15 %
20 %
5 %
10 %
58
-------
While the 1.0 % casein B. atrophaeus Bl shows promise, variability was a problem as evidenced by the
high survival rates in Test H and Test I. Additional tests discussed in Section 3.9 were conducted to
identify (and remove) preventable sources of variability.
3.9 Variability Investigation
A number of additional fumigations and tests were performed to better understand the variability resulting
from the Bl tests. The majority of these tests were performed using B. atrophaeus Bis with and without
CSN burden.
3.9.1 Spore Preparations
Tests O, P and Q were designed to investigate whether the source of Bl variability might arise from
unavoidable variations in fumigation conditions or from unavoidable variations in spore preparations. A
single batch of Bis for all three tests was prepared from two different sources of spores, Raven and
Yakibou. Other than the source of the liquid inoculum, Bis were prepared identically by Yakibou.
Test O was aborted early due to CI02 generator error, so only the five hour time point was valid. The
results are shown in Figure 3-23.
Survival was aided with increasing CSN concentration. Surprisingly, the Raven spore preparation was
more resistant to fumigation than the Yakibou spore preparation, in contrast to the Raven B. atrophaeus
Bis, which had typically been less resistant to fumigation than Yakibou Bis (Sections 3.5 and 3.6).
Test O Survival Rates
120
100
_ 80
£
I 60
">
^ 40
20
0
0.8 0.85 0.9 0.95 1 1.05 1.1 1.15
Casein Concentration (%)
Figure 3-23. Survival (5000 ppm*hours, 1000 ppm CI02) of Two Spore Preparations (n=10)
as a Function of CSN Concentration (Test O)
-Yakibou
Raven
59
-------
Figures 3-24 and 3-25 shows the survival rates for Test P Bis with casein burden with Yakibou and
Raven spore preparations, respectively.
120
100
so"
80
"ni
>
60
>
3
CO
40
20
0
Test P Survival Rates for Yakibou
Spore Bis
2000 4000 6000
CT (ppm*hours)
8000 10000
CSN
Concentration
0.85 % CSN
0.90 % CSN
0.95 % CSN
1.00 % CSN
1.05 % CSN
1.10% CSN
Figure 3-24. Survival (1000 ppm CI02) of Yakibou Spore Bis (n=20) with CSN Burden (Test
P)
Test P Survival Rates for Raven Spore
Bis
120
100
so"
80
"S
>
60
>
3
l/l
40
20
0
2000 4000 6000
CT (ppm*hours)
8000
10000
CSN
Concentration
0.85 % CSN
0.90 % CSN
0.95 % CSN
1.00 % CSN
1.05 % CSN
1.10% CSN
Figure 3-25. Survival (1000 ppm CI02) of Raven Spore Bis (n=20) with CSN Burden (Test
P)
60
-------
Again, the Raven spore preparation was hardier than the Yakibou spore preparation. The nine-hour time
point for the Yakibou spores showed unexpected behavior, with higher concentrations ofCSN burden
demonstrating lower survival rates than lower concentrations. Test Q survival rates were similar to Test P.
These results suggest differences in spore preparation procedures may have a significant impact on Bl
survival. Such results draw into scrutiny the utility of Bis for evaluating fumigation efficacy, as subtle
differences in between-batch or between-vendor spore preparation conditions could significantly alter the
outcome of these indicators.
D-values were calculated using the Most Probable Number method proposed by Stumbo [4], The D-
values for Test P and Test Q are shown in Figure 3-26.
D-values for Test P and Test Q Bis
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
* ft 1 1
0.8 0.9 1 1.1
CSN Concentration (%)
1.2
Spore Source
and Test ID
~ Raven Test Q
Yakibou Test Q
X Raven Test P
A Yakibou-Test P
Figure 3-26. Calculated Average D-Values for Test P and Test Q Bis
Average D-values of Yakibou Bis are inelastic to CSN concentration, whereas increasing burden
concentration increases D-values for the Raven Bis. The inelasticity of the Yakibou Bis is contrary to
previous Tests E, F, and G.
Figure 3-27 shows the calculated D-values of Test P as a function of exposure time. The D-values of a Bl
with linear response should be the same at all time points. A negative slope of D-value over time (sloping
down) is indicative of a time lag or a shoulder. Such a Bl would have no response to fumigation until after
a certain minimum exposure has been reached. The Bis in Figure 3-27 have a D-value overtime with a
positive slope. Similar to the findings of Rastogi et al (2010) [1], these data suggest that kill curves are not
first order reactions and that the survivors are the result of tailings.
61
-------
D-Values as a Function of Exposure Time (Test P)
1.50
1.40
1.30
1.20
"wT
0 1.10
-C
0)
"I 1.00
>
1
~
0.90
0.80
0.70
0.60
3456789 10
Exposure Time (Hours)
Figure 3-27.Calculated D-Values of Yakibou Bis per Exposure Time
Tests O, P, and Q demonstrate that spore preparations can react quite differently to similar fumigation
conditions and can contribute to the survival rates of an organism or Bl. There are no publicly available
differences in the spore preparation procedures used by different laboratories to explain different survival
rates. However, spore preparations have been shown by other researchers to affect resistance. Young
and Setlow [4] determined that "spores prepared at higher temperatures were more resistant". Bloomfield
and Arthur [5] showed that both spore coat and the cortex affects the resistance of B. subtilis spores to
chlorine-releasing agents. Future investigations could help develop protocols which would remove this
variability.
However, spore preparation does not account for all of the variability, perhaps best shown in a
comparison of Test U and Test V. Survival rates are shown in Figure 3-28.
5?
Burden
o>
LO
00
0
1
6 CSN
-¦-0.90 °/
6 CSN
-A-0.95 °/
6 CSN
o>
O
o
t-H
t
6 CSN
—*—1.05 °/
6 CSN
o>
0
1
rH
1
6 CSN
62
-------
Survival Rates of Various Bis during Test U and
Test V
120
100
80
"to
">
3
l/l
60
40
20
Burden and
Test ID
-1% CSN Test V
0 % CSN Test V
-1% CSN
0% CSN
5000 10000 15000
CT (ppm*hours)
20000
25000
Figure 3-28. Survival (1000 ppm CI02) of Test U and Test V Bis (n=30)
The Bis used for Test U and V were prepared on different dates approximately five months apart, but with
the same stock solution. The difference in response of the 0% casein is remarkable. Some unknown
difference in the preparation of the Bis may account for the moderate survival rates even after long
fumigations.
3.9.2 Effect of Vortex Mixing Bis during Analysis Procedures
During incubation, some burdens would form a layer which would encapsulate the Bl growth. One cause
of the variability could be the difficulty in recognizing the growth of a Bl when the encapsulation was
happening. Test J included Bis that were vortex-mixed, using the highest setting on the vortex mixer,
before plating the seven-day incubated broth in addition to Bis that were analyzed with the previously
used procedure without vortex mixing. The number of Bis per type varied between two and five replicates
based on availability. The results, shown in Table 3-16, suggest that the use of vortex mixing did not
systematically affect survival rates.
63
-------
Table 3-16. Effect of Vortex Mixing on Survival Rate Determination
B. atrophaeus Bis with GEL Burden
B. atrophaeus Bis with CSN Burden
Survival Rate (%)
Survival Rate (%)
Bl ID
Vortex
Mixed
Not Vortex
Mixed
Bl ID
Vortex
Mixed
Not Vortex
Mixed
GEL-025-286+V-7-01
0
0
CSN-010-239+V-7-01
0
0
GEL-050-286+V-7-01
0
0
CSN-010-286+V-7-01
33
0
GEL-075-286+V-7-01
0
0
CSN-050-239+V-7-01
0
0
GEL-100-117+V-7-01
100
33
CSN-050-239+V-9-01
0
0
GEL-100-286+V-7-01
0
0
CSN-090-117+V-5-01
0
20
GEL-200-239+V-9-01
100
100
CSN-090-117+V-7-01
0
0
CSN-090-117+V-9-01
0
0
CSN-100-117+V-5-01
100
100
CSN-100-117+V-7-01
100
100
CSN-100-117+V-9-01
100
100
CSN-120-117+V-7-01
100
100
CSN-120-117+V-9-01
100
100
CSN-120-239+V-7-01
0
0
CSN-120-239+V-9-01
0
0
CSN-1000-286+V-9-01
100
100
3.9.3 Effect of RH
The effect of RH during fumigation was not in the original test matrix, but an operator error led to an RH of
60 % in the fumigation chamber for Test H. While all B. atrophaeus and G. stearothermophilus Bis
survived the lower RH fumigation, some Bis did not survive the fumigation at 75 % RH (Table 3-17). The
survival rates for the higher RH test are shown in Figure 3-29 and Figure 3-30 for the GEL burden and
CSN burden Bis, respectively. These results may corroborate previous reports [6, 7, 8] of the sensitivity of
Bis to RH during CI02 fumigation.
64
-------
120
100
so"
80
o\
"ni
>
60
>
3
CO
40
20
0
Survival Rate of B. atrophaeus Bis with GEL
Burden at 75 % RH
Burden
¦0 % GEL
0.8% GEL
0.9% GEL
¦1% GEL
2000 4000 6000
CT (ppm*hours)
8000
10000
Figure 3-29. Survival (1000 ppm CI02) of B. atrophaeus Bis (n =30) with GEL Burden at
75% RH* (Test I)
* All Bis survived fumigation at 60 % RH
Survival Rate of B. atrophaeus Bis with CSN
Burden at 75 % RH
2000 4000 6000
CT (ppm*hours)
8000
10000
Burden
Concentration
0% CSN
0.9% CSN
1% CSN
1.05% CSN
1.1% CSN
1.15% CSN
1.2% CSN
Figure 3-30. Survival (1000 ppm CI02) of B. atrophaeus Bis (n=30) with CSN Burden at
75% RH* (Test I)
* All Bis survived fumigation at 60 % RH
65
-------
Table 3-17. Survival of Bis after 60% RH and 75% RH Fumigations
Survival
Survival
Burden
%
Concentration
Hours at
1000 ppm
cio2
(%) after
75% RH
fumigation
(Test I)
(%) after
60% RH
fumigation
(Test H)
1
0
0
0.00
5
0
0
7
0
0
9
0
0
1
100
100
5
100
100
0.80
7
100
100
8
90
100
Gelatin
9
100
100
1
100
100
5
100
100
0.90
7
100
100
8
100
100
9
90
100
1
100
100
5
100
100
1.00
7
100
100
8
97
100
9
100
100
1
0
0
0.00
5
0
0
7
0
0
9
0
0
1
100
100
0.90
5
60
100
7
50
100
8
17
100
9
17
100
CSN
1
100
100
1.00
5
100
100
7
100
100
8
100
100
9
100
100
1
100
100
1.05
5
100
100
7
100
100
8
100
100
9
83
100
1.10
1
100
100
66
-------
Burden
%
Concentration
Hours at
1000 ppm
cio2
Survival
(%) after
75% RH
fumigation
(Test I)
Survival
(%) after
60% RH
fumigation
(Test H)
5
100
100
7
100
100
8
100
100
9
97
100
1
100
100
1.15
5
100
100
7
100
100
8
100
100
9
97
100
1
100
100
1.20
5
100
100
7
100
100
8
100
100
9
100
100
3.9.4 Quantitative Analysis
Tests R, S, T, U and V were conducted to quantify the spores remaining after fumigation and to correlate
D-values calculated from qualitative results to quantitative log reductions (LRs). Test R included Bis that
were analyzed in two ways. For each time point and Bl type, half were analyzed qualitatively (Section
2.4.1) like most Bis in this study, and the other half were analyzed quantitatively (Section 2.4.3) to
determine the number of spores generating the "growth" result. Figure 3-31 shows the quantitative results
from Test R. Figure 3-32 shows the same data, but the quantitative data are interpreted as Growth/No
Growth and presented in the same format of "Survival (%)' as many of the other figures in this text. For
example, any replicate with one or more detected viable spores would be interpreted as "Positive Growth"
for that replicate. The percentage of replicates with >1 CFU is therefore reported as "Survival (%)".
67
-------
Quantitative Results from Test R
Fumigation
ii
-a
ai
*_
ai
>
o
<
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
1.00E-01
Burden
Concentraion
—~—0 % CSN
0.5 % CSN
—A— 1.0% CSN
2000 4000 6000 8000
CT (ppm*hours)
10000 12000
Figure 3-31.Quantitative Analysis of CFU (n-5) following 1000 ppm CI02 fumigation (Test
R)
Qualitative Analysis of Quantitative
Bis - Test R
120
100
NO
80
"ni
>
60
>
3
40
CO
20
0
Burden
Concentration
—~—0 % CSN
—A— 0.5 % CSN
1.0% CSN
2000 4000 6000 8000
CT (ppm*hours)
1 1
10000 12000
Figure 3-32.Qualitative Interpretation of Figure 3-31 Quantitative Results
As expected, unprotected B. atrophaeus Bis (0% CSN) did not survive fumigation conditions longer than
three hours. As seen in Figure 3-31, the unprotected spores in 0% CSN B. atrophaeus Bis also
demonstrated the expected kill curve or decay rate. While the spores are being killed on the Bl, until
complete kill (inactivation of all spores on the Bl), the Bl itself would still present a "Positive Growth" result
if analyzed qualitatively. As a comparison to Figure 3-32, Figure 3-33 shows the survival rate of the Bis
that were analyzed qualitatively.
68
-------
Qualitative Analysis of Bis - Test R
120
100
Burden
Concentration
3 60
0 % CSN
>
3
CO
0.5 % CSN
40
1.0% CSN
20
0
2000 4000 6000 8000 10000 12000
CT (ppm*hours)
Figure 3-33. Test R Qualitative Results from Qualitative Bis (n=5)
The quantitative results allowed both D-value methods (Sections 1.4.1 and 1.4.2) to be calculated for Test
R. The D-values, calculated by each of the methods, are shown in Figure 3-34.
2.00
-5- 1.50
3
O
Q 0.50
0.00
D-values of Test R Bis
X
x
T*
i
—i—
4
Fumigation Time (Hours)
X
10
Burden Concentration
and D-value method
AO % CSN SMC
¦ 0.5% CSN SMC
~ 1% CSN SMC
X0 % CSN Quantitative
0.5 % CSN Quantitative
X1 % CSN Quantitative
Figure 3-34.D-Values (1000 ppm CI02) of B. atrophaeus Bis versus Fumigation Time (Test
R)
69
-------
Figure 3-34 shows the sigmoidal response of the Bis with the strong relationship of the D-value to
fumigation time. Figure 3-34 also shows no sensitivity to the concentration of the CSN burden, but does
show a good correlation of the two methods of D-value estimation.
The similarities of Figure 3-32 and Figure 3-33, and the similar values for D-values as shown in Figure
3-34, suggest that both qualitative and quantitative Bl methods can be used to assess the efficacy of a
fumigation, suggesting in turn that the survival rate of qualitatively analyzed Bis can hinge on the
presence or absence of very few spores. Designing a Bl and predicting the response of 99 % or even
99.9 % of the spores on the Bl is rather easy, but predicting the response of just a few protected spores
with special circumstances is exceedingly difficult. This second population of resilient spores drives the
Growth/No Growth response.
3.9.5 Fumigation Repeatability (Tests S, T, U and V)
Tests S and T were originally designed to be triplicate fumigations of the same set of Bis, designed to
detect differences in D-value and survival rates of the Bis due to variations in fumigations. Only two of
these tests were performed; the third test was performed for longer exposure times. Like Test R
described in Section 3.9.4, this test included Bis to be analyzed both quantitatively and qualitatively.
Unlike some of the previous differences in survival rates of burdened and unburdened Bis from previous
fumigations, the results of these two replicate fumigations were very similar. Figures 3-29 and 3-30 show
a comparison of Bl results between the two fumigations.
Survival Rates of Test S and Test T
B. atrophaeus Bis
120
Burden
Concentration
—Test S - 0 % CSN
—Test T -1 % CSN
)K Test S -1 % CSN
Test T - 0 % CSN
and Test ID
0
3000 6000 9000 12000
CT( ppm*hours)
Figure 3-35. Survival (1000 ppm CI02) from Test S and Test T Bis (n=20)
70
-------
2.00
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
D-value (hours) of 1 % CSN B. atrophaeus Bl
i "
x
~
¦
X
X
x
X
—I—
10
Test ID and D-value
method
~ Tests Quantitative D-value
Test S SMC Analysis
XTestT Quantitaitive D-value
A Test T SMC Analysis
12
Hours at 1000 ppm CI02
Figure 3-36.Two D-Value Methods of 1 % CSN B. atrophaeus Bis from Two Fumigations
The positive slope of D-values over time again suggests that the survival curve of the 1 % casein Bl has a
tailing. Survival rates at long fumigation times are driven by a subset of very resistant spores. This subset
of spores may possess an intrinsic resistance to fumigation, or may be protected due to location or
proximity to other spores.
Tests U and V investigated longer fumigation times (up to 20 hours) to determine the length of the tailing.
The survival rates of 1% casein Bis over the four tests are shown in Figure 3-37.
71
-------
Survival Rates of 1 % CSN
B. atrophaeus Bl
120
100
S? 80
60
40
20
0
5000 10000 15000
CT (ppm*hours)
20000
Test ID
-Test S
TestT
Test U
-Test V
25000
Figure 3-37.Survival (1000 ppm CI02) of 1% Casein Bis (Qualitative Analysis)
While there are some obvious differences in the survival rates, the Tests S and U showed a lag time of
greater than one hour and Tests S, T, and U all had survival rates under 10 % after ten hours. In contrast,
the Test V batch of Bis had a time lag less than one hour and had a tailing with survival rates near 20 %
for fumigation times between eight and twenty hours. Differences in production may have more of an
effect than differences in fumigation parameters
3.9.6 Effects of Spore Population Density
A series of tests (Tests W, X and Y) was performed to determine if the tailing effects were due to a
relatively small number of surviving spores protected by clumping or some other mechanism due to the
large spore population on the Bl. This series of tests used two populations of B. atrophaeus (1.2 x 103
and 1.1 x 105 CFU) rather than the approximately 2 x 106 population inoculated onto previous tests.
Figure 3-38 shows the survival rates of the lowest inoculum Bis for all three fumigations.
72
-------
Test W 1 x 103 B. atrophaeus Bis
120
100
% Casein
80
0%
60
1%
40
to
2%
20
5%
0
0
2000
4000
6000
8000
CT (ppm*hours)
10000
12000
14000
Test X 1 x 103 B. atrophaeus Bis
120
Test Y 1 x 103 B. atrophaeus Bis
120
100
% Casein
—0%
60
1%
2%
20
5%
2000
4000
6000
8000
CT (ppm*hours)
10000
12000
14000
Figure 3-38.Survival (1000 ppm CI02) of 103CFU Inoculum B. atrophaeus Bis (n=10)
73
-------
A clear relationship between survival rates and casein concentration can be seen in Figure 3-38. Higher
burden concentrations impart increased chances for survival by increasing the time lag. These data do
not suggest that the burden also changes the D-value once the time lag has been met, though more data
points would be necessary to fully test that hypothesis. Again, Bis with a 1% - 2% casein burden show
promise, with resistance to CI02 fumigation for four to six hours, and with high probability of deactivation
at nine hours. Such results may warrant further investigation of this Bl.
A second batch of the low inoculum Bis were produced for the CT investigation described in Section
3.9.7. The survival rates for Test AA are shown in Figure 3-39. This second batch of low inoculum Bis
had a response similar to the prior batch.
Test AA 1 x 103 B. atrophaeus Bis
*_
iTi 40
% Casein
0%
1%
2%
2000 4000 6000 8000 10000 12000 14000
CT (ppm*hours)
Figure 3-39. Survival (1000 ppm CI02) for Low inoculum Bis (n=20) in Test AA
While further testing is needed, the 1 % and 2 % casein Bis with a 1 x 103 inoculum could be an excellent
model for B. anthracis spores. Side-by-side testing of the two species could provide information for a
confidence model, including guidance on the number of replicates needed and whether one concentration
or a combination of both burden concentrations offers a better prediction.
Figure 3-40 shows the response of Bis with 100x more spores (i.e., 1x105 B. atrophaeus) in the original
inoculum (see Figure 3-38). Clearly, the higher inoculum levels provide a greater chance for survival.
Interestingly, the unburdened Bis in some cases had higher survival rates than Bis with CSN. Behavior of
these Bis was not unlike previous tests with the higher 2 x 106 inoculum.
74
-------
Test W 1 x 105 B. atrophaeus Bis
120
100
80
2000 4000 6000 8000
CT (ppm*hours)
10000 12000
14000
% Casein
—~—0%
—2%
-*-5%
Test X 1 x 105 B. atrophaeus Bis
2000 4000 6000 8000
CT (ppm*hours)
10000
12000
14000
Test Y 1 x 105 B. atrophaeus Bis
a-
120
100
ST 80
60
2000 4000 6000 8000
CT (ppm*hours)
10000
12000
% Casein
0%
1%
2%
5%
% Casein
0%
1%
2%
5%
14000
Figure 3-40.Survival (1000 ppm CI02) of 10s CFU Inoculum B. atrophaeus Bis (n=10)
75
-------
The W. X, and Y fumigations were not of sufficient duration to determine if tailings might be more likely
with the higher inoculum, though tailings with the higher inoculum are certainly suggested with the 0%
casein results.
3.9.7 CT Investigation of Low Inoculum
Tests Z, AA, and AB used the low inoculum Bis discussed in Section 3.9.6 at three CI02 concentrations,
500 ppm, 1000 ppm, and 2000 ppm. For these tests, exposures were targeted towards the same CT for
the different concentrations. The target times and the actual CTs are listed in Table 3-18.
Table 3-18. Target and Actual CT Exposure for Tests Z, AA, and AB
Target CT
(ppm*hours)
2000 ppm
1000 ppm
500 ppm
Test Z
Test AA
Test AB
Exposure
Time
(hours)
Actual CT
(ppm*hours)
Exposure
Time
(hours)
Actual CT
(ppm*hours)
Exposure
Time
(hours)
Actual CT
(ppm*hours)
6000
3
6470
6
5980
12
5840
9000
4.5
9720
9
8980
18
9410
12000
6
13070
12
12070
24
13350
Most of the B. atrophaeus Bis with no burden or 1 % casein burden were deactivated by the 6000
ppm*hour CT for all three fumigations. The survival rate of the 2 % casein Bl is shown in Figure 3-41,
plotted against both CT and fumigation time.
The response of this Bl to CT exposure looks similar at 2000 ppm and 1000 ppm, but does not look
similar for the 500 ppm fumigation. The long fumigation times required to reach target CT at 500 ppm
proved more effective than the shorter fumigation times at the two higher concentrations, possibly related
to increased permeability of the spore coat by extended exposure to high RH, as there seemed to be no
benefit to raising the CI02 concentration from 500 ppm to 1000 ppm at a 12 hour fumigation time. This is
not to say that the Bis were insensitive to increased concentration, and the 2000 ppm fumigation
conditions provided a similar decontamination efficacy in a shorter amount of time.
The material coupons (See Section 2.1.6) that were included in this test series responded similarly to the
2% casein Bis. Table 3-19 shows the CFU recovered from coupons from Test AA and Test AB. The
longer fumigation at 500 ppm was more effective than the shorter fumigation at 1000 ppm, though both
were effective at providing a 6 LR for at least some coupon types.
76
-------
Survival Rate of 2 % CSN B. atrophaeus
Bl
120
100
so"
80
"ni
>
60
>
3
CO
40
20
2000 ppm
A 1000 ppm
X500 ppm
X X/ X
5000 10000 15000
CT (ppm*hours)
Survival Rate of 2 % CSN B. atrophaeus
Bl
120
100
so"
80
"ni
>
60
>
3
CO
40
20
0
~
X V X
I 1 1 7\ 1 1 1
5 10 15 20 25 30
Exposure (hours)
2000 ppm
A 1000 ppm
X500 ppm
Figure 3-41. Survival of 2 % CSN B. atrophaeus Bl (n=20) at Three CI02 Concentrations
(Test Z, AA, and AB)
77
-------
Table 3-19. Recovery (CFU) from Test AA and Test AB Coupons
CT
(ppm*hours)
0
6000
9000
12000
Aluminum
6.03E+06
ND
43
50
500
Carpet
4.87E+06
73
ND
ND
ppm
Wood
6.99E+06
ND
ND
ND
Aluminum
2.41E+07
109
10
ND
1000
Carpet
6.98E+06
316
113
164
ppm
Wood
6.96E+06
6
7
ND
Carpet coupons were the most difficult to decontaminate, with spores surviving a 12-hour fumigation at
1000 ppm CI02 (12,000 ppm*hours).The carpet coupons did not show growth after an 18-hour fumigation
at 500 ppm (9,000 ppm*hours).
3.9.8 Age of B!
Test J was conducted with a constricted test matrix due to the limited availability of Bis remaining from
prior tests. Bis of various batches and ages were subjected to the same fumigation (1000ppm, 75% RH,
25°C) during Test J to identify any effect of age on Bl survival rates. For all Bis, survival rates following
fumigation of aged Bis were lower than rates from the original fumigation. Figure 3-42 shows the change
in the survival rates as a function of the age of Bis with various concentrations of CSN burden. A change
of 100% means that 100% of the Bis survived the original fumigation (when new) and 0 % of the same
batch survived the fumigation when old.
120
a>
+¦»
(0
100
DC
"ai
80
">
60
3
to
40
c
-------
In general, there was a change in survival rates for CSN-burdened Bis, but there does not appear to be a
trend with respect to the age of the Bl, suggesting that Bis with burdens may have a relatively stable shelf
life, though the number of variations, including differences in burden concentration, fumigation conditions,
and Bl batches, lessen the confidence in this conclusion. No age-related trends were detected in either
gelatin (GEL) or CSN B. atrophaeus Bis.
The Test WXY series also permitted triplicate fumigations of the same batch of Bis. Fumigation conditions
are shown in Table 3-20.
Table 3-20. Fumigation Conditions for Low Inoculum Tests
Test W
Test X
Test Y
Average RH (%)
75.1
75.4
75.0
SD RH
0.0
0.5
0.2
Average Temp
(°C)
22.9
23.5
23.7
SD Temp
0.2
0.4
0.1
Average mSM
4500-CI02 (mg
cio2/l)
2.5
2.8
2.9
Average
Photometer (mg
cio2/l)
2.4
2.8
2.8
6 Hour ppm*hours
5410
6150
6290
D-values (Table 3-21) were calculated for all Bis that demonstrated partial survival; D-values cannot be
calculated for conditions where all Bis show growth or all Bis show no growth. The Pearson correlations
between the D-values and fumigation conditions were calculated and are shown in Table 3-22.
Table 3-21. D-Values (hours) for Bl Types during Low Inoculum Tests
Bl
Test
W
Test X
TestY
1 % CSN 102 CFU
0.96
1.47
2.27
2% CSN 102CFU
2.09
2.06
2.71
0 % CSN 104CFU
1.01
1.52
1.81
1 % CSN 104CFU
1.06
1.27
1.71
79
-------
Table 3-22. Correlation between D-Values of Selected Bis and Fumigation Conditions
Bl
RH
corr
T
corr
4500
corr
EMS
corr
ppm*hours
corr
Age of Bl
corr
1 % CSN 102 CFU
-0.16
0.90
0.82
0.72
0.87
1.00
2 % CSN 102 CFU
-0.57
0.62
0.49
0.36
0.58
0.92
0 % CSN 104 CFU
0.12
0.99
0.95
0.89
0.98
0.95
1 % CSN 104 CFU
-0.23
0.87
0.78
0.67
0.84
1.00
Corr = Correlation.
The strongest correlation with D-value was the age of the Bl, with increasing resistance to fumigation as
the Bl aged on the shelf. Unlike Test J, this series was conducted with much shorter shelf lives, on the
order of weeks rather than months. Coincidentally, the actual fumigation concentration also increased
with increasing age of the Bl. Further testing should be conducted before drawing conclusions about shelf
stability, though shelf stability would be essential for a custom Bl.
There was no change in D-value for the series of Test S, Test T, and Test U as a result of age.
80
-------
4 Quality Assurance
This project was performed under two approved Category III Quality Assurance Project Plans (QAPP)
titled Decontamination Process Indicators. Part 1 - Biological Indicators. Part 2 - Process Parameter
Correlations (December 2009) and Part III - Determination of the Effect of Spores Storage Time on
Susceptibility to Inactivation by CI02 (September 2010)..
4.1 Sampling, Monitoring, and Analysis Equipment Calibration
Documented operating procedures were used for the maintenance and calibration of all laboratory and
NHSRC RTP Microbiology Laboratory equipment. All equipment was certified by the manufacturer as
calibrated or had the calibration verified by EPA's Air Pollution Prevention and Control Division (APPCD)
on-site (RTP, NC) Metrology Laboratory prior to use. Standard laboratory equipment such as balances,
pH meters, BSCs and incubators were routinely monitored for proper performance. Calibration of
instruments was done at the frequency shown in Table 4-1. If deficiencies were noted, the instrument was
adjusted to meet calibration tolerances and recalibrated within 24 hours. If tolerances were not met after
recalibration, additional corrective action was taken, including recalibration and/or replacement of the
equipment.
Table 4-1. Sampling and Monitoring Equipment Calibration Frequency
Equipment
Calibration/Certification
Expected Tolerance
Thermometer
Compare to independent NIST thermometer (this is a
thermometer that is recertified annually by either
NIST or an International Organization for
Standardization (ISO)-17025 facility) value once per
quarter
± 1°C
Stopwatch
Compare against NIST Official U.S. time at
http://nist.time. qov/timezone.cqi?Eastern/d/-5/iava
once every 30 days.
± 1 min/30 days
Clock
Compare to office U.S. Time (3). time.aov everv 30
days.
± 1 min/30 days
pH meter
Compare to NIST-traceable buffer solutions daily
± 0.2 units
Micropipettes
All micropipettes will be certified as calibrated at time
of use. Pipettes are recalibrated by gravimetric
evaluation of pipette performance to manufacturer's
specifications every year.
± 5%
BSC
The BSC will be verified to be within certification dates
at the time of use. BSC are adjusted yearly to be within
flow tolerances established by the manufacturer.
± 10%
Titration Equipment
Titration equipment and reagents will be calibrated
weekly against a known standard solution of 1000
ppmv chlorite.
± 15%
Scale
Compare reading to Class S weights
± 1%
The metering device used for CI02 extractive sample collection was calibrated annually by the APPCD
Metrology Laboratory.
81
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4.2 Data Quality
The data quality objectives (DQOs) of this project are three-fold:
• Collect data to permit development of a custom Bl that matches B. anthracis response to
fumigation. (Part 1)
• Collect data to examine the sensitivity of a fumigation process to variability in environmental
factors, specifically temperature and relative humidity. (Part 2)
• Collect data to determine the effect of Bl storage time on susceptibility to inactivation by CI02.
(Part 3)
The objective of this project was to develop a custom Bl that would provide reliable results of No Growth
after fumigation with 9000 ppm*hours CI02, while at the same time providing Growth results at fumigation
conditions unlikely to deactivate B. anthracis spores. This section discusses the QA/QC checks (Section
4.3) and Acceptance Criteria for Critical Measurements (Section 4.4) considered critical to accomplishing
the DQOs.
4.3 QA/QC Checks
Uniformity of the test materials was a critical attribute for assuring reliable test results. Uniformity was
maintained by obtaining a large enough quantity of material that multiple material sections and carriers
could be constructed with presumably uniform characteristics. Samples and test chemicals were
maintained to ensure their integrity. Samples were stored away from standards or other samples which
could cross-contaminate them.
Supplies and consumables were acquired from reputable sources and were NIST-traceable when
possible. Supplies and consumables were examined for evidence of tampering or damage upon receipt
and prior to use, as appropriate. Supplies and consumables showing evidence of tampering or damage
were not used. All examinations were documented and supplies were appropriately labeled. Project
personnel checked supplies and consumables prior to use to verify that they met specified task quality
objectives and did not exceed expiration dates.
Quantitative standards do not exist for biological agents. Quantitative determinations of organisms in this
investigation did not involve the use of analytical measurement devices. Rather, CFU were enumerated
manually and recorded. QC checks for critical measurements/parameters are shown in Table 4-2.
Acceptance criteria (see Section 4.4) were set at the most stringent level that could be routinely achieved.
Positive controls and procedural blanks were included along with the test samples in the experiments.
Other background checks were also included as part of the standard protocol. Replicate Bis were
included for each set of test conditions. Operating procedures were performed by qualified, trained and
experienced personnel were used to ensure data collection consistency. The confirmation procedure,
controls, blanks, and method validation efforts were the basis of support for biological investigation
results. If necessary, training sessions were conducted by knowledgeable parties, and in-house practice
runs were used to gain expertise and proficiency prior to initiating the research.
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Table 4-2. QA/QC Sample Acceptance Criteria
QC Sample
Information Provided
Acceptance Criteria
Corrective Action
Negative Control Bl
(coupon or Bl without
biological agent)
Controls for sterility of
materials and methods
used in the procedure.
No observed CFU
Reject results, identify and
remove source of
contamination.
Positive control
(Bl or inoculated material
not fumigated)
Shows ability of incubation
tubes to support and show
growth
Growth.
Reject results. Identify and
correct problem.
Turbidity Control
(Bl with material or burden,
not inoculated but
fumigated)
Provides information about
the tendency towards false
positives of candidate Bl
Both outcomes were
accepted.
Materials or burdens which
show growth were rejected
for subsequent study.
Performance Control (Bl
with burden spiked after
fumigation)
Provides confirmation that
the fumigated burden is
compatible with bacterial
growth
Growth
Reject burden.
Blank tryptic soy agar
Sterility Control
(plate incubated, but not
inoculated)
Controls for sterility of
plates.
No observed growth
following incubation.
All plates are incubated, so
any contaminated plates
were discarded.
In addition, Appendix C contains the DTRL - QC Checklist for Data Reviewers, which was used to review
the data presented in this report.
4.4 Acceptance Criteria for Critical Measurements
DQOs are used to identify the critical measurements needed to address the stated objectives and specify
tolerable levels of potential errors associated with simulating the prescribed decontamination
environments. The following measurements were deemed to be critical to accomplish the project
objectives:
• Real-time fumigant concentrations
• Temperature
• RH
• Fumigation time sequence
• Determination of spore survival for selected samples
The Data Quality Indicators (DQIs) listed in Table 4-3 are specific criteria used to quantify how well the
collected data met the DQOs. The accuracy of the real-time CI02 monitors as assessed with respect to
the mSM 4500-CI02 Methods. Precision of the EMS real-time CI02 monitor could not be accessed due to
unavailability of a constant-concentration source and the feedback nature of their operation in this specific
testing. The accuracy of the extractive methods was assessed using standards of known concentration.
RH sensors were compared to a calibrated standard humidity sensor and a standard saturated salt
83
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solution producing a 75 % RH atmosphere. Failure to provide a measurement method or device that met
these goals resulted in the rejection of results derived from the critical measurement. For instance, if the
plated volume of a sample was not known (i.e., is not 100% complete), then that sample was deemed
invalid.
Table 4-3. Accuracy and Completeness DQIs for Critical Measurements
Measurement Parameter
Analysis Method
Accuracy
Detection
Limit
Completeness
%
Real-time CIO2
concentration inside the test
chamber (high concentration
tests)
ClorDiSys EMS monitor
(0.1 -30 mg/L)
15% of
mSM-4500-E
0.1 mg/L
36 ppmv
100
Extracted CIO2, high
concentration
mSM 4500-CI02
5% of
Standard
solution
0.1 mg/L
(solution)
90
RH
RH probes (0-100%)
± 5 % of 75%
standard salt
solution
NA
95
Differential time
Computer clock
1 % of
reading
0.5 sec
95
Temperature inside the test
chamber
Thermocouple
± 2 C
NA
90
Bacterial Growth
NHSRC RTP
Microbiology Laboratory
MOP 6560. 6566
Categorical
(presence or
absence)
1 CFU
99
Table 4-4 lists how well the critical measurements collected during testing met the completeness criteria
described in Table 4-3.
84
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Table 4-4. Completeness of DQIs
Test ID
Real-time CI02
Concentration
Inside the Test
Chamber
Extracted
cio2
RH
Differential
Time
Temperature
Inside the
Test
Chamber
DQI Completeness Goals
100%
90%
95%
95%
90%
Test A
100.0
0
100
100
100
Test B
90.0
0
100
100
100
Test C
90.2
0
100
100
100
Test D
92.8a
0
100
100
100
Test E
0.0
0
100
100
100
Test F
100.0
0
100
100
100
Test G
97.2
0
100
100
100
Test H
97.0
100
0
100
100
Test I
94.5
100
100
100
100
Test J
97.9
100
100
100
100
Test K
100.0
100
100
100
100
Test L
99.9
0
100
100
100
Test M
100.0
0
100
100
100
Test N
51.7
0
100
100
100
Test O
95.3
100
100
63.7
100
Test P
74.1
0
100
100
100
Test Q
87.4
0
100
100
100
Test R
100.0
0
100
100
100
Test S
100.0
0
100
100
100
Test T
99.4
0
100
100
100
Test U
94.9
0
100
100
100
Test V
100.0
0
100
100
100
Test W
100.2
100
100
100
100
Test X
98.1
0
100
100
100
Test Y
99.8
0
100
100
100
Test Z
100
0
100
100
100
Test AA
100
0
100
100
100
Test AB
10.0b
0
100
100
100
aThe unavailability of primary photometer data records required the real-time CI02 concentration data recorded by the CI02
generator (secondary photometer) be used.
b During Test AB, approximately eight hours into the 24 hour exposure, the primary photometer began to fail, resulting in 10%
completeness for the category
In most instances, when the real-time CI02 concentration measurement did not meet the completeness
goal, fluctuations with the ClorDiSys primary photometer were the cause. However, in Test E, a ClorDiSys
EMS monitor malfunction resulted in 0% completion of the real-time CI02 concentration DQI. A CI02
generator data file was also unavailable for this test. Extracted CI02 samples were therefore used to
85
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monitor the CI02 concentration. Also, screenshots of the real-time plot generated by the CI02 generator
provide a graphical representation of CI02 concentrations throughout exposure time. Records for the
validation of the titration equipment could not be found for many tests
The completeness for the Test H RH was 0 % because the RH during the exposure time was lower than
intended. Since the acceptance criteria for precision were met, the data set was used as a low relative
humidity test for comparison with the other relatively high RH tests.
The DQIs for bacterial growth are determined by the presence of turbidity associated with the growth of
target microorganisms within TSB media. Every TSB media tube is visually inspected prior to the addition
of any Bl and is inspected again 7-9 days after a Bl has been aseptically transferred to a sterile TSB tube,
and the tube has been allowed to incubate at the temperature most favorable for growth of the target
microorganism. Visual inspection of the tubes, both before and after the addition of the Bl samples, is 100
%. The accuracy and ability to detect and allow for growth and proliferation of the target organism in the
TSB media (to determine the presence or absence of viable microorganisms) is 1 CFU.
Further, all TSB media tubes that were found to be negative for turbidity (growth) were subjected to
homogenization by either inversion of the sample tube or by vortex mixer and were directly plated (either
by sterile loop or pipette) to confirm the absence of growth (especially by the target microorganism). Also,
10 % of all samples that were turbid upon visual inspection and therefore indicative of growth were plated
(either by sterile loop or pipette) to confirm that the growth was consistent with the colony morphology for
the target microorganism. MOPs 6566 and 6566 rev 1 were followed to complete the analytical methods
for processing the Bl samples.
The quantitative acceptance criteria were associated with targeted setting conditions in the test chambers
during the entire exposure time. These acceptance criteria are listed in Table 4-5.
Table 4-5. Precision Acceptance Criteria for Critical Measurements
Measurement Parameter
Analysis Method
Precision
RSD (%)
Real-time CIO2 concentration inside the
test chamber
ClorDiSys EMS monitor
(0.1-30 mg/L)
+ 10%
Extracted CIO2 inside the test chamber
mSM 4500-CI02
± 15%
RH inside the test chamber
RH probes (0-100%)
± 15%
Temperature inside the chamber
Thermistor
O
0
C\J
+l
Incubator temperature
Type K thermocouple
0
0
C\J
+l
Refrigerator temperature
Type K thermocouple
± 2 °C
Plated media (incubated before
inoculation)
Visual
NA
Microbiological material blank
Visual
NA
Positive Control Bis
Visual
NA
86
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Table 4-6 details the precision of the critical measurements for each test.
Table 4-6. Observed Precision of Critical Measurements
Test ID
Real-time CI02
Concentration
Inside the Test
Chamber
Extracted CI02,
High
Concentration
RH
Temperature
Inside the Test
Chamber
Precision RSD Acceptance Criteria Goals
±10%
±15 %
±15 %
l+
ro
o
O
Test A
3.8
1.5
0.1
0.8
Test B
12.4
14.2
0.1
1.2
Test C
5.6
13.8
1.1
2.7
Test D
1.7
3.6
0.3
1.6
Test E
NA
2.2
0.4
0.2
Test F
2.0
3.0
0.2
1.5
Test G
6.6
5.1
0.2
0.5
Test H
5.4
4.3
0.5
2.6
Test 1
13.6
11.7
1.4
2.7
Test J
3.5
6.4
2.4
2.6
Test K
3.1
3.5
0.1
2.9
Test L
3.8
3.7
0.4
0.6
Test M
14.7
2.5
1.2
1.2
Test N
35.8
13.4
1.9
1.1
Test O
16.4
15.7
0.3
1.0
Test P
4.6
2.9
0.9
0.5
Test Q
17.8
2.9
0.5
0.5
Test R
3.3
2.2
0.3
0.4
Test S
3.5
3.2
0.1
0.4
Test T
3.2
5.5
0.1
1.5
Test U
5.4
4.6
0.1
1.2
Test V
3.0
2.9
0.1
1.9
Test W
5.0
5.8
0.1
0.4
Test X
6.5
7.6
0.6
5.4
TestY
8.9
3.5
1.3
0.6
Test Z
3.4
9.1
3.3
1.1
Test AA
3.3
4.6
0.3
1.4
Test AB
20.1
32.1
3.3
0.3
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During Test E, all Bis experienced a spike in CI02 concentration to 6.6 mg/L at the beginning of
this fumigation. They were tested at 1, 5, 7 and 9 hours. The primary photometer failed to report
data, so the only photometer data available are manually recorded values.
The RH during Test H was incorrectly controlled due to operator error, leading to an average RH of 60%.
Several anomalies were encountered during the Test I fumigation due to equipment failure, leading to a
spike of nearly twice the target concentration.
The photometer reading of the GMP chlorine dioxide generator during Test O was erratic, leading to the
cancellation of the test and higher than anticipated exposure.
Instances when the real-time CI02 concentration measurement was not within QA specifications can be
attributed to mechanical failures which, in some cases, were resolved by performing regular equipment
maintenance. The majority of the tests that did not meet the real-time CI02 concentration inside the test
chamber precision requirement met the precision requirement for extracted CI02, further indicating
mechanical failure for the real-time measurements that did not meet the acceptance criteria. The two
instances (i.e. Test O and Test AB) where both the real-time and extracted CI02 measurements are
indicative of a poorly controlled fumigation which should be considered when examining the results. The
temperature measurements for consecutive tests, Test H, Test I, Test J and Test K, were only slightly
outside of the precision requirement.
As before mentioned, data from both photometers were unavailable for Test E. Therefore, a value for
precision could not be applied
Test N had a target CI02 concentration of 250 ppm. However, the operating range for the CI02 generator
is ± 35 ppm, and the generator is not optimized to control CI02 levels below 300 ppm. As a result, the
acceptance criteria for the CI02 concentration tests were not met.
Plated volume critical measurement goals were 100 % completion. All pipettes are calibrated yearly by an
outside contractor (Calibrate, Inc., Carrboro, NC, USA) and verified gravimetrically at the conclusion of
testing.
Plates were quantitatively analyzed (CFU/plate) using a manual counting method. For each set of results
(per test), a second count was performed on 25 % of the plates with significant data (data found to be
between 30-300 CFU). All second counts were found to be within 10 %of the original count.
Many QA/QC checks are used to validate microbiological measurements. These checks include samples
that demonstrate the ability of the NHSRC RTP Microbiology Laboratory to culture the test organism, as
well as to demonstrate that materials used in this effort do not themselves contain spores. The checks
include
• Field blank samples: sterile Bis or coupons sampled at the same time as inoculated Bis or
coupons.
88
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• Laboratory Media and Supplies: includes all materials, individually, used by the NHSRC RTP
Microbiology Laboratory in sample analysis.
4.5 Data Quality Audits
This project was assigned QA Category III and did not require technical systems or performance
evaluation audits.
4.6 QA/QC Reporting
QA/QC procedures were performed in accordance with the QAPP for this investigation.
4.7 Amendments to Original QAPP
All amendments to the original QAPP were submitted by e-mail to the EPA QA officer for formal approval.
89
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5 Summary
Biological indicators (Bis) have often been used to indicate the efficacy of a sterilization technique,
especially in cases such as fumigations where distribution may not be uniform. Many COTS Bis and
modifications to Bis were tested under this multi-year investigation for their suitability to determine the
efficacy of a CI02 fumigation for B. anf/?rac/'s-contaminated building materials. These modifications
included chemical burdens, changes in coupon material (carriers), and physical barriers. Based on
previous data, a kill point of 9,000 ppm*hours exposure to CI02 gas was used as the target exposure to
model B. anthracis spore kill on building material surfaces. No modification was identified capable of
achieving a repeatable and precise (± 500 ppm*hours) Bl deactivation at 9000 ppm*hours, but not before.
However, results from tests investigating the sources of variance may be used to focus future
decontamination and sterilization research.
Burdens can have the effect of increasing survival rates and D-values of Bis. Burdens that seemed to
increase survival rates of seven-hour fumigations and yet did not provide protection such that all Bis
survived nine-hour fumigations included cellobiose, dithiothreitol, carrageenan, gelatin, and casein, all of
which could be further evaluated. Most promising was 1 % casein as a burden on low inoculum (103 CFU)
B. atrophaeus Bis. Results were variable, however, with large variations between batches and
fumigations due to unidentified factors seemingly most related to production. Such results draw into
question the utility of Bis for evaluating fumigation efficacy, as subtle differences in between-batch or
between-vendor spore preparation conditions could significantly alter the outcome of these indicators.
While coupon materials did affect the survival rates of Bis, none of the carriers showed promise, providing
either too much or too little protection. Unlike burdens, carriers could not be tested at different
concentrations, therefore making it more difficult to adjust kill points. Fumigated wooden carriers would
not support growth of the target organism and were therefore not a suitable carrier material. Carriers
made of rubber were highly resistant to inactivation by CI02, suggesting this material may be difficult to
decontaminate.
Either semi-permeable barriers or lumens can be used as physical barriers, and types of both were
demonstrated to extend the survival rates of spores. More research should be conducted on semi-
permeable membranes, as incorporation of membranes into Bl manufacture would be easy to implement.
Bis incorporating tortuous paths such as lumens should also be further investigated.
Various COTS Bis were investigated, including B. atrophaeus Bis from Apex Laboratories, Raven Labs,
and three variations from Mesa Laboratories. Some were more hardy and some were less hardy than the
target 9000 ppm*hourfull kill. Moreover, the spore preparation was found to have an impact on spore
survival rates. Because spore preparations exhibit this variability, the behavior of Bis can fluctuate from
batch to batch, though there is also variability between batches using the same spore preparation, which
suggests some other production factor may be causing the variation in survival rates. Future research
should focus on removing variation from spore preparations or focus on species that are more easily
destroyed, thus removing the significance of spore variation. Regardless, the apparent variability in kill
points amongst COTS Bis suggests that Bl variability may be unavoidable and therefore to some degree
acceptable.
90
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Two COTS Bis (Apex and Raven stainless steel B. atrophaeus) behaved very similarly on a
concentration*time (CT) basis for two fumigations at two different fumigant concentrations (250 ppmv and
1000 ppmv CI02). This similar behavior is promising because field conditions in an actual event are
expected to affect the maximum concentration any one fumigation technology can meet, and others have
demonstrated that B. anthracis spore kill is more dependent upon the product of concentration and time
(CT) than either concentration or time alone. Further, the spacing (distance between) of Bis within the
exposure chamber did not affect kill kinetics in the current study. This is another important finding that
can inform field-use of Bis.
Consistent with previous reports, the current study found that D-values are not linear over the duration of
the fumigation. Many studies [9, 10, 11] have found curvilinear responses to chemical sterilizations, rather
than a generally linear response to thermal sterilizations. Many Bis resilient enough to survive a seven-
hour fumigation would also tend to survive a nine-hour fumigation. Put another way, a subpopulation of
spores on a Bl may have higher resistance than the main population, due either to a protective location or
inherent hardiness, producing a biphasic response with most spores deactivated in an early portion of the
sterilization cycle and a subset deactivated in a much later portion of the cycle. While a Bl with very hardy
spores may predict the behavior of bacterial spores in an actual event, a Bl with overly hardy spores may
also falsely predict the negative outcome of what in reality was a successful fumigation. One possible
explanation of hardy spores was the protective bio-burden of clumping in high-inoculum Bis. Lower
inoculum Bis were tested and showed a trend of lower survival rates. Further testing was conducted on
these lower inoculum Bis, suggesting a lower tendency towards the long-surviving tail and an increasing
resistance with increasing casein burden.
This work identified several techniques to allow Bis to survive longer fumigation durations. These
techniques may be used to tune a Bl to better model the inactivation of any target organism. To produce
a good model of Bacillus anthracis with CI02 fumigations, the authors would recommend side-by-side
comparisons of Bacillus anthracis to Bis with low inoculum and 1 % and 2 % casein burden.
Testing with COTS Bis showed that perceived issues (tailing effects) during custom Bl developmental
tests are not uncommon, as such effects are observed for COTS Bis as well. This effect has been
observed during Bl exposure to gaseous fumigation technologies, and may be less prevalent in
heat/steam-based sterilization technologies such as autoclaving (personal communication, Joe Dalmasso
- Yakibou Labs, Inc.). Tailing effects can have significant implications for Bl use during real-world
decontaminations, where Bis may be used as partial evidence to suggest fumigant effectiveness against
an infectious agent. Designing a Bl and predicting the response of 99 % or even 99.9 % of the spores on
the Bl is rather easy, but predicting the response of just a few protected spores with special
circumstances is exceedingly difficult. This second population of resilient spores drives the Growth/No
Growth response.
91
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References
1. Rastogi, V.K., et a I., Systematic Evaluation of the Efficacy of Chlorine Dioxide in Decontamination of
Building Interior Surfaces Contaminated with Anthrax Spores. Applied and Environmental
Microbiology, 2010. 76(10): p. 3343-3351.
2. Bartram, P.W., J. T. Lynn, L. P. Reif, M. D. Brickhouse, T. A. Lalain, S. Ryan, B. Martin, and D.
Stark, Material demand studies: interaction of chlorine dioxide gas with building materials U.S.
Environmental Protection Agency, 2008. EPA/600/R-08/091.
3. S.A.I.C., Compilation of available data on building decontamination alternatives.. U.S.
Environmental Protection Agency, 2005. EPA 600-R-05-03.
4. Stumbo, C.R., A technique for studying resistance of bacterial spores to temperature in higher range.
Food Tech., 1948. 2: p. 228.
5. Young, S.B. and P. Setlow, Mechanisms of killing of Bacillus subtilis spores by hypochlorite and
chlorine dioxide. Journal of Applied Microbiology, 2003. 95(1): p. 54-67.
6. Bloomfield, S.F. and M. Arthur, Interaction of Bacillus subtilis spores with sodium hypochlorite, sodium
dichloroisocyan urate and chloramine-T. J Appl Bacterid, 1992. 72(2): p. 166-72.
7. Jeng, D.K. and A.G. Woodworth, Chlorine Dioxide Gas Sterilization under Square-Wave
Conditions. Appl Environ Microbiol, 1990. 56(2): p. 514-9.
8. Agalloco, J.P.C., Frederick J., Validation of Pharmeceutical Processes. 3rd ed. 2007.
9. Fujikawa, H. and T. Itoh, Tailing of thermal inactivation curve of Aspergillus niger spores. Applied
and Environmental Microbiology, 1996. 62(10): p. 3745-3749.
10. Head, D.S., et al., Effects of superheated steam on Geobacillus stearothermophilus spore viability.
Journal of Applied Microbiology, 2008. 104(4): p. 1213-1220.
11. Rajan, S., et al., Inactivation of Bacillus stearothermophilus spores in egg patties by pressure-assisted
thermal processing. Lwt-Food Science and Technology, 2006. 39(8): p. 844-851.
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Appendix A: Miscellaneous Operating Procedures
MOP 6535a Serial Dilution: Spread Plate Procedure to Quantify Viable Bacterial Spores
MOP 6560 Biological Indicator (Bl) Tests using Nutrient Broth and Analysis of Results
MOP 6562 Preparing Pre-Measured Tubes with Aliquoted Amounts of Phosphate Buffered
Saline with Tween20 (PBST)
MOP 6566 Culturing of Apex Laboratories Tyvek Packaged Biological Indicators (Rev 0)
and
Culturing Biological Indicator Strips (Rev 1)
MOP 6570 Use of STERIS Amsco Century SV 120 Scientific Prevacuum Sterilizer
MOP 6576 Determination of Spore Thermal Challenge (Heat Shock) Resistance
-------
MOP 6535a
Revision 4
January 2013
Page 1 of 8
Miscellaneous Operating Procedure (MOP) 6535a:
Serial Dilution: Spread Plate Procedure to Quantify Viable
Bacterial Spores
Prepared by:
IS Work Assignment Leader
Reviewed by:
Nicole Gnllin Ga
Date: 2/11/2013
Dahman Towkti, ARCADlS-PrCjectManager
Date: 2/11/2013
' /
Approved by:
Worth Calfee, EPA Work Assignment Manager
Date: 2/11/2013
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
a
ftKUVUD
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
-------
MOP 6535a
Revision 4
January 2013
Page 2 of 8
MOP 6535a
TITLE:
SCOPE:
PURPOSE:
Materials:
• Liquid suspension of bacterial spores
• Sterile centrifuge tubes
• Diluent as specified in QAPP or Test Plan (e.g., sterile water, Phosphate Buffered Saline with
Tween 20 (PBST))
• Media plates as specified in QAPP or Test Plan (e.g., Trypticase Soy Agar (TSA) plates)
• Microliter pipettes with sterile tips
• Sterile beads placed inside a test tube (used for spreading samples on the media surface
according to MOP 6555 {Petri Dish Media Inoculation Using Beads) or cell spreaders
• Vortex mixer
1.0 PROCEDURE (This protocol is designed for 10-fold dilutions.)
1. For each bacterial spore suspension to be tested label microcentrifuge tubes as follows: 10"1,
10"2, 10"3, 10"4, 10"5, 10"6... (The number of dilution tubes will vary depending on the
concentration of spores in the suspension). Aseptically, add 900 uL of sterile diluent to each
of the tubes.
2. Label three media plates for each dilution that will be plated. These dilutions will be plated in
triplicate.
3. Mix original spore suspension by vortexing thoroughly for 30 seconds. Immediately after the
cessation of vortexing, transfer 100 uL of the stock suspension to the 10"1 tube. Mix the 10"1
tube by vortexing for 10 seconds, and immediately pipette 100 uL to the 10"2 tube. Repeat
this process until the final dilution is made. It is imperative that used pipette tips be
exchanged for a sterile tip each time a new dilution is started.
SERIAL DILUTION: SPREAD PLATE PROCEDURE TO QUANTIFY
VIABLE BACTERIAL SPORES
Determine the abundance of bacterial spores in a liquid extract
Determine quantitatively the number of viable bacterial spores in a liquid
suspension using the spread plate procedure to count colony-forming units (CFU)
4. To plate the dilutions, vortex the dilution to be plated 10 seconds, immediately pipette 100
uL of the dilution onto the surface of a media plate, taking care to dispense all of the liquid
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MOP 6535a
Revision 4
January 2013
Page 3 of 8
from the pipette tip. If less than 10 seconds elapses between inoculation of all replicate
plates, then the initial vortex mixing before the first replicate is sufficient for all replicates of
the sample. Use a new pipette tip for each set of replicate dilutions.
5. Carefully and aseptically spread the aliquotted dilution on the surface of the media either by
use of glass beads (MOP 6555) or cell spreader (the method used may be directed in the
QAPP or Test Plan) until the entire sample is distributed on the surface of the agar plate.
Repeat for all plates.
6. Incubate the plates for the optimum time period at the optimum growth temperature for the
target organism (incubation conditions will vary depending on the organism's optimum
growth temperature and generation time. This information can be found in Bergev's Manual
of Determinative Bacteriology or it will be provided with the ATCC certification.
7. Manually enumerate the colony forming units (CFU) on the media plates by manually
counting with the aid of a plate counting lamp and a marker (place a mark on the surface of
the Petri dish over each CFU when counting, so that no CFU is counted twice). A hand held
tally counter or an electronic counting pen may be used to assist the person counting, but
may not be used as the primary source for the count.
Quality control (QC) requirements for bacterial enumeration will be addressed per QAPP or
test plan. However, in general, the following QC practices should always be adhered to:
a. The arrangement of plates and tubes, and the procedure for preparing dilutions and
enumerating CFU should be done the exact same way each time. This helps prevent
systematic errors and often helps determine the cause of problems when a discrepancy is
found.
b. A visual check of the graduated pipette tip should be made during each use to ensure the
pipette is pulling properly.
c. Samples should acclimate to room temperature for 1 hour prior to plating.
d. Samples should be processed (extracted and plated) from the least contaminated to the
most contaminated.
e. When a target range of CFU is known, three dilution factors are plated to bracket the
expected results (0,-1, and -2, if the -1 dilution factor was the target).
f. Enumerated colonies and results should be verified that the results are the target
organism, and that second counts have been performed. Second counts must be
completed on 25% of significant data, and must be within 10% of the first count. If CFUs
are found to have more than a 10% difference between first and second counts, then a
third count is to be completed.
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MOP 6535a
Revision 4
January 2013
Page 4 of 8
g. Pictures should be taken of any plates that are contaminated or have results out of the
normal
8. Record all quantitative data in the "Serial Dilution/Plating Results Sheet". Target range for
statistically significant counts is 30-300 CFU. Data that fall out of the 30-300 CFU range are
addressed in MOP 6584 (Procedure for Replating Bacteria Spore Extract Samples) and MOP
6565 (Filtration and Plating of Bacteria from Liquid Extracts).
2.0 CALCULATIONS
Total abundance of spores (CFU) within extract:
(Avg CFU / volume (mL) plated) x (1 / tube dilution factor) x extract volume
For example:
Tube Dilution Volume plated
10"3 100 |iL(0.1 mL)
10"3 100 |iL(0.1 mL)
10"3 100 |iL(0.1 mL)
Replicate
CFU
1
150
2
250
3
200
Extract total volume = 20 mL
(200 CFU / 0.1 mL) x (1/10 3) x 20 mL =
(2000) x (1000) x 20 = 4.0 x 107 CFU
Note: The volume plated (mL) and tube dilution can be multiplied to yield a 'decimal factor'
(DF). DF can be used in the following manner to simplify the abundance calculation.
Spore Abundance per mL = (Avg CFU) x (1 / DF) x extract volume
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MOP 6535a
Revision 4
January 2013
Page 5 of 8
Serial Dilution/Plating Results Sheet Page 1 of
1 KSI INFORMATION
EPA Project No.
PI
Technician Name
Test Date
Technician Signature
Test No.
m:si i i s
\ (ilium- I'hik-d:
TiiIk- Dilution
Siiinplc II)
I'lillC
Kcpl.
10"
10 1
I02
10 s
I04
10 5
10"
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
NOTES:
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MOP 6535a
Revision 4
January 2013
Page 6 of 8
Page 2 of
Siimple II)
I'hiu-
Ki'pl.
10"
I01
lo2
10'
I04
I0?
10"
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
NOTES:
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MOP 6535a
Revision 4
January 2013
Page 7 of 8
Page of
Siimple II)
I'hiu-
Ki'pl.
10"
I01
lo2
10'
I04
I0?
10"
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
NOTES:
-------
MOP 6535a
Revision 4
January 2013
Page 8 of 8
Page of
Siimple II)
I'hiu-
Ki'pl.
10"
I01
lo2
10'
I04
I0?
10"
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
NOTES:
-------
MOP 6560
Revision 1
November 2012
Page 1 of 4
Miscellaneous Operating Procedure (MOP) 6560:
Biological Indicator (BI) Tests Using Nutrient Broth and Analysis
of Results
Prepared by:
Nicole Griffin Gati
Date: 11/15/2012
S Work Assignment Leader
Reviewed by:
Date: 11/15/2012
DahmanyTouati, ARCADT? Project Manager
/
Approved by:
Date: 11/15/2012
Worth Cflfee, pPA Work Assignment Manager
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
-------
MOP 6560
MOP 6560
Revision 1
November 2012
Page 2 of 4
TITLE: BIOLOGICAL INDICATOR (BI) TESTS USING NUTRIENT BROTH
AND ANALYSIS OF RESULTS
SCOPE: This MOP provides the procedure for testing biological indicators (Bis) in 25
mL nutrient broth tubes and then obtaining results from the Bis in the tubes.
PURPOSE: This procedure will ensure that that the Bis are properly placed in 25 mL
nutrient broth tubes aseptically and that the results obtained are accurate.
1.0 PREPARING 25 ML NUTRIENT BROTH TUBES
Refer to MOP 6556 from Biolab Facility Manual.
2.0 PLACING BI's IN NUTRIENT BROTH TUBES
Always note which flat the 25mL nutrient broth tubes are coming from and make certain that
if more than one batch of tubes (from one bottle) is used, several (preferably three) tubes are
taken from each as negative material controls to test the sterility of the broth. These tubes
will serve as negative controls.
Sample sets should have positive controls as well as negative controls. The positive controls
will not be subjected to any testing variable and will be placed straight into the tubes. The
positive controls should come from the same batch as the negative controls. At least three
positive controls should be placed with a sample set or test.
1. Prepare the hood by wiping down with ethanol and a clean Kimwipe. Then stock the
hood with the following items if they are not already there:
The flats of 25mL nutrient broth tubes
Sharpie marker
The BI samples
Tweezers
Ethanol
Burner and striker
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MOP 6560
Revision 1
November 2012
Page 3 of 4
2. Label the 25 mL tubes with a Sharpie marker before you start. Put all pertinent
information, such as the sample number and the date on the tubes. Each BI will go into
one tube.
3. Light the burner and adjust the flame for a width adequate to flame tweezers if needed.
4. Unscrew the caps to the nutrient broth tubes and place the lid top side down on the
benchtop. Quickly open the BI (usually one side says "peel" or "open here") and without
touching the BI, let it fall into the 25mL nutrient broth tube. If the BI is paper, due to
static electricity, this technique may not work, so instead, hold the open BI while with a
free hand place the tweezers into ethanol and flame them. Then use the disinfected
tweezers to promptly extract the BI from the lining and drop it into the tube.
5. Replace the cap to the tube immediately.
6. Repeat with all samples.
7. Place all tubes into proper incubator for 7-9 days. (BI's of Bacillus subtilis, Bacillus
atrophaeus or Bacillus anthracis Sterne go into the 32 °C incubator, while those of
Geobacillus stearothermophilus go into the 60 °C incubator). Seven days is preferable,
but if the sample pull falls on a weekend, 9 days is fine for recovery.
3.0 ANALYSIS OF RESULTS FROM INCUBATED Bis IN NUTRIENT BROTH
TUBES
1. Carefully take Bis in 25 mL nutrient broth tubes out of the incubator in which they were
placed. Try not to stir up the contents of the tubes.
2. Look at the negative controls by holding them up to light and gently swirling the tube.
The tube should not have any turbidity or debris in it. Record results.
3. Look at the positive controls by holding them up to light and gently swirling the tube.
The tube should be turbid and should have growth/debris indicative of the BI that was
used. The positive controls will be the basis fo comparison to positive samples. Record
results.
4. All test samples will then be resulted in the same visual manner. Make certain that the
growth in each tube resembles the same type of growth as seen in the positive control.
5. After all tubes are viewed and the results of turbidity recorded, the tubes must be plated.
Using tryptic soy agar petri dishes, label one plate for each tube.
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MOP 6560
Revision 1
November 2012
Page 4 of 4
6. Prepare a biohood by wiping down with ethanol and a clean Kimwipe. Then stock the
hood with the following items if they are not already there:
The labeled plates
The BI samples in 25 mL tubes
Sterile swabs
Vortexer
7. Using the vortexer, vortex each tube prior to opening it.
8. Open the tube and lay the cap top side down on the benchtop.
9. Using the sterile swab and making certain it does not touch anything but the inside of the
25 mL tube, place the swab into the broth with the BI.
10. Take the swab and make a zigzag motion on the appropriately labeled petri dish agar.
11. Throw the swab away in the appropriate receptacle.
12. Replace the cap on the tube.
13. Put all plates and tubes in the appropriate incubator. Save the tubes until after the plate
results are considered valid and finalized.
14. After 12 to 24 hours, check the plates for growth and record results. The results should
be consistent with the visual turbidity results.
15. If further investigation of the plate growth is needed, complete gram stain or other
physiological tests.
16. Report results.
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MOP 6562
Revision 1
February 2013
Page 1 of 6
Miscellaneous Operating Procedure (MOP) 6562:
Preparing Pre-Measured Tubes with Aliquoted Amounts of
Phosphate Buffered Saline with Tween 20 (PBST)
Prepared by:
Date: 2/12/2013
Nicole Griffin G^tch^fian,
'IS Work Assignment Leader
Reviewed by:
Date: 2/12/2013
Dahman Xeruati, ARCADI5 Project Manager
Approved by:
Date: 2/12/2013
Worth Calfee, EpA Work Assignment Manager
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
O* \1\ rv*"K
r:' r\ fU
ARCAD1S U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
-------
MOP 6562
Revision 1
February 2013
Page 2 of 6
MOP 6562
TITLE: PREPARING PRE-MEASURED TUBES WITH ALIQUOTED AMOUNTS
OF PHOSPHATE BUFFERED SALINE WITH TWEEN 20 (PBST)
SCOPE: This MOP provides the procedure for preparing PBST.
PURPOSE: This procedure will ensure that that the PBST is prepared correctly and that all
measured tubes are filled aseptically.
1.0 PREPARING STERILE PHOSPHATE BUFFERED SALINE WITH TWEEN 20
(PBST)
Phosphate Buffered Saline with Tween 20 (PBST) is prepared 1 L at a time in a 1 L flask.
1. Add 1 packet of SIGMA Phosphate Buffered Saline with Tween 20 (P-3563) to 1 L of
deionized (DI) water.
2. Shake vigorously to mix until dissolved.
3. Label bottle as "non-sterile PBST" and include date and initials of person who made
PBST.
4. Filter sterilize into two 500 mL reagent bottles using 150 ml bottle top filter (w/ 33mm
neck and .22 |im cellulose acetate filter) for sterilization. Complete this by pouring the
liquid into the non-sterile PBST into the top portion of the filtration unit 150 ml at a time,
while using the vacuum to suck the liquid through the filter. Continue to do this until 500
ml have been sterilized into a 500 ml bottle. Change bottle top filter units between each
and every 500 ml bottle.
5. Change label to reflect that the PBST is now sterile. Include initials and date of
sterilization. The label should now include information on when the PBST was initially
made and when it was sterilized and by whom.
6. Each batch of PBST should be used within 90 days.
2.0 PREPARING 20 ML/5 ML PBST TUBES FOR USE DURING
EXPERIMENTATION
Twenty (20) ml or five (5) ml of the prepared PBST will be added to each sterile 50-ml
conical tube as detailed below. Each flat of conical tubes contains 25 tubes, so one 500 ml
sterile bottle of PBST should fill approximately one flat when 20 ml tubes are needed and
four flats when 5 ml tubes are needed.
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MOP 6562
Revision 1
February 2013
Page 3 of 6
1. Prepare the hood by wiping down with ethanol, followed by bleach, followed by DI water
and a clean Kimwipe or Techwipe. Then stock the hood with the following items if they
are not already there:
The flats of sterile conical tubes you need to fill with PBST.
Sufficient bottles of sterile PBST to fill these tubes.
Ample 25 ml serological pipettes (at least 3 per flat) for 20 ml transfers and 10
ml serological pipettes for the 5 ml transfers.
Serological pipetter (automatic, hand-held pipette).
Burner and striker.
2. Light the burner and adjust the flame for a width adequate to flame the lips of the PBST
bottles.
3. Take one flat of sterile conical tubes and loosen each cap on the outside edges (about V2
turn).
4. Open a serological pipette and insert into the serological pipetter, taking care to not touch
the tip to any surface.
5. Hold the pipetter with the first three fingers of your right (or dominant) hand. With your
left hand (or non-dominant hand), pick up a bottle of the PBST and use the bottom of
your right hand to unscrew the lid. Place the lid upside down on the benchtop and quickly
flame the lip of the bottle. Turn the bottle and repeat, taking care to thoroughly flame the
lip without getting the glass so hot that it shatters.
6. Inset the tip of the pipette into the bottle and fill to the 20 ml line. Flame the bottle lip and
place the bottle on the benchtop.
NOTE: If the tip of the pipette touches the outside of the bottle or any other
surface in the hood, consider it contaminated. Discard the pipette
and reload a new one.
7. Quickly pick up one of the tubes that you have loosened the cap on, and use the bottom
of your right hand to remove the cap. Completely discharge the entire pipette into the
tube, taking care to not touch anything with the tip of the pipette. Recap the tube and
place back into the flat (the lid does not have to be tight - you will tighten the lids after
you have completed filling the 10 outside tubes).
NOTE: If the tip touches the outside or rim of the tube (or any other surface
in the hood), consider the tube and pipette contaminated. Discard
both the tube and the pipette.
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MOP 6562
Revision 1
February 2013
Page 4 of 6
8. Pick up the PBST bottle and flame the lip. Repeat Steps 6 and 7 until all 10 of the tubes
on the outside of the flat have been filled. Flame the lip of the PBST bottle and replace
the cap. Slide the used pipette back into the plastic sleeve and put to the side of the hood
for disposal. Then tighten the lid of each tube you just filled. But rather than placing it
back into its original spot in the flat, switch it for the empty tube from the next row.
When this has been completed, go around the outside of the flat again and loosen the lids
of these 10 tubes. Repeat steps 4 through 7 to fill and cap these tubes.
9. This same procedure is used to fill the middle row of tubes from the flat, and if more
than one flat of tubes is being filled, can be done at the same time as the outside rows of a
second flat.
10. When all tubes have been filled, label each flat as follows, and place on the shelf in room
E390B:
"PBST Tubes (20 ml or 5 ml)"
Date prepared
Your initials
11. These tubes should be made at least 14 days before they need to be used so that they can
be verified as sterile. Any tubes that are cloudy or that have any floating matter/turbidity
should be discarded. The tubes are stable for and should be used within 90 days.
3.0 CLEANUP FOR 20 ML/5 ML PBST TUBES
1. Dispose of the used pipettes in the nonregulated waste.
2. Plug in the serological pipetter so that it can recharge.
3. Replace any unused PBST in the liquid containment on the shelf. Make sure that the
bottle is labeled as having been opened (date opened and initials of whomever used it).
4. Turn off the burner.
5. Wipe down the hood benchtop with ethanol, followed by bleach, followed by DI water
and a clean Kimwipe or TechWipe.
4.0 PREPARING 900jiL PBST TUBES FOR USE DURING EXPERIMENTATION
1. Prepare the hood by wiping down with ethanol, followed by bleach, followed by DI water
and a clean Kimwipe or Techwipe. Then stock the hood with the following items if they
are not already there:
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MOP 6562
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February 2013
Page 5 of 6
A sterile beaker of microcentrifuge tubes.
Sufficient tubes of sterile PBST to fill these tubes (PBST may be aseptically
transferred to 50 ml conical tubes for an easier aseptic transfer to the
microcentrifuge tubes- it is easier than working from a 500 ml reagent bottle.
Make certain that these 50 ml conical tubes are labeled to when the PBST was
made, sterilized, etc.).
1000 |iL micropipette.
1000 |iL sterile pipette tips
Microcentrifuge tube racks.
Labeled beaker or waste container used to hold non-regulated waste, such as
tips, under the hood.
2. Carefully remove the microcentrifuge tubes one at a time from the beaker and close the
top on each one before placing it in the tube rack. Place the tubes in the rack skipping
every other row. Fill up two racks doing this.
3. Add 900 |iL of PBST to the microcentrifuge tubes by aseptically transferring the PBST
from the sterile 50 ml conical tube containing the PBST. Do this by using the 1000 |iL
micropitte and tips. Change tips whenever after two rows of tubes are completed or
whenever a contamination event (such as touching the outside of the 50 ml tube or the
microcentrifuge tube) occurs. Put the dirty tips in the beaker or container used to contain
waste (tips, tubes) in the hood. If any 900 |iL tubes are contaminated during the transfer,
dispose of them in the waste container used to hold tips under the hood. If a new box of
tips has to be opened, make certain the date it was opened and initials of the person who
opened it are clearly labeled on the box.
4. After both racks are full, carefully move all the tubes from one rack to fill in the empty
rows on the other rack. In this manner, one rack should be completely filled with tubes at
this point.
5. Label the rack of tubes as "Sterile 900 |iL PBST Tubes", along with the name of the
person who completed the transfer, along with the date. Also, include the date that the
original stock of PBST was made and the date it was sterilized, along with the initials of
the person who completed those steps.
5.0 CLEANUP FOR 900jiL PBST TUBES
1. Dispose of the waste that was put in the labeled beaker or waste container (micropipette
tips and tubes) in the nonregulated waste. Then, place this beaker in the "To be
decontaminated via sterilization- contaminated glassware" bin or if it is a disposable
container, then it can be put in the non-regulated waste container.
2. Put the unused sterile tips and the micropipetter back in its original location.
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MOP 6562
Revision 1
February 2013
Page 6 of 6
3. Replace any unused 50 ml corneals of PBST in the liquid containment on the shelf. Make
sure that the tube is labeled as having been opened (date opened and initials of whomever
used it). If the tube could possibly be contaminated in any way, dispose of it in non-
regulated waste.
4. Wipe down the hood benchtop with ethanol, followed by bleach, followed by DI water
and a clean Kimwipe or TechWipe.
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MOP 6566
Revision 0
October 2009
Page 1 of 3
MOP 6566
TITLE:
SCOPE:
PURPOSE:
Materials:
• PPE (gloves, lab coat, safety goggles)
• Vortex mixer
• Sterile forceps (optional)
• Culture tubes with presterilized bacterial growth media (Tryptic Soy Broth or Nutrient roth
as determined by study)
• Incubator set to appropriate growth temperature for indicator organism (i.e., 55 - 60°C for
Geobacillus stearothermophilus)
• Biological Safety Cabinet (Class II)
1.0 PROCEDURE (without the use of forceps)
1. Label all culture tubes, prior to start, with date, sample ID, organism, and processors initials.
2. Begin by donning PPE (gloves, lab coat, and protective eyewear).
3. Clean the workspace (biological safety cabinet) by wiping surfaces with pH-adjusted bleach,
next with diL^O, and lastly with a 70-90 % solution of denatured ethanol. Make sure the
workspace is clean and free of debris. Gather all necessary items to perform the task, place
these items within arms reach of the biological safety cabinet so that, once the procedure has
begun, the task may be performed without interruptions and travel about the laboratory.
4. Discard gloves and replace with fresh pair.
CULTURING OF APEX LABORATORIES TYVEK PACKAGED
BIOLOGICAL INDICATORS
This MOP outlines the procedure for culturing Apex Laboratory Biological
Indicators (Bis)
Repeatable, aseptic transfer of Bis to growth media to determine viability
5. Using proper aseptic techniques, loosen the lid of the first sample's culture tube, careful not
to completely remove the lid.
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MOP 6566
Revision 0
October 2009
Page 2 of 3
6. Locate the corresponding first biological indicator (still concealed in a Tyvek envelope),
confirm that the label of the biological indicator matches that of the culture tube; then, grasp
the offset tabs at the end of the indicator with thumb and index finger of both hands.
7. Open the pouch slowly, with equal force, by pulling the two tabs apart until the enclosed
indicator coupon is just visible. Peeling the envelope too far may result in the indicator
coupon falling from the packaging and becoming contaminated.
NOTE: If an indicator coupon should come in contact with any surface other than
the untouched inner portion of the Tyvek envelope or the inside of the culture
tube, the indicator coupon must be discarded, and noted in the laboratory
notebook.
8. Once the Tyvek envelope has been peeled open, and the indicator coupon slightly exposed;
gently grip the coupon by squeezing the outside of the Tyvek envelope between the thumb
and index finger of one hand, (take extreme care not to touch the coupon with fingers).
9. Being careful not to touch the coupon to any surface, including the exterior of the culture
tube, transfer the coupon to the inside of the culture tube by removing the culture tube lid
with one hand, holding the envelope and pouch inverted above the open culture tube, and
then releasing the coupon from the Tyvek envelope. Immediately replace the culture tube
lid, confirm again that label of the indicator matches that of the culture tube. Save all empty
Tyvek envelopes, and catalog them by date of experiment.
NOTE: Sterile forceps can be utilized in this procedure to transfer the indicator
coupon from the Tyvek envelope to the culture tube. If using forceps, it is
important to remember that when working with bacterial spores, forceps and
other items are not readily sterilized with an ethanol soak followed by
flaming. More stringent measures should be utilized to prevent cross
contamination if reusing forceps or other items, (i.e., bleach soak 10 minutes,
followed by dTbO rinse, followed by ethanol soak for 1 minute, and flamed to
remove ethanol). Alternately, a new disposable sterile forceps can be utilized
for each sample.
10. Repeat procedure (from step 4) for remaining samples. Periodically replace gloves,
especially any time gloves come into contact with items outside the biosafety cabinet.
11. Once all samples have been successfully transferred to culture tubes, place tubes in incubator
at appropriate temperature.
12. After one (1) and seven (7) days, check the growth status of each tube (Or other interval as
determined by Quality Assurance Practice Plan (QAPP). Record results and observations in
laboratory notebook. Growth is deemed 'positive' if the media is visibly turbid. Growth is
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MOP 6566
Revision 0
October 2009
Page 3 of 3
deemed 'negative' if the growth media is lucid, displays no turbidity, and is indistinguishable
from that of the negative controls.
NOTE: Turbidity is best detected by holding each culture tube above eye-level,
bringing the tube between a light source and the technician's eyes, and then
swirling the tube. Turbidity can be characterized by clearly visible clumps of
cells, or by faintly visibly rolling clouds of cells.
13. Optional: To confirm the turbidity of the culture tube is from growth of the test organism, a
loop-full of the culture may be streaked onto an agar plate and incubated overnight to
confirm colony morphology. To confirm the absence of growth in tubes that display no
visible turbidity, the entire contents of the culture tube can be filtered using a 0.2 or 0.45 |im
pore-size analytical filter, and the filter subsequently placed on the surface of an agar plate
(collection side up) and incubated overnight. Step 12 is to be used at the discretion of the PI,
or as outlined in the QAPP.
-------
MOP 6566
Revision 1
May 2013
Page 1 of 4
Miscellaneous Operating Procedure (MOP) 6566:
Culturing Biological Indicator Strips
Prepared by:
Nicole Griffin GatisJia^an, AR^OIS Work Assignment Leader
Date: 5/16/2013
Reviewed by:
/J
Dahman Tduati, A RC A DiS Proj ect Manager
Approved by:
WjGptii Calfee, EPA Work Assignment Manager
Date: 5/16/2013
Date: 5/16/2013
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
-------
MOP 6566
Revision 1
May 2013
Page 2 of 4
MOP 6566
TITLE: CULTURING OF BIOLOGICAL INDICATOR STRIPS
SCOPE: This MOP outlines the procedure for culturing biological indicator (Bis) strips.
PURPOSE: Repeatable, aseptic transfer of Bis to a growth media to determine viability.
Materials:
• PPE (gloves, lab coat, safety goggles)
• pH-amended bleach
• DI water
• 70% Ethanol
• Vortex mixer
• Sterile thumb forceps
• Culture tubes with -10 mL of pre-sterilized bacterial growth media as determined by
manufacturer or QAPP (i.e.,Tryptic Soy Broth, Nutrient Broth)
• Incubator set to appropriate growth temperature for indicator organism as determined by
manufacturer or QAPP (i.e., 55 - 60°C for Geobacillus stearothermophilus)
• Class II Biological Safety Cabinet (BSC)
• Quality Assurance Project Plan (QAPP) or Test Plan
1.0 PROCEDURE
1. Prior to start, label all culture tubes with sample ID as determined by QAPP or Test Plan,
along with the date. Sort the culture tubes in logical manner, so that it will be easy to place
them into tubes. They must be placed in tubes from least possibly contaminated to most
possibly contaminated.
2. Begin by donning PPE (gloves, lab coat, and protective eyewear).
3. Clean the workspace (BSC) by spraying surfaces with pH-amended bleach and allow it to sit
for 3 minutes minimum. Next, spray the surfaces with DI water, and then wipe it clean with a
KimWipe. Lastly, spray the surfaces with 70% ethanol and wipe it clean with a KimWipe.
Make sure the workspace is clean and free of debris. Gather all necessary items to perform
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MOP 6566
Revision 1
May 2013
Page 3 of 4
the task, place these items on a cart beside the BSC so that, once the procedure has begun,
the task may be performed without interruption.
4. Discard gloves and replace with fresh pair.
5. Place the prelabeled culture tubes in the BSC. Make certain they are in a logical order so that
placing them into the culture tubes progresses from least contaminated to most contaminated.
6. Locate the first BI strip and its corresponding culture tube. Confirm that the label of the BI
matches that of the culture tube.
7. Place the culture tube in a rack, and aseptically remove its cap. Immediately grasp the offset
tabs at the end of the corresponding BI with both the thumb and index finger of both hands.
Separate the packaging containing the BI and allow the BI to aseptically free fall into the
tube's culture media.
NOTE: Open the pouch slowly, with equal force, by pulling the two tabs apart until
the enclosed indicator coupon is just visible. Peeling the envelope too far may result
in the indicator coupon falling from the packaging and becoming contaminated.
If an indicator coupon should come in contact with any surface other than the
untouched inner portion of the BI packaging or the inside of the culture tube make a
note of it on both the tube and in the laboratory notebook.
8. Immediately replace the culture tube lid, confirm again that label of the indicator matches
that of the culture tube. Discard all empty BI packaging into non-regulated waste.
NOTE: Sterile disposable thumb forceps can be utilized in this procedure to transfer
the indicator coupon from BI packaging to the culture tube, especially in the
event that the transfer is difficult due to BI packaging issues. Use a new pair
of sterile disposable thumb forceps for each sample and dispose of all sterile
thumb forceps in non-regulated waste.
9. Repeat steps 6-8 for remaining samples.
10. Once all samples have been successfully transferred to culture tubes, place tubes in incubator
at appropriate temperature as determined in the QAPP or Test Plan.
11. Check the growth status of each culture tube at an interval as determined by the QAPP or
Test Plan. Record results and observations in laboratory notebook. Growth is deemed
'positive' if the media is visibly turbid. Growth is deemed 'negative' if the growth media is
lucid, displays no turbidity, and is indistinguishable from that of the negative controls.
NOTE: Turbidity is best detected by holding each culture tube above eye-level and
bringing the tube between a light source and the technician's eyes, and then
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swirling the tube. Use the positive and negative controls to aid in determining
turbidity.
To confirm that all 'negative' samples are free of growth and to confirm that all 'positive'
turbidity from the culture tubes are from growth of the target organism, the culture tubes must be
plated and any and all colony morphology observed. These steps, however, are to be directed by
the QAPP or Test Plan.
1. Agitate the culture tube with the vortex mixer for 3 to 5 seconds, and then immediately
aseptically transfer 100 |iL from the culture tube to prelabeled media plates. Incubate for
appropriate time and temperature, as determined either by the manufacturer or the QAPP or
Test Plan.
2. To further confirm the absence of growth in tubes that display no visible turbidity, the entire
contents of the culture tube can be filtered using a 0.2 or 0.45 |im pore-size analytical filter,
and the filter subsequently placed on the surface of an agar plate (collection side up) and
incubated for appropriate time and temperature.
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MOP 6570
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Miscellaneous Operating Procedure (MOP) 6570:
Use of Steris Amsco Century SV 120 Scientific Prevacuum
Sterilizer
Prepared by:
Qli&AvDt
Nicole Griffin Gati
Date: 3/21/2013
Work Assignment Leader
Reviewed by:
Dahman^Rj'uati, ARCADtS Frofect Manager
Approved by:
Worth Calfee, EPA Work Assignment Manager
Date: 3/21/2013
Date: 3/21/2013
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
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MOP 6570
MOP 6570
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TITLE: USE OF STERIS AMSCO CENTURY SV 120 SCIENTIFIC
PREVACUUM STERILIZER
SCOPE: Basic instructions for use of the large Steris autoclave.
PURPOSE: To outline proper procedural use of the autoclave, using preprogrammed
cycles, to effectively sterilize items, while complying with quality control
standards.
Materials:
• Amsco Century SV 120 Scientific Prevacuum Sterilizer
• Items to be sterilized (liquids, solids, waste, etc)
• Pouches to contain materials during sterilization and maintain sterility until use
• Aluminum foil
• Autoclave indicator tape
• Sterilization verification ampoules (such as Raven ProSpore Ampoules)
• Thermally resistant gloves
• De-Ionized (DI) water
1.0 PROCEDURE
1.1 Start Up
1. Turn on the autoclave. The power switch is located behind the door in the top right
corner. The digital touch screen on the front of the unit will power up and indicate
that a memory test is in progress.
2. After the memory test is complete, the device will request that it be flushed. This
should be conducted daily to minimize scaling inside the boiler. The flush valve is
located behind the door on the bottom, left of the device (yellow handle). Move the
valve to the open position and then press the "Start Timer" button on the touch
screen. The flush will run for 5 minutes and will alert at completion with a single
chime.
3. Once the flush is complete, close the flush valve and press the "Continue" button on
the touch screen. The screen should then return to its default menu which has 2
choices "Cycle Menu" and "Options"
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1.2 Basic Operation
1. Prepare any items that need to be sterilized. The items must be carefully wrapped or
sealed in sterilization pouches in order to maintain sterility when removed from the
autoclave. Examples of this include: wrapping any orifices with aluminum foil,
placing whole items in autoclave pouches, loosely applying a cap on a bottle (to allow
for the pressure changes inside).
2. Once prepared, each item should be outfitted with a sterility indicator such as a small
piece of autoclave indicator tape; or by utilizing an autoclave pouch with a built-in
sterility indicator strip. These indicators provide a visual verification that the
sterilizing temperature (121°C) was reached.
3. To add items to the autoclave, open the autoclave door by pressing down on the foot
pedal on the bottom right corner on the front of the device.
4. Place items that need to be sterilized into the autoclave, adding or moving racks to
accommodate the load. If liquids are being autoclaved, then they must have secondary
containment (usually a large plastic autoclave-safe tray) to contain any fluids in the
event of a leak, spill or boil-over. Add an indicator ampoule to the first autoclave
cycle of the day, regardless of the type of cycle.
5. Once the autoclave is loaded, press the foot pedal to close the autoclave door.
6. Once the door is sealed, a menu of the cycles can be seen by pressing the button on
the touch screen labeled "Cycle Menu". Then choose the appropriate cycle by
touching the corresponding button. If the cycle chosen is the one desired for the
sterilization process, press the "Start Cycle" button. Otherwise, press "Back" to return
to the prior menu screen.
7. After the cycle has started, the type of cycle, the number of the cycle, the items
placed in the autoclave during the cycle, the time, whether or not an indicator
ampuole was included in the load, and the initials of the person starting the cycle
must be recorded in the autoclave log book, located in the drawer across form the unit
labeled "Autoclave Supplies."
8. Quality control (QC) indicator ampoules, usually Raven ProSpore Ampoules with
Geobacillus stearothermophilus (at a concentration 10E6), are added to one cycle
each day to ensure that the autoclave is functioning properly. These ampoules are
used according to manufacturer's instructions. These ampoules must be properly
labeled with the date in which they were autoclaved and the initials of the individual
that completed the cycle. At the beginning of each week, a positive control ampoule
must be processed, where the ampoule is placed directly into the 55°C water bath,
without being autoclaved. The positive control indicator ampoule should change from
purple to yellow in color, indicating growth. All test ampoules should be placed in a
water bath following the end of the cycle in which they are run. These ampoules
should not change color (from purple to yellow, but instead should remain a purple to
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MOP 6570
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purple-brown color). Ampoules should be checked at both 24 and 48 hour intervals
for growth and then finally recorded and disposed of after 48 hours. All QC
information concerning ampoules should be recorded in the autoclave notebook.
9. Upon completion of any cycle, the autoclave will alarm with a repeating beep for
approximately one minute. Any time after this alarm starts, it is safe to open the main
door (take caution because the steam escaping the chamber will be very hot when the
door is opened). The contents from the autoclave will be very hot; use protection to
remove items from the autoclave (thermally resistant gloves).
10. Place the contents of the autoclave in an appropriate place to cool, and close the
autoclave door using the foot pedal.
1.3 Cycles
1.3.1 Gravity Cycles
Gravity cycles are used to sterilize glassware and other utensils, which are not submerged
in nor contain any volume of liquid. These cycles are typically used for "dry" materials.
Currently there are two different gravity cycles programmed for daily operations: a 1-
hour cycle and a 30-minute cycle. The time that the chamber is held at the sterilization
temperature (121 °C) is the only difference between these two cycles. The different
sterilization times allow for the compensation of the various sizes of materials and more
resilient organisms. The 30-minute cycle is primarily used for a small quantity of
material. The 1 hour cycle is used for large loads or items containing a large amount of
contamination. The 1 hour cycle is recommended for inactivation of gram positive spore-
forming bacteria.
1.3.2 Liquid Cycles
Liquid cycles are used to sterilize a variety of liquids and solutions. The solutions are
typically mixed prior to sterilization. It is important to have secondary containment to
contain any fluids in the event of a leak, spill or boil-over. The 30-minute liquid cycle is
used to sterilize small volumes of liquid (usually less than 2L total). When attempting to
sterilize any volume larger than 2L, the 1-hour liquid cycle should be used to ensure
complete sterilization. The 1-hour liquid cycle is the preferential cycle used as the
destruction cycle for waste. In the event of materials (liquid or otherwise) being
contaminated/exposed microorganisms, the 1-hour liquid cycle will be used as the initial
means of decontamination. When completing a decontamination cycle, if there is no
liquid inside of a container, then deionized water must be added to the container or the
item must be submerge prior to the start of the cycle. Only items that are being
decontaminated can go in destruction cycles. Decontamination cycles cannot be mixed
with sterilization cycles.
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MOP 6576
Revision 3
June 2013
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Miscellaneous Operating Procedure (MOP) 6576:
Determination of Spore Thermal Challenge (Heat Shock) Resistance
Nicole Gnllin Ga
Prepared by:
Reviewed by:
Dahman Toydti, ARCADIS'Pfoject Manager
Date: 6/12/2013
S Work Assignment Leader
Approved by: / /
Worth Calffee, I'PA Work Assignment Manager
Date: 6/12/2013
Date: 6/12/2013
Prepared for
National Homeland Security Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
Prepared by
ARCADIS U.S., Inc.
4915 Prospectus Drive, Suite F
Durham, NC 27713
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MOP 6576
Revision 3
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Page 2 of 3
MOP 6576
TITLE:
SCOPE:
PURPOSE:
Materials:
• PPE (gloves, lab coat, safety goggles)
• Biological Safety Cabinet (Class II)
• Liquid suspension of bacterial spores
• Sterile microcentrifuge tubes
• Diluent (sterile deionized water, BPW, PBS, or PBST)
• Vortex mixer
• Water Bath (set to 80°C)
• Micropipette
• Sterile pipette tips
• NIST Traceable timer
1.0 PROCEDURE
1. Determine the titer of the spores suspension to be tested using BioLab MOP 6535a.
2. Using fresh, sterile diluent, prepare 1.0 ml aliquots of the spore suspension in
microcentrifuge tubes, each containing between 1E4 and 1E5 CFU per ml (calculations to
determine volume of spore suspension vs diluents based upon results from step 1). Use the
following equation:
Target spore titer (i.e. 2E4) X 1 ml = Spike volume (in ml)
Determined Stock Titer (i.e, 1E8)
(Add this spike volume to enough diluent to total 1 ml)
DETERMINATION OF SPORE THERMAL CHALLENGE (HEAT SHOCK)
RESISTANCE
This MOP outlines the procedure for determining the ability of a spore suspension
to withstand a thermal challenge (heat shock)
A quality control measure to ensure spores used for experimentation display the
robust phenotype (heat resistance) characteristic of bacterial spores.
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MOP 6576
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NOTE: Vortex stock samples 20 seconds prior to pulling spike volume to ensure
homogeneity.
3. Prepare six 1 ml sample tubes for each spore stock to be analyzed. Three will be exposed to
the treatment (heat), three will be controls (unexposed). Label tubes accordingly.
4. Once all tubes are assembled, and the temperature (80 ± 2°C) of the water bath has been
verified and recorded; insert all treatment tubes into the water bath, using floating or
submersible racks as necessary. Be sure that all of the liquid in the tube is below or equal
with the level of the water in the water bath. Start the NIST Traceable timer. Keep control
(unexposed) samples refrigerated or on ice.
5. Exactly following 20 minutes of exposure, remove treatment tubes, and place on ice (or
refrigerate) for 5 minutes.
6. Determine spore titer in each sample (exposed and unexposed) using BioLab MOP 6535a.
7. The mean log titer of exposed samples should be not be greater than 0.5 log less than the
mean log titer of unexposed samples (Equation 1). Data should be reported as the mean log
of exposed samples minus the mean log of unexposed samples (Equation 1). Note: it is not
uncommon to observe greater than 100% titer (ratio of exposed to unexposed samples), since
heat shock may increase germination efficiency of bacterial endospores.
Logio (Mean Unexposed Tubes) - Logio (Mean Exposed Tubes) < 0.5 (1)
8. In addition, data should be reported as CFU/ml for all samples and as percent survival
according to equation 2:
Mean titer of exposed samples X 100
Mean titer of control samples
(2)
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Appendix B: DTRL - QC Checklist for Data Reviewers
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DTRL -QC CHECKLIST FOR DATA REVIEWERS
Spreadsheet checks:
n Before beginning QC, make sure the WAL (or Task Leader as appropriate) has briefed you on the
task. DQI goals should be noted and understood by both parties.
n Create a QC tab in the spreadsheet. Notes should be recorded in this QC tab as described in
these checks. When a QC reviewer adds information directly to a data file, it should be noted in
the QCtab.
n Column headers present and sufficiently detailed.
n Units noted in each column/row, as required.
n Spot check that raw data were entered correctly (10%). Make sure digitally recorded data has
the correct time stamp. For recorded data such as Labview files, simply sourcing the data to its
original file is sufficient.
n Proper CFU acceptance criteria used, and all required re-plates and filter plates done.
Note: It is the responsibility of the QC reviewer to make sure they are using the correct criteria.
Currently, the following general rules apply:
Acceptable plate counts are from 30 to 300 CFU (unless noted).
There should be no colored cells in the spreadsheet, unless noted as to why this is
acceptable. If the replicate counts on a dilution plate don't agree within 50% of each
other, the ID cell turns blue. If the RSD is outside of 50%, the cell turns yellow. Once
an acceptable count agreement/RSD has been obtained by a re-plate, the cell will no
longer be blue/yellow.
Filter plates or higher volume plates must be run when there are between 0 and 29
CFU at the zero dilution. For filter plate data, the highest volume should be used for 0
counts. All non-zero filter data must be listed.
If reportable counts seem like an outlier, these results are turned to text by the
enterer, and become left aligned (rather than right aligned).
n Check the accuracy of each formula in one cell. Then randomly click other cells in that
row/column to verify that the formula was copied correctly. Check for cells indicated by Excel
with a green corner as inconsistent formulae.
n Document the source of any constants in formulae, if not apparent.
n The location of the status worksheet is noted (or the location of the raw data files, if a status
worksheet is not used).
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n Project deviations are noted and detailed.
n There are no links to external spreadsheets. These should be turned to fixed numbers, and have
the source listed in a comment or other method. The absolute path (including sheet and cell
reference) to the value should be noted in a comment for easy updating using macros. These
macros can be found in DTRL/Facility/Macros.
n Add name and date to data spreadsheet as QC Reviewer.
n Check results against DQI goals for the project. For any DQI goal not met, specify the failure in
the QC section.
n Ensure all necessary calibrations are performed according to the QAPP. It is also important to
check that these calibrations (pre and post as required) have been appropriately applied to all
test data collected.
n Witness (sign and date) each page of the laboratory notebook associated with this data.
Project Documentation:
Depending on the project in question, "Project Documentation" can involve any or all of the following:
• Raw data files
• Chain of Custody
• Data collection forms
• Laboratory notebooks
• Living documents
• QAPP
• MOPs
• Calibration files
The QC check will usually require that you at least look through the laboratory notebook to see if there
were any deviations that need to be detailed in the data spreadsheet. The other documents may be
needed for project details, procedures, instrumentation, etc.
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&EPA
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
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