EPA/600/R-16/149 | July 2016

www.epa.gov/homeland-security-research

Evaluation of Sporicidal Wipes and 

Liquid Agents for Decontamination of 

Anthrax-Contaminated Surfaces by 

Robotic Cleaners 

Office of Research and Development

National Homeland Security Research Center

 

EPA/600/R-16/149 

July 2016 

Evaluation of Sporicidal Wipes and 

Liquid Agents for Decontamination of 

Anthrax-Contaminated Surfaces by 

Robotic Cleaners 

National Homeland Security Research Center 

Office of Research and Development 

U.S. Environmental Protection Agency 

Research Triangle Park, NC 27711 
 

 

 

 

Disclaimer 

The U.S. Environmental Protection Agency (EPA), through its Office of Research and Development’s 

National Homeland Security Research Center, funded and managed this investigation through Contract 

No. EP-C-09-027, Work Assignment 72 with ARCADIS U.S., Inc. (ARCADIS), and Contract No. EPC-15-008 with Jacobs Technology Inc. (Jacobs). This report will be peer and administratively reviewed 

and will be approved for publication as an EPA document. This report does not necessarily reflect the 

views of the EPA. No official endorsement should be inferred. This report includes photographs of 

commercially available products. The photographs are included for the purposes of illustration only and 

are not intended to imply that EPA approves or endorses the products or their manufacturers. EPA does 

not endorse the purchase or sale of any commercial products or services. 

Questions concerning this document and its application should be addressed to the following individual: 

Sang Don Lee, Ph.D. 

U.S. Environmental Protection Agency 

Office of Research and Development 

National Homeland Security Research Center 

109 T.W. Alexander Dr. (E343-06) 

Research Triangle Park, NC 27711 

Telephone No.: (919) 541-4531 

Fax No.: (919) 541-0496 

e-Mail Address: lee.sangdon@epa.gov 

i 
Acknowledgments 

The following individuals and organizations are acknowledged for review of this document: 

United States Environmental Protection Agency 

Leroy Mickelsen 

Lukas Oudejans 

Doris Betancourt 

ii 
Table of Contents 

Disclaimer ..................................................................................................................................................... i 

Acknowledgments ...................................................................................................................................... ii 

Figures ........................................................................................................................................................iv 

Tables ........................................................................................................................................................iv 

Acronyms and Abbreviations .................................................................................................................... v 

Executive Summary...................................................................................................................................vi 

1 Introduction ......................................................................................................................................... 1 

2 Experimental Approach...................................................................................................................... 2 

Project Objectives and Robots Tested ......................................................................................... 2 

Experimental Methods and Materials ........................................................................................... 3 

2.2.1 Test Coupon Preparation and Sterilization...................................................................... 4 

2.2.2 B. atrophaeus Spore Preparation .................................................................................... 4 

2.2.3 Coupon Inoculation.......................................................................................................... 5 

2.2.4 Robotic Cleaner Decontamination Procedures ............................................................... 7 

2.2.4.1 R1 Decontamination Procedure ...................................................................... 7 

2.2.4.2 R2 Decontamination Procedure ...................................................................... 7 

Data Reduction ............................................................................................................................. 8 

3 Sampling Approach ............................................................................................................................ 9 

Sampling Site Environmental Conditions ..................................................................................... 9 

Sampling Media ............................................................................................................................ 9 

Wipe Sampling Procedures .......................................................................................................... 9 

Sampling Frequency...................................................................................................................10 

Critical Measurements ................................................................................................................11 

Prevention of Cross-Contamination of Samples during Sampling .............................................11 

Collection of Representative Samples........................................................................................12 

Sample Storage and Preservation..............................................................................................12 

4 Analytical Procedures for Microbiological Analyses....................................................................13 

5 Test Results.......................................................................................................................................14 

R1 (Mint Automatic Floor Cleaner 4200) Test Results ...............................................................14 

R2 (iRobot Scooba® 390) Test Results ......................................................................................16 

6 Quality Assurance and Quality Control..........................................................................................20 

Criteria for Critical Measurements/Parameters ..........................................................................20 

Quality Control Checks ...............................................................................................................21 

6.2.1 Integrity of Samples and Supplies .................................................................................21 

6.2.2 NHRSC Biolab Control Checks .....................................................................................21 

QA/QC Sample Acceptance Criteria...........................................................................................21 

6.3.1 QA/QC Test Results Validation .....................................................................................23 

6.3.2 Sample Holding Time ....................................................................................................23 

7 Summary............................................................................................................................................24 

8 References.........................................................................................................................................25 

Appendix A: Spatial Sampling Data ...................................................................................................... A-1 

Appendix B: Sample Holding Time ....................................................................................................... B-1 

iii 
 

 

 

Figures 

Figure 2-1. R1 (Left) and R2 (Right) Robotic Cleaners ................................................................................ 2 

Figure 2-2. ADA ............................................................................................................................................ 5 

Figure 2-3. MDI and MDI Actuator ................................................................................................................ 5 

Figure 2-4. Large Coupon Hot Spot Test Inoculation Area (Section e) ........................................................ 6 

Figure 2-5. Large Coupon Broad Area Test Inoculation Areas (Sections a, e, and i) ..................................6 

Figure 4-1. Dilution Plate (Left) and Filter Plate (Right) Showing Colonies of B. atrophaeus ....................13 

Figure 5-1. R1 Vinyl Hot Spot Test Results, Replicate 1 of (a) Hype-WipeTM Cloth and 

(b) pAB Cloth........................................................................................................................15 

Figure 5-2. Highest R2 Hot Spot LR Results for: (a) pAB on Vinyl, Replicate 2; and (b) SporKlenz® on Vinyl, Replicate 1, and (c) pAB on Laminate.......................................................17 

Figure 5-3. Highest R2 LR Results, Broad Area Inoculation ......................................................................19 

Tables 

Table 2-1. Commercially Available Robotic Cleaners Evaluated.................................................................. 2 

Table 2-2. Test Matrix for Robotic Cleaners ................................................................................................. 3 

Table 2-3. Robotic Cleaner Decontamination Test Coupon Material Specifications ....................................4 

Table 3-1. Sampling Frequency..................................................................................................................10 

Table 3-2. Critical and Noncritical Measurements ......................................................................................11 

Table 5-1. R1 Decontamination Results Summary.....................................................................................14 

Table 5-2. R2 Decontamination Results Summary.....................................................................................16 

Table 6-1. Critical Measurement Criteria ....................................................................................................20 

Table 6-2. QA/QC Sample Acceptance Criteria..........................................................................................22 

Table 6-3. QA/QC Test Results ..................................................................................................................23 

iv 
Acronyms and Abbreviations 

ADA aerosol deposition apparatus 

Anthrax Bacillus anthracis 

ATCC American Type Culture Collection 

B. Bacillus 

Biolab National Homeland Security Research Center Biocontaminant Laboratory 

CFU colony forming unit(s) 

DQI data quality indicator 

DQO data quality objective 

EPA U.S. Environmental Protection Agency 

EtO ethylene oxide 

ft2 square foot/feet 

HSPD Homeland Security Presidential Directive 

H2O2 hydrogen peroxide 

Lowe’s Lowe’s Home Improvement 

LR log reduction 

MDI metered-dose inhaler 

mL milliliter(s) 

MOP miscellaneous operating procedure 

NHSRC National Homeland Security Research Center 

NIST National Institute of Standards and Technology 

PAA peracetic acid 

pAB pH-adjusted bleach 

PBST phosphate-buffered saline with 0.05% Tween® 20 

PRB polyester rayon blend 

QA quality assurance 

QAPP quality assurance project plan 

QC quality control 

RH relative humidity 

STD standard deviation 

STS sodium thiosulfate 

TSA tryptic soy agar 

VHP® vaporized hydrogen peroxide 

v 
 

 

 

 

 

 

 

 

 

 

 

 

 

Executive Summary 

In 2001, U.S. Postal Service system facilities were contaminated through the introduction of a few letters 

containing Bacillus anthracis (anthrax) spores. The likelihood of occurrence of another such incident has 

prompted the U.S. Environmental Protection Agency (EPA) to develop strategies, guidelines, and plans 

for decontaminating persons, equipment, and facilities to mitigate the risks of contamination after a 

biological weapons attack. This work is being conducted in accordance with Homeland Security 

Presidential Directives (HSPDs)-5, -7, -8, and -10 in a coordinated effort with other federal agencies. 

During the response to the U.S. Postal Service incident, fumigation was used primarily in heavily 

contaminated facilities. Other cleaning methods were used in (1) secondarily contaminated areas (areas 

handling cross-contaminated letters potentially in contact with the letters containing the anthrax spores 

and areas where anthrax contamination was tracked from primarily contaminated areas), and (2) primarily 

contaminated areas showing a minimal presence of anthrax spores. These other cleaning methods 

included combinations of disposal of contaminated items, vacuuming, and the use of liquid sporicides 

such as a pH-adjusted (pAB) bleach solution. 

The use of commercially available robotic cleaners is a potentially effective decontamination technique for 

the complete removal or inactivation of Bacillus (B.) spores. This report discusses a project that 

investigated the ability of robotic cleaners to apply sporicidal liquid/wipe to floor surfaces and 

inactivate/remove B. atrophaeus spores, formerly known as B. globigii, as a surrogate for anthrax spores. 

Specifically, this report addresses the operational aspects of decontamination using robotic cleaners for 

large surface areas tested under conditions that mimic their realistic field use. 

This project evaluated the robotic cleaners for deactivation of B. atrophaeus on two test material surfaces: 

laminate and vinyl. The decontamination procedures were performed using the following two robotic 

cleaners: 

• Mint Automatic Floor Cleaner 4200 supplied with a North Star navigation system to track the

area cleaned tested with ready-to-use Hype-WipeTM cloths or the manufacturer’s cloth wetted

with a decontamination solution of pAB;

• iRobot Scooba® 390 that uses a four-stage, hard-floor cleaning process (preparation,

washing, scrubbing, and using squeegees) tested with pAB or Spor-Klenz® directly sprayed

onto the floor surface and then vacuumed back up.

The Mint Automatic Floor Cleaner 4200 tests using both the Hype-WipeTM cloths and the pAB-treated 

cloths demonstrated a very low decontamination efficacy, with an overall log reduction (LR) in viable 

spores of less than 1. Furthermore, a set of experiments was conducted to determine the extent of the 

potential spreading of contamination by the Mint Automatic Floor Cleaner 4200 robotic cleaner using both 

types of cloths, and results show that not only is the robotic cleaner ineffective at decontaminating a hotspot area, the Mint Automatic Floor Cleaner 4200 robotic cleaner also increases the total area 

contaminated by spreading the contamination. 

The iRobot Scooba® 390 tests showed better decontamination efficacy, with Spor-Klenz® showing better 

results than pAB in limiting the spread of contamination outside the inoculation zone. However, the iRobot 

Scooba® decontamination efficacy was below a 3 LR in viable spores overall regardless of the type of 

liquid decontaminant used. For laboratory based testing, demonstration of a 6 LR is the target for an 

vi 
 

 

effective sporicide under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). 

In summary, neither robotic cleaner successfully showed a decontamination efficacy of a 6 LR, in total 

spore count nor significantly reduced the surface contamination amount. The maximum result was a 2.9 

LR for the iRobot Scooba® 390 using Spor-Klenz® on vinyl. However, the average efficacy result for all 

three replicates was only a 2.29 LR. Spor-Klenz® appears to be more successful at decontamination than 

pAB on these surfaces with this application type, and increasing the reservoir volume of the iRobot 

Scooba® 390 may increase the LR. Spor-Klenz® did help limit the spread of contamination to new areas, 

although some spread was still observed. 

vii 
 

 

 

 

 

 

 

 

 

 

1 Introduction 

In 2001, the introduction of a few letters containing Bacillus anthracis (anthrax) spores into the U.S. 

Postal Service system resulted in the contamination of several facilities. Although most of the facilities in 

which these letters were processed or received were heavily contaminated, the facilities were 

successfully remediated using approaches such as fumigation with chlorine dioxide or Vaporous 

Hydrogen Peroxide (VHP®). The likelihood of occurrence of another such incident has prompted the U.S. 

Environmental Protection Agency (EPA) to develop strategies, guidelines, and plans for decontaminating 

persons, equipment, and facilities to mitigate the risks of contamination after a biological weapons attack. 

This work is being conducted in accordance with Homeland Security Presidential Directives (HSPDs)-5, -

7, -8, and -10 in a coordinated effort with other federal agencies. It is well agreed that available, effective, 

and economical decontamination methods that can be used over wide areas (outdoors and indoors) are 

required to increase preparedness to respond to such releases. 

During the response to the U.S. Postal Service incident, fumigation was used primarily in heavily 

contaminated facilities. Other cleaning methods were used in (1) secondarily contaminated areas (areas 

handling cross-contaminated letters potentially in contact with the letters containing the anthrax spores 

and areas where anthrax contamination was tracked from primarily contaminated areas) and (2) primarily 

contaminated areas showing a minimal presence of anthrax spores. These other cleaning methods 

included combinations of disposal of contaminated items, vacuuming, and the use of liquid sporicides 

such as a pH-adjusted (pAB) bleach solution. 

The use of commercially available robotic cleaners is a potentially effective decontamination technique for 

the complete removal or inactivation of Bacillus (B.) spores. This report discusses a project that 

investigated the ability of robotic cleaners to apply sporicidal liquid/wipe to floor surfaces and 

inactivate/remove B. atrophaeus spores, formerly known as B. globigii, as a surrogate for anthrax spores. 

Specifically, this report addresses the operational aspects of decontamination using robotic cleaners for 

large surface areas tested under conditions that mimic their realistic field use. 

The primary objective of this project is to provide agencies responding to occurrences of toxic biological 

contamination with operational criteria that would lead to safe and efficacious decontamination of 

bacterial agents on various materials and surfaces. This report specifically addresses the use of two 

commercially available robotic cleaners in the removal or inactivation of B. anthracis spores under various 

conditions (type of sporicide and type of surface). 

Consistent with sporicidal efficacy tests used to register sporicides under FIFRA, the current study utilized 

the generally accepted criterion of 6 Log Reduction to consider an approach effective. Tests were 

conducted using commonly used surface materials and environmental conditions. Recovery of no viable 

spores following treatment was considered highly effective. 

Section 2 of this report discusses the experimental approach, Section 3 discusses the sampling 

approach, Section 4 discusses analytical procedures for microbial analysis, Section 5 presents the test 

results, and Section 6 discusses quality assurance (QA) and quality control (QC) for the project. 

1 
 

 

 

 

 

Robot No. Model Manufacturer Cleaning type 

Applicable 

Surface 

Mint Automatic Floor Evolution Robotics, Pasadena, Sweep and R1 Hard floor Cleaner 4200 CA mop 

R2 iRobot Scooba® 390 iRobot, Bedford, MA Wet vacuum Hard floor 

Table 2-1. Commercially Available Robotic Cleaners Evaluated 

2 Experimental Approach 

Testing was conducted at EPA’s facility at Research Triangle Park, NC. This section describes the project 

objectives and robots tested, experimental methods and materials, and the test organism and spore 

preparation. 

Project Objectives and Robots Tested 

The project objective was to test commercially available, off-the shelf robotic cleaners equipped with 

commercially available wipe cloths and decontamination solution for decontaminating indoor flooring 

surface materials (laminate and vinyl) contaminated with B. atrophaeus spores (surrogate for anthrax). 

The two robots used this study have been previously evaluated to determine their effectiveness for 

sampling B. atrophaeus spores [1]. These two selected robots use liquid or wet wipes for floor cleaning, 

and these approaches are commonly used for surface decontamination. These robots were chosen to 

represent two major groups: cloth-wipe type and spray type. 

Table 2-1 lists the two types of robotic cleaners evaluated under this project. Figure 2-1 shows each type 

of robot tested. 

Figure 2-1. R1 (Left) and R2 (Right) Robotic Cleaners 

2 
 

 

 

 

 

The R1 robotic cleaner is the Mint Automatic Floor Cleaner 4200 supplied with a North Star navigation 

system to track the area cleaned. This robotic cleaner uses dry or wet disposable cleaning cloths or 

reusable microfiber cleaning cloths. The North Star navigation system tracks the area cleaned so that the 

cleaner will not miss a spot. For this project, the R1 used ready-to-use Hype-WipeTM cloths or the 

manufacturer’s cloth wetted with a decontamination solution of pH adjusted bleach (pAB). 

The R2 robotic cleaner is the iRobot Scooba® 390, which uses a four-stage, hard-floor cleaning process 

(preparation, washing, scrubbing, and using squeegees). For this project, the R2 used pAB or SporKlenz® in the cleaner’s reservoir directly sprayed onto the floor surface and then vacuumed back up. The 

amount of liquid remaining in the R2 reservoir was measured as an indication of further cleaning capacity. 

Table 2-2 summarizes the test matrix. This matrix was tested in triplicate. 

Table 2-2. Test Matrix for Robotic Cleaners 

Surface Material 

Laminate 

Vinyl 

Vinyl 

Laminate 

Test Area Size 

42 by 336 inches, 

L shape 

Robot 

R1 

R2 

R1 

R2 

R1 

R2 

R1 

R2 

Decontamination Wipe 

or Solution Inoculated Area 

pAB 

Hot spot test area measuring 12 by 12 

inches inoculated at minimum target 

concentration of 107 spores per square 

foot (ft2) 

Hype-WipeTM 

pAB 

Spor-Klenz® 

pAB 

Hype-WipeTM 

pAB 

Spor-Klenz® 

pAB 

Broad area inoculation test areas include 

12- by 12- inch sections inoculated at a 

minimum target concentration of 106 

spores/ft2 

Hype-WipeTM 

pAB 

Spor-Klenz® 

pAB 

Hype-WipeTM 

pAB 

Spor-Klenz® 

Experimental Methods and Materials 

This section describes the experimental testing and materials, including test coupon preparation and 

sterilization, B. atrophaeus spore preparation, coupon inoculation, and the robotic cleaner 

decontamination procedures. Digital video was collected during inoculation and decontamination. 

Photographs were taken of selected material coupons with any visible change due to the decontamination 

procedure. 

3 
 

 

 

 

 

 

 

 

 

 

2.2.1 Test Coupon Preparation and Sterilization 

The representativeness and uniformity of test materials are essential in achieving defensible evaluation 

results. Materials are considered representative if they are typical of materials commonly found in facilities 

and buildings in terms of quality, surface characteristics, and structural integrity. Material uniformity 

means that all test materials of each type are equivalent. Uniformity was maintained by obtaining a 

quantity of material sufficient to allow the preparation of multiple test coupons with presumably uniform 

characteristics (that is, test coupons were cut from the interior rather than the edge of a large piece of 

material). 

For testing the robotic cleaners, two coupon materials were used: laminate and vinyl. Materials were 

purchased locally. The test coupons were L-shaped and had a surface area of approximately 100 ft2 (42 

by 336 inches). The positive and negative control coupons were 42 by 42 inches. The vinyl coupon was 

made of one continuous piece of vinyl. Each laminate coupon had two layers of substrate with joints 

offset. Joints of laminate were typical of office construction in accordance with the manufacturer’s 

recommendations. A 6-inch-high border was installed around each coupon to allow the robotic cleaner to 

navigate as it would in an indoor area. Table 2-3 lists the coupon specifications. 

Table 2-3. Robotic Cleaner Decontamination Test Coupon Material Specifications 

Material 

Laminate 

Flooring 

Vinyl 

Flooring 

Description 

7-5/8- by 50-3/4-inch Laminate Wood Locking 

Flooring (# 103553) 

12-foot-wide River Park Staggered Slate 

Brown Multi Vinyl Sheet (# G4820) 

Manufacturer or Supplier 

Name 

Coupon Surface 

Size 

Lowe’s store, Mooresville, NC 42 by 336 

Armstrong World Industries Inc. inches , 

Lancaster Township, PA 

The test coupons were sterilized before inoculation using Dispatch® wipes (Clorox® Company, Oakland 

CA). After 5 minutes, each coupon was wiped with a wipe soaked with 3% sodium thiosulfate (STS) 

solution to neutralize or remove any remaining hypochlorite and then an alcohol wipe (Catalog No. 

21910-110; VWR® Premoistened Clean-WipesTM with isopropyl alcohol/deionized water, VWR, 

Radnor, PA). 

2.2.2 B. atrophaeus Spore Preparation 

The test organism for this project was a powdered spore preparation consisting of a mixture of B. 

atrophaeus (formerly B. globigii) spores (American Type Culture Collection [ATCC] 9372) and silicon 

dioxide particles. The preparation was obtained from the U.S. Army Dugway Proving Ground Life Science 

Division. The preparation procedure is fully reported in Brown et al. [2] and is summarized as follows. 

After 80 to 90% sporulation, the suspension was centrifuged to generate a preparation of approximately 

20% solids. The final product was a powdered matrix containing approximately 1 x 1011 viable spores per 

gram prepared by dry blending and jet milling the dried spores with fumed silica particles (Degussa, 

Frankfurt am Main, Germany). 

The powdered preparation that was received was loaded into metered-dose inhalers (MDIs) at the EPA 

facility in Research Triangle Park in accordance with a proprietary protocol. Control checks for each MDI 

were included in each batch of coupons contaminated using a single MDI. The initial weight of each MDI 

was verified using an Ohaus Adventurer Pro balance ARC120 (Ohaus Corporation, Parsippany, NJ). 

4 
 

 

 

 

 

 

Ongoing control checks for each MDI were included in the batches of coupons contaminated using a 

single MDI. The ongoing checks were performed using a Mettler-Toledo PL303 balance (Mettler-Toledo, 

Inc., Columbus, OH). 

2.2.3 Coupon Inoculation 

Both the test and positive control coupons were inoculated independently with spores of B. atrophaeus in 

a separate dosing chamber called an aerosol deposition apparatus (ADA) designed to fit one 14- by 

14-inch coupon of any thickness [3]. The ADA consisted of a stainless-steel hood sized to exactly cover 

the square test coupon. In the center at the top of the hood was an opening to attach an MDI and an MDI 

actuator. ADAs were sterilized with a 250-part-per-million, four-hour VHP® sterilization cycle, and MDI 

actuators were sterilized with ethylene oxide (EtO). Figures 2-2 and 2-3 show the ADA and MDI and MDI 

actuator used in this project. 

Figure 2-2. ADA Figure 2-3. MDI and MDI Actuator 

The MDI was discharged a single time into the ADA. The MDIs are claimed to provide 200 discharges per 

MDI. The number of discharges per MDI was tracked so that use did not exceed this value. Additionally, 

the weight of each MDI was determined after completion of the contamination of each coupon. The 14- by 

14-inch stainless steel contamination control coupons were inoculated as the first and last coupons within 

a single group of coupons inoculated by any one MDI within a single test. Spores were allowed to settle 

onto the coupon surfaces for a minimum of 18 hours. 

For the robotic cleaner hot spot tests, coupons were divided into eight areas (42- by 42-inch per area, 

Areas A through H), each area containing nine 14- by 14-inch sections (sections a through i). Section e of 

Area A was inoculated as shown in Figure 2-4. 

5 
 

 

 

 

 

 

 

 

Figure 2-4. Large Coupon Hot Spot Test Inoculation Area (Section e) 

The minimum target concentration was 107 spores/ft2. The robotic cleaner decontamination test was 

started in section c of Area B. 

For the robotic cleaner broad area inoculations, coupons were divided into 42- by 42-inch areas (Areas A 

through H), each containing nine 14- by 14-inch sections (sections a through i). Sections a, e, and i of 

Areas A, B, C, and D were inoculated as shown in Figure 2-5. 

Figure 2-5. Large Coupon Broad Area Test Inoculation Areas (Sections a, e, and i) 

The minimum target concentration was 106 spores/ft2 for this test. The robotic cleaner decontamination 

test was started in section c of Area B with the robot facing Area C. Routinely, the depositions exceeded 

107 spores/ft2. In one of the broad area replicate tests, this minimum target concentration was not 

reached. However, the results for this test did not differ significantly from results for the other two replicate 

tests. Similar sections of all replicate test and positive control coupons were inoculated before moving on 

to the next section. All section “a” areas were dosed before the section “e” areas, which were dosed 

before the section “i” areas. 

A log was maintained for each set of coupons dosed. Each record in this log recorded the unique coupon 

identifier, the MDI unique identifier, the date, the operator, the weight of the MDI before dissemination into 

the ADA, the weight of the MDI after dissemination, and the difference between these two weights. The 

coupon codes were pre-printed on the log sheet before coupon inoculation (dosing) began. 

6 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.2.4 Robotic Cleaner Decontamination Procedures 

The robotic cleaner decontamination procedures were performed using R1 and R2. R1 used pAB and 

Hype-WipeTM cloth wipes for decontamination, and R2 used pAB and Spor-Klenz® in the cleaning 

reservoir. The pAB solution consisted of approximately 6,000 parts per million hypochlorite (measured as 

free available chlorine) adjusted to a pH of 7 with acetic acid. Ready-to-Use Spor-Klenz®, an off-the-shelf 

product, is an aqueous solution of hydrogen peroxide (H2O2) and peracetic acid (PAA). Three R1 units 

and three R2 units were tested, one for each replicate test. The decontamination procedures are 

discussed below for each robotic cleaner unit. 

2.2.4.1 R1 Decontamination Procedure 

1.	 The Mint Automatic Floor Cleaner 4200 was removed from the box and placed where contamination 

was unlikely. 

2.	 The R1 unit was checked to make certain that its batteries were fully charged. If necessary, the 

batteries were charged to full capacity using the supplied cable. According to the manufacturer’s 

instructions, the batteries should be fully charged before any cleaning cycle begins. Battery installation 

and removal procedures are discussed in the user’s manual. 

3.	 The gray mopping cloth (8 by 11 inches) was sterilized over an 18-h cycle using an EtO sterilization 

cabinet (EOGas 333, Anderson Products, Haw River, NC) before being secured to the cleaning pad. 

Each mopping cloth was placed in a surgical EtO sterilization flat paper bag and then transferred to a 

larger EOGas bag along with an EtO cartridge, a Humidichip®, and an EtO dosimeter. 

4.	 Directly before sampling, 60 milliliters (mL) of decontamination liquid was pipetted uniformly across the 

gray mopping cloth. 

5.	 For Hype-WipeTM testing, the decontamination wipes were used directly from their individual sachets 

(each wipe was folded to fit the Mint cleaner pad [8 by 6 inches]). No additional decontamination liquid 

was added. 

6.	 The R1 unit was deployed in the test area. 

2.2.4.2 R2 Decontamination Procedure 

1.	 The iRobot Scooba® 390 was removed from the box and placed where contamination was unlikely. 

2.	 The R2 was inspected to make certain all parts were connected and installed in a functional manner 

(such as making sure the filter was in its proper position). 

3.	 The R2 unit was checked to make certain that its batteries were fully charged. If necessary, the 

batteries were charged to full capacity using the supplied cable. According to the manufacturer’s 

instructions, the batteries should be fully charged before any cleaning cycle begins. Battery installation 

and removal procedures are discussed in the user’s manual. 

4.	 Directly before sampling, 335 mL of the decontamination solution was loaded into the R2 reservoir using 

the procedures summarized below. 

a.	 The reservoir was opened to remove the R2 tank. 

b.	 Using a serological pipette, 335 mL of the decontamination liquid was aseptically added to the tank. 

c.	 The tank was closed and tightly capped. 

d.	 The tank was inserted into the reservoir by pressing down on the tank. When a click was heard, the 

tank was installed correctly. 

5.	 The R2 unit was deployed in the test area. 

7 
 

 

 

 

 

 

Data Reduction 

Data reduction was performed on measurements of the total spores (CFU) recovered from each replicate 

coupon, average recovered CFU, and standard deviation (STD) for each group of coupons. The groups of 

coupons included the following for each combination of material type and extracted sample type: 

• Positive control areas (replicate CFU, average recovered CFU, and STD);

• Test areas (replicate CFU, average recovered CFU, and STD);

• Procedural blank coupons.

Efficacy is defined as the extent (by LR) to which the agent recovered from the surface of the coupons 

after decontamination has been reduced compared to the positive control areas (not decontaminated). 

Efficacy was calculated using Equation 2-1, below, for each material within each combination of 

decontamination procedure (i) and test material (j). 

LR = ∑log( CFU ) / N −∑log( CFU ) / N	 [2-1] ij ijc ijc ijk ijk c=1 k =1 

Where, 

LRi = Average LR of spores on a specific material surface 

Average of the logarithm of the number of viable spores (determined ∑log( ) / CFUijc NC = by CFU) recovered on the control coupons [C = control, j = coupon c=1 number, and NC = number of coupons (1, j)] 

Average of the logarithm of the number of viable spores (determined 

by CFU) remaining on the surface of a decontaminated coupon [S = ∑log( ) / 

k 

CFU ijk Nijk =	 decontaminated coupon, k = coupon number, and Nt = number of

coupons tested (1, k)] 

When no viable B. atrophaeus spores were detected, the detection limit of the sample was used, and the 

efficacy was reported as greater than or equal to the value calculated using Equation 2-1. 

The pooled standard error for the LR was calculated using Equation 2-2 as follows: 

[2-2] 

Where, 

SSUUUU =	 STD of LR values for untreated carriers (positive controls) 

SSTTTT = STD of LR values for treated carriers (post-decontamination samples) 

NNUUUU = Number of control samples 

NNTTTT = Number of post-decontamination samples 

8 
 

 

 

 

 

 

 

 

3 Sampling Approach 

This section discusses the sampling approach, including sampling site environmental conditions, 

sampling media, wipe sampling procedures, sampling frequency, critical measurements, prevention of 

cross-contamination during sampling, collection of representative samples, sample storage and 

preservation, and sample holding time. Digital video was collected during sampling. Photographs were 

taken of selected material coupons with any visible change due to the sampling procedure. 

Sampling Site Environmental Conditions 

Ambient environmental conditions such as temperature, relative humidity (RH), and barometric pressure 

can affect the evaporation rate of liquids from surfaces. All tests were conducted at room temperature, 

ambient RH, and ambient barometric pressure. RH and temperature were monitored during testing, and 

tests were conducted on days when environmental conditions were within 35 to 60% RH and 20 to 25 oC. 

All coupons were conditioned at ambient conditions for one week before use. 

Sampling Media 

Polyester rayon blend (PRB) 2- by 2-inch wipe (CurityTM All Purpose Sponges, Covidien, Mansfield, MA) 

samples were collected from each 42- by 14-inch section (three linear 14- by 14-inch sections labeled a, 

b, and c in Figure 2-4) of the positive control and test coupons. The PRB wipe samples were collected 

from coupon surfaces after the surfaces had dried following decontamination. A typical drying time of 30 

minutes was used unless some combination of surface and decontamination method was still visually wet 

after that time. 

The used cloths from R1 and liquid from the reservoirs from R2 were collected, neutralized, and plated as 

samples for initial tests. Wipe sampling always began in Area H. The robot decontamination always 

started in Area B (section g), but the robot always followed a random path. Stainless-steel coupons (14 by 

14 inches) were sampled to provide results indicating the MDI stability during inoculation. 

Wipe Sampling Procedures 

Wetted wipe sampling is typically used for small sample areas on nonporous smooth surfaces such as 

ceramics, vinyl, metals, painted surfaces, and plastics. The general approach is to use a moistened, 

sterile, nonwoven PRB cloth to wipe a specified area to recover bacteria, viruses, and biological toxins. 

The protocol used for this project was adapted from that provided by Brown et al [2]. The wetted wipe was 

used to sample a 14- by 42-inch area. Wipe samples were extracted in 20 mL of Phosphate Buffered 

Saline with 0.05% TWEEN® 20 (PBST), sonicated, vortexed, and subjected to serial 10-fold dilution and 

spread-plating. The sampling sequence started from Area H (sections c, f, and i or strip H3) and ended in 

Area A (sections a, d, g or strip A1). 

LR was determined using the sum of viable spores collected on wipes from all sections of each coupon 

compared to the number of viable spores collected on wipes from positive control coupons not subjected 

to decontamination. 

9 
 

 

 

 

Sampling Frequency 

Table 3-1 lists the sampling frequency for all samples collected. 

Table 3-1. Sampling Frequency 

Sample Type or 

Measurement Quantity Frequency Location or Condition Purpose 

Test Coupon 

Samples 

Three samples per 

coupon section for a 

total of 24 wipe 

samples (8 sections) 

One set per test Decontaminated 

To determine the number of 

viable spores after 

decontamination 

Negative Control 

Coupon Samples 

One per test coupon 

replicate for a total 

of 3 samples 

One per 

decontamination 

Not 

decontaminated 

To determine extent of 

cross-contamination and the 

sterility of coupons 

Positive Control 

Coupon Samples 

One per test coupon 

replicate for a total 

of 3 samples 

One set per 

material 

Not 

decontaminated 

To determine the number of 

viable spores recoverable 

from the coupons 

MDI Control 

Coupon (stainless 

steel) Samples 

At least two per 

inoculation event as 

the first and the last 

actuation of the MDI 

One set per 

inoculation 

Not 

decontaminated 

To determine the number of 

viable spores deposited 

onto the coupons, and to 

assess the stability of the 

MDI 

Laboratory Material 

Blank Samples Three per material One per use of 

material Not applicable 

To demonstrate sterility of 

extraction and plating 

materials 

Material Sterility 

Check (swab) 

Samples 

One per sterilized 

batch of ADAs, 

coupons, and robots 

Once before 

testing Not applicable To demonstrate sterility of 

ADAs 

Decontamination 

Liquid Test-dependent Once before and 

after each test Not applicable 

To determine the 

concentration of active 

ingredients in the solution, 

and to verify the solution is 

within the target 

concentration 

RH and 

Temperature 1 Logged every 10 

seconds Glovebox 

To determine environmental 

conditions during 

decontamination 

10 
 

 

 

 

 

 

 

 

 

Critical Measurements 

Table 3-2 lists critical and noncritical measurements for each relevant sample type. 

Table 3-2. Critical and Noncritical Measurements 

Sample Type Critical Measurements Non-critical 

Measurements 

Test Coupon 

Samples 

Plated volume, incubation temperature, extracted volume, and colony 

forming units (CFU) 

Storage time and 

temperature 

Negative Control 

Coupon Samples Plated volume, incubation temperature, extracted volume, and CFU Storage time and temperature 

Positive Control 

Coupon Samples Plated volume, incubation temperature, extracted volume, and CFU Storage time and temperature 

Field Blank 

Coupon Samples Plated volume, incubation temperature, extracted volume, and CFU Storage time and temperature 

Laboratory Blank 

Coupon Samples Plated volume, incubation temperature, extracted volume, and CFU Storage time and temperature 

Laboratory Material 

Blank Samples Plated volume, incubation temperature, extracted volume, and CFUs Storage time and temperature 

pAB pH, hypochlorite concentration, and storage time Temperature 

Spor-Klenz® pH, H2O2 concentration, PAA concentration, and storage time Temperature 

Prevention of Cross-Contamination of Samples during Sampling 

Sampling posed a potentially significant opportunity for cross-contamination of samples. To minimize the 

possibility of cross-contamination, the management controls summarized below were followed. 

•	 Lines were drawn with a permanent marker to delimit the sampling sections, and sampling was 

contained within these lines. 

•	 In accordance with aseptic technique, a two-person sampling team was used, consisting of a 

“sampler” and a “sample handler.” 

•	 The sample handler was designated as the only person to operate the automated dosing apparatus 

(ADA) and handle the test coupons during the sampling event. 

•	 The sampler handled only the sampling media (wipes and liquids) and performed the surface 

sampling of the test coupons. 

•	 At the completion of each sampling event, each member of the sampling team changed gloves in 

preparation for working with the next sample. 

As a further precaution to avoid cross-contamination of samples, the order of coupon sampling was from 

coupons expected to be the least contaminated with B. atrophaeus to those expected to be most 

contaminated as follows: (1) all blank coupons, (2) all decontaminated coupons, and (3) positive control 

coupons. 

The National Homeland Security Research Center (NHSRC) Biocontaminant Laboratory (biolab) located 

at the EPA facility in Research Triangle Park, NC, analyzed the samples with strict adherence to aseptic 

laboratory techniques to recover, plate, culture, and analyze samples. The order of analysis was the 

same as the order of sampling as follows: (1) all blank coupons, (2) all decontaminated coupons, and (3) 

all positive control coupons. 

11 
 

 

Collection of Representative Samples 

The representativeness and uniformity of test coupon materials was considered essential in achieving 

defensible evaluation results. Material representativeness was achieved by using materials typical of 

those currently used in buildings and facilities in terms of quality, surface characteristics, and structural 

integrity. The materials conformed to industry standards or specifications for indoor use. Material 

uniformity was achieved by obtaining a quantity of material sufficient to allow multiple test samples to be 

prepared with presumably uniform characteristics. 

Sample Storage and Preservation 

After sample collection, sample integrity was accomplished through triple containment of the samples in: 

(1) a sample collection container, (2) a sterile bag with the exterior sterilized during the sample packaging 

process, and (3) a clean container holding all samples from a test. All individual sample containers 

remained sealed in the coupon decontamination laboratory and during transport to the NHSRC 

Biocontaminant Laboratory. The sampling person did not handle any samples after they were 

relinquished to the sample handler during placement into the primary sample container. 

All samples received were stored in a refrigerator at 4 °C ± 2 °C until analysis. All samples were allowed 

to stabilize at room temperature for one hour before analysis. 

12 
 

 

 

 

 

 

4 Analytical Procedures for Microbiological Analyses 

This section discusses analytical procedures for microbiological analyses, including the filtration and 

plating of bacteria from the PRB wipe samples used to determine the spores remaining on the coupons. 

The NHSRC Biocontaminant Laboratory analyzed all samples for spore presence (sterility check 

samples) and to quantify the CFU per sample. A laboratory notebook was used to document the details of 

each sampling event (or test). 

Spores were extracted from the PRB wipe samples by adding 20 mL PBST to each tube, then agitating 

the tubes using a vortex mixer (set to maximum rotation) for wo minutes in ten-second intervals. For all 

sample types, after extraction, the liquid extracts were serially diluted ten-fold (in PBST). The PBST buffer 

used was subjected to a five-stage serial dilution (10-1 to 10-5). Then 0.1 mL of the extract was spreadplated onto tryptic soy agar (TSA) plates in triplicate and incubated overnight (18 to 24 hours) at 35 °C ± 2 

°C. CFU were enumerated visually. Only dilution plates containing between 30 and 300 CFU were used 

for recovery estimates. Extracts were diluted and replated if none of the 10-fold dilutions resulted in all 

three plates containing CFU counts within the acceptable range. 

Total spore recovery was calculated by multiplying the mean CFU counts from triplicate plates by the 

inverse of the volume plated, by the dilution factor, and finally by the total volume of the extract. Any 

samples with CFU counts below the acceptable range (30 to 300 CFU) on the primary dilution plates 

were subsequently filter-plated through 0.2-micrometer-pore-size filters (Nalgene, Rochester, NY), with 

the filters placed onto TSA plates, followed by incubation at 35 °C ± 2 oC for 18 to 24 hours. The CFU 

counts from these plates were used to calculate recovery in these circumstances. Figure 4-1 shows a 

dilution plate and a filter plate with colonies of B. atrophaeus. 

Figure 4-1. Dilution Plate (Left) and Filter Plate (Right) Showing Colonies of B. atrophaeus 

13 
 

 

 

 

 

 

 

 

 

5 Test Results 

This section discusses data reduction and the results of the tests evaluating the R1 and R2 robotic 

cleaners for inactivating B. atrophaeus on two test material surfaces (laminate and vinyl). Appendix A to 

this report provides a complete set of spatial sampling data for each test condition. 

R1 (Mint Automatic Floor Cleaner 4200) Test Results 

The first set of R1 tests were conducted following a hot spot inoculation (14- by 14-inch section, e, of 

Area A) on a vinyl coupon (42 by 336 inches) with a Hype-WipeTM cloth mounted to the robotic cleaner. 

Table 5-1 summarizes the results. The positive controls column shows the total number of CFU recovered 

from the positive control coupon, and the test coupon column shows the total number of CFU recovered 

from the entire 42- by 336-inch test coupon surface for each replicate experiment (3 replicates total for 

each test). The LR calculated using Equation 2-1 shows that none of the tests were effective at removing 

spores below a single LR, and the average of all three replicates gives a sample LR of 0.67, with a 

sample pooled standard error of 0.17. This average is well below the 6 LR set as the target for laboratory 

based testing if sporicidal decontaminants. Further, the results suggest strongly that the R1 robotic 

cleaner would not be effective overall. It was initially decided to run only a single test using a pAB-treated 

cloth to determine if additional testing would be needed. Table 5-1 also summarizes the results of this 

test, which showed only a 0.73 LR in spore count. Therefore, it was decided to halt experiments using the 

R1 robotic cleaner because it proved ineffective under the best circumstances. 

Table 5-1. R1 Decontamination Results Summary 

Test Area Surface 

Type Cloth Type Average Recovery (CFUs) LR Average 

Positive Controls Test Samples 

Hot Spot 

A 

Vinyl Hype-WipeTM 1.30 ± 0.31 x 107 3.06 ± 0.18 x 107 0.67 ± 0.17 

pAB 1.30 107 2.40 107 0.73 

Figure 5-1 shows the results of the spatial sampling for: (a) replicate 1 of the Hype-WipeTM on vinyl test, 

and (b) the single pAB-treated cloth on vinyl. Areas A through H in the figure represent the 42- by 42-inch 

test coupon areas, P represents the full positive control coupon, and M represents the stainless-steel test 

coupon used to check the sampling efficiency of the test swabs. The color scale indicates the order of 

magnitude of the total number of CFU recovered from each sampling area. 

The initial deposition was only in section A2 of the large test coupon. Figure 5-1 shows that a significant 

number of spores were recovered far from the inoculation zone, indicating not only that the R1 robotic 

cleaner was ineffective at decontaminating a single area. In addition it might also spread the spores. 

Since the current test area (100 ft2) is within the manufacturer’s recommended wet mopping area (~150 

ft2), this spore spread during R1 robotic cleaner operation may be due to the insufficient decontamination 

agent deployment. The positive control sampling results should have shown deposition only in the P2 

zone. The CFU in the P1 and P3 zones likely are attributable to human error from sampling overlap into 

zone P2. The CFU in the P1 and P3 zones were added to the P2 sample data to determine the total 

spore inoculation. No broad-area inoculation tests were conducted using the R1 robotic cleaner. 

14 
E-01 E+00 E+01 E+02 E+03 E+04 E+05 E+06 E+07 E+08 

(a) 

 

 

(b)

Figure 5-1. R1 Vinyl Hot Spot Test Results, Replicate 1 of (a) Hype-WipeTM Cloth and (b) pAB Cloth 

15 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

R2 (iRobot Scooba® 390) Test Results 

The R2 tests were conducted on both hot spot and broad area inoculations on vinyl and laminate 

coupons using both pAB and Spor-Klenz® in the reservoir of the cleaner. Table 5-2 summarizes the 

results. The positive controls column shows the total number of CFU recovered from the positive control 

coupon, and the test coupon column shows the total number of CFU recovered from the entire 14- by 

336-inch test coupon surface for each replicate experiment. The hot spot inoculation tests showed a 

maximum LR of 2.91 for Spor-Klenz® on vinyl. The average LR of the Spor-Klenz® hot spot 

decontamination experiments was 2.29, with a sample pooled standard error of 0.56. The pAB hot spot 

on vinyl test was not as successful as the Spor-Klenz® on vinyl test. Replicate 2 of the pAB hot spot tests 

on vinyl showed an LR of 2.71, which was much higher than the other two replicates. The overall average 

LR of the pAB on vinyl hot spot tests was 1.17, with a sample pooled standard error of 0.70. Only a single 

test was conducted with pAB on laminate, and result showing a 1.50 LR was consistent with the vinyl test. 

Therefore, no more hot spot tests were conducted. 

Table 5-2. R2 Decontamination Results Summary 

Test Area Surface 

Type 

Decontaminant 

Type 

Average Recovery (CFUs) 

LR Average 

Positive Controls Test Samples 

Vinyl pAB 1.57 x 107 ± 5.73 x 106 3.43 x 107 ± 3.16 x 106 1.17 ± 0.70 

Hot Spot A Vinyl Spor-Klenz® 1.47 x 107 ± 1.63 x 106 1.57 x 107 ± 5.73 x 106 2.29 ± 0.56 

Laminate pAB 1.08 x 107 ± 0.00 x 100 3.46 x 107 ± 0.00 x 106 1.50 ± 0.00 

Broad Area 

Vinyl pAB 9.41 x 107 ± 8.14 x 107 1.57 x 107 ± 5.73 x 106 0.84 ± 0.48 

Vinyl Spor-Klenz® 1.96 x 108 ± 9.83 x 107 2.05 x 107 ± 2.03 x 107 1.10 ± 0.33 

Figure 5-2 shows the spatial distribution of CFU left after decontamination for (a) replicate 2 of pAB on vinyl, 

(b) replicate 1 of Spor-Klenz® on vinyl, and (c) the single pAB on laminate test. Here, (a) and (b) represent 

the replicates with the highest LR for their respective test conditions. Areas A through H in the figure 

represent the 42- by 42-inch test coupon areas, P represents the full positive control coupon, and M 

represents the stainless-steel test coupon used to check the sampling efficiency of the test swabs. The color 

scale indicates the order of magnitude of the total number of CFU recovered from each sampling area. 

Spor-Klenz® was more successful than pAB at decontamination and much more successful at limiting 

spread of the inoculum to previously uncontaminated areas (Areas B through H). Figures A-3 and A-4 in 

Appendix A show the spatial sampling data for each triplicate of the pAB on vinyl and Spor-Klenz® on vinyl 

tests, respectively. These replicates also show the limited spread of inoculum in the Spor-Klenz® tests 

compared to the pAB tests. Although the Spor-Klenz® tests showed promise, all of the LR results were well 

below the 6 LR target used to define effective sporicides in laboratory based testing. 

However, to be thorough, two broad area decontamination tests were conducted for pAB and Spor-Klenz® 

on vinyl. These tests showed an LR below 2 in surface spores. 

Figure 5-3 shows the best decontamination replicate results from the two broad area inoculation tests for (a) 

replicate 2 of pAB on vinyl and (b) replicate 2 of Spor-Klenz® on vinyl. In this case, as opposed to the hot 

spot area tests, the total initial inoculation was calculated by sampling the positive control coupon and 

multiplying the results by 4 because Areas A through D were inoculated. Spor-Klenz® is, on average, 

significantly better than pAB at limiting the spread of inoculum to areas outside the initial contamination zone 

(Areas A through D), although significant spread is shown in Figure A-6 (c) in Appendix A. 

16 
E-01 E+00 E+01 E+02 E+03 E+04 E+05 E+06 E+07 E+08 

(a) 

(b) 

Figure 5-2. Highest R2 Hot Spot LR Results for: (a) pAB on Vinyl, Replicate 2; and (b) Spor-Klenz® on Vinyl, Replicate 1, and (c) pAB on 

Laminate 

17 
(c) 

Figure 5-2. [Continued] 

18 
(a) 

(b) 

Figure 5-3. Highest R2 LR Results, Broad Area Inoculation 

19 
 

 

 

 

 

6 Quality Assurance and Quality Control 

This project was performed under the approved Category III quality assurance project plan (QAPP), 

“Evaluation of Sporicidal Wipes and Liquid Agents for Decontamination of Anthrax-Contaminated 

Surfaces by Hand and Robotic Cleaners,” November 2014. 

All test activities were documented via narratives in laboratory notebooks and the use of digital 

photography. The documentation included, but was not limited to, a record for each decontamination 

procedure, any deviations from the QAPP, and physical impacts on materials. All tests were conducted in 

accordance with established Decontamination Technologies Research Laboratory (DTRL) and NHSRC 

Biocontaminant Laboratory procedures to ensure repeatability and adherence to the data quality 

validation criteria set for this project. 

Criteria for Critical Measurements/Parameters 

The data quality objectives (DQOs) are used to determine 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 critical to 

accomplish part or all of the project objectives: 

• Weight of the wipes; 

• Sample volume collected; 

• Plated volume; and 

• Counts of CFU. 

The data quality indicators (DQIs) for the critical measurements listed in Table 6-1 were used to 

determine if the collected data met the quality assurance objectives. If a measurement method or device 

resulted in data that did not meet these goals, the data derived from the critical measurement were 

rejected. For instance, if the plated volume of a sample was not known (i.e., was not 100% complete), 

then that sample was declared invalid. If a collected sample was lost or did not meet the criteria for other 

reasons, then another sample was collected to take its place. 

Table 6-1. Critical Measurement Criteria 

Critical Measurement Measurement Device Accuracy Precision Detection Limit 

Sample volume Serological pipette Subdivision 0.5 mL + 0.2 mL + 0.1 mL 

Plated volume Pipette ± 2% ± 1% NA 

CFU/plate Counting ± 10% (between 2 counters) ± 5 1 CFU 

Weight Scale 0.004 g 0.0001 g NA 

NA = not applicable 

20 
Quality Control Checks 

Many QA/QC checks were used in this project to ensure that the data collected met all the critical 

measurements listed in Table 6-1. The measurement/parameter criteria were set at the most stringent 

level that can routinely be achieved. The integrity of the sample during collection and analysis was 

evaluated. Control samples and procedural blanks were included along with the test samples so that wellcontrolled quantitative values were obtained. Background checks for the presence of bacterial spores 

were included as part of the standard protocol. Replicate coupons were included for each set of test 

conditions. Specific quality control checks that were performed in this project are described in the 

following sections. 

6.2.1 Integrity of Samples and Supplies 

Samples were carefully maintained and preserved to ensure their integrity. Samples were stored away 

from standards or other samples which could possibly cross-contaminate them. 

Project personnel carefully checked supplies and consumables prior to use to verify that they met 

specified project quality objectives. All pipettes were calibrated yearly by an outside contractor (Calibrate, 

Inc.), incubation temperature was monitored using NIST-traceable thermometers, and balances were 

calibrated yearly by the EPA Metrology Laboratory. 

6.2.2 NHRSC Biolab Control Checks 

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, the CFU were 

enumerated manually and recorded. If the CFU count for bacterial growth did not fall within the target 

range, the sample was either filtered or re-plated. For each set of results (per test), a second count was 

performed on 25 percent of the plates within the quantification range (plates with 30 - 300 CFU). All 

second counts were found to be within 10 percent of the original count. 

QA/QC Sample Acceptance Criteria 

The acceptance criteria for the critical CFU measurements were set at the most stringent level that could 

be achieved routinely. Positive controls and procedural blanks were included along with the test samples 

in the experiments so that well-controlled quantitative values were obtained. Background checks were 

also included as part of the standard protocol. Replicate coupons were included for each set of test 

conditions. Further QC samples were collected and analyzed to check the ability of the NHSRC Biolab to 

culture the test organism, as well as to demonstrate that materials used in this effort did not themselves 

contain spores. The checks included the following: 

 

•	 Negative control coupons: sterile coupons that underwent the same sampling process without 

spore deposition. 

•	 Field blank coupons: sterile coupons carried to the decontamination location but not 

decontaminated. 

•	 Laboratory blank coupons: sterile coupons not removed from NHSRC Biolab. 

•	 Laboratory material coupons: includes all materials, individually, used by the NHSRC Biolab in 

sample analysis. 

•	 Stainless steel positive control coupons: coupons inoculated but not decontaminated. 

21 
 

 

 

 

 

 

QA/QC acceptance criteria are shown in Table 6-2. These provide assurances against crosscontamination and other biases of microbiological samples. 

Table 6-2. QA/QC Sample Acceptance Criteria 

Sample Type Purpose Acceptance Criteria Corrective Actions Frequency 

Negative control 

coupons 

Determine extent of 

cross-contamination in 

test area. 

No detectable 

spores. 

No corrective actions 

were taken. Values 

on coupons were 

considered to have 

resulted from crosscontamination. 

One per test 

Field blank 

coupons 

Verify the presence of 

coupons does not 

introduce contamination into samples. 

No detectable 

spores. 

Determine source of 

contamination and 

remove. 

One per sample 

type per test 

Laboratory blank 

coupons 

Verify the sterility of 

coupons using swabs 

No detectable 

spores. 

Determine source of 

contamination and 

remove. 

One per test per 

coupon type 

Laboratory 

material coupons 

Verify the sterility of 

materials used to 

analyze viable spore 

count. 

No detectable 

spores. 

Determine source of 

contamination and 

remove. 

Three per material 

per test 

Blank TSA 

sterility control 

(plate incubated, 

but not 

inoculated) 

Controls for sterility of 

plates. 

No observed growth 

following incubation. 

All plates are 

incubated prior to 

use, so any 

contaminated ones 

were discarded. 

Each plate 

Positive control 

coupons 

Used to determine the 

extent of inoculation 

on the coupons. 

5 x 106 CFU, ± 0.5 

log or 5 x 104 CFU, 

± 0.5 log. 

Outside target range: 

discuss potential 

impact on results 

with EPA WACOR; 

correct loading 

procedure for next 

test and repeat 

depending on 

decided impact. 

One per test 

Inoculation 

control coupons 

Used to determine 

drift in the MDI. 

CFU recovered from 

the first coupon 

must be ±0.5 log of 

the last coupon. 

Reject results and 

repeat test. 

Two per inoculation 

Replicate plating 

of diluted 

microbiological 

samples 

Used to determine 

variability in CFU 

counts. 

Reportable CFU of 

triplicate plates 

must be within 

100%. Reportable 

CFU are between 

30 and 300 CFU per 

plate. 

Re-plate sample. Each sample 

22 
 

 

 

6.3.1 QA/QC Test Results Validation 

The QA/QC test results for the whole sampling campaign are shown in Table 6-3. All field blanks and 

inoculum blanks were found to be non-detects. However, some of the negative control blanks (2 out of 17) 

were found to have been on the order of 1 x 103 CFU per coupon. Although some of the negative controls 

were detects, they do not have a significant bearing on the overall results since decontamination results of 

no more than 3 LR were found. 

Table 6-3. QA/QC Test Results 

Sample Robot Material Inoculation 

Type 

Decontamination 

Type 

Average Recovery (CFU per coupon) 

Negative 

Control Field Blank 

Inoculum 

Control 

Blank 

Replicate 1 

Mint 

Vinyl 

Hot-Spot A Hype-WipeTM 

9 ND 1 

Replicate 2 83 ND ND 

Replicate 3 671 ND ND 

Replicate 1 pAB 375 ND ND 

Replicate 1 

Scooba 

Broad Area 

pAB 

2 ND ND 

Replicate 2 ND ND ND 

Replicate 3 7 ND ND 

Replicate 1 

Spor-Klenz® 

49 ND ND 

Replicate 2 6 ND ND 

Replicate 3 26 ND ND 

Replicate 1 

Hot-Spot A 

pAB 

33 ND ND 

Replicate 2 956 4 ND 

Replicate 3 22 ND 2 

Replicate 1 

Spor-Klenz® 

ND ND ND 

Replicate 2 4 ND ND 

Replicate 3 32 ND ND 

Replicate 1 Laminate pAB 4 ND ND 

6.3.2 Sample Holding Time 

After sample collection for a single test was completed, all biological samples collected for that test were 

immediately transported to the NHSRC biolab along with the appropriate chain of custody form(s). The 

QAPP for this project stated that samples were to be stored no longer than five days before beginning 

primary analysis. However, given the volume of samples generated over a short period of time, the 

samples had to be stored in the refrigerator for longer than five days before primary analysis began. 

Therefore, tests were conducted to evaluate the potential reduction of bacterial spores in the wipe sample 

containers as a function of time. The results from the sample holding tests showed no reduction of 

bacterial spores. Details of this testing and the results are provided in Appendix B. 

23 
 

 

 

7 Summary 

The R1 tests using either Hype-WipeTM or pAB treated wipe mounted to the robot demonstrated an 

overall log reduction of less than 1. The results of a set of experiments conducted to determine the extent 

of potential spreading of the contamination, using the R1 with either Hype-WipeTM or a regular mopping 

cloth impregnated with 60 mL of pAB, show that the R1 was not effective (less than 1 log reduction) 

enough to decontaminate a hot spot area and R1 operation increases the total area contaminated by 

spreading. This may be due to the insufficient liquid decontamination agent available for the entire test 

surfaces. The robot is programmed to cover the entire test area (100 ft2) which is within the manufacture 

estimated coverage area (150ft2), so it is highly possible that the decontamination agent from the robot 

might have run out before completion. 

The R2 tests resulted in a better decontamination efficacy, with Spor-Klenz faring better than pAB in 

limiting the spread of the contamination and less spreading of the contamination outside the inoculation 

zone. However, a sub 3 log reduction overall decontamination efficacy was found for this robot, 

independent of the type of liquid decontaminant used. For laboratory based testing, demonstration of a 6 

LR is the target for an effective sporicide under the Federal Insecticide, Fungicide, and Rodenticide Act 

(FIFRA). The low efficacy may be due to the lack of contact time between decontamination agent and 

surface since the R2 is designed to apply the liquid and retrieve it in a relatively short period of time (less 

than 5 seconds). The previous field study used the 10 min contact time of pH-amended bleach on all 

surfaces as application procedure and this application method was highly efficacious.4 

In summary neither robot successfully performed a 6 Log reduction in total spore count nor significantly 

reduced the spores loading on the material surfaces. Spor-Klenz appears to be more successful than 

pAB and increasing the reservoir for the Scooba robot may increase the log reduction. Spor-Klenz did 

help limit the spread of the contaminant to new areas though some spread of viable spores was still seen. 

24 
 

 

 

 

 

 

8 References 

1.	 Lee, S.D., Calfee, M.W., Mickelsen, L., Clayton, M., and Touati, A. Scenario-based evaluation of 

commercially available cleaning robots for collection of Bacillus spores from environmental 

surfaces. Remediation, 2014, 24(2):123–133. 

2.	 Brown, G.S., Betty, R.G., Brockmann, J.E., Lucero, D.A., Souza, C.A., Walsh, K.S., Boucher, R.M., 

Tezak, M.S., Wilson, M.C., Rudolph, T., Lindquist, H.D., and Martinez, K.F. Evaluation of rayon 

swab surface sample collection method for Bacillus spores from nonporous surfaces. Journal of 

Applied Microbiology, 2007, 103(4):1074–1080. 

3.	 Lee, S.D., Ryan, S.P., and Snyder, E.G. Development of an aerosol surface inoculation method for 

Bacillus spores. Applied and Environmental Microbiology, 2011, 77(5):1638–1645. 

4.	 U.S. EPA. Bio-Response Operational Testing and Evaluation (BOTE) Project - Phase 1: 

Decontamination Assessment. U.S. Environmental Protection Agency, Washington, DC, 

EPA/600/R-13/168, 2013. 

25 
Appendix A: Spatial Sampling Data 

E-01 E+00 E+01 E+02 E+03 E+04 E+05 E+06 E+07 E+08 

(a) 

(b) 

Figure A-1. R1 Test Results on Vinyl, Hype-WipeTM Cloth, Hot Spot Inoculation 

A-1 
(c) 

Figure A-1 [continued]. R1 Test Results on Vinyl, Hype-WipeTM Cloth, Hot Spot Inoculation 

A-2 
Figure A-2. R1 Test Results on Vinyl, pAB Cloth, Hot Spot Inoculation 

A-3 
(a) 

(b) 

Figure A-3. R2 Test Results on Vinyl, pAB Solution, Hot Spot Inoculation 

A-4 
(c) 

Figure A-3 [continued]. R2 Test Results on Vinyl, pAB Solution, Hot Spot Inoculation 

A-5 
(a) 

(b) 

Figure A-4. R2 Test Results on Vinyl, Spor-Klenz® Solution, Hot Spot Inoculation 

A-6 
(c) 

Figure A-4. [continued] R2 Test Results on Vinyl, Spor-Klenz® Solution, Hot Spot Inoculation 

A-7 
(a) 

(b) 

Figure A-5. R2 Test Results on Vinyl, pAB Solution, Broad Area Inoculation 

A-8 
 

(c) 

Figure A-5 [continued]. R2 Test Results on Vinyl, pAB Solution, Broad Area Inoculation 

A-9 
(a) 

(b) 

Figure A-6. R2 Test Results on Vinyl, Spor-Klenz® Solution, Broad Area Inoculation 

A-10 
(c) 

Figure A-6 [continued]. R2 Test Results on Vinyl, Spor-Klenz® Solution, Broad Area Inoculation 

A-11 
 

 

 

 

 

 

 

 

 

 

 

Appendix B: Sample Holding Time 

The QAPP for this project stated that samples were to be stored no longer than five days before 

beginning primary analysis. However, given the volume of samples generated over a short period of time, 

the samples had to be stored in the refrigerator for longer than five days before primary analysis began. 

Therefore, the tests described here were conducted to evaluate the potential reduction of bacterial spores 

in the wipe sample containers as a function of time. 

In this testing sequence, the following hypochlorite sporicidal wipes were tested on large coupon surfaces 

to estimate the occurrence and potential reduction of bacterial spores as a function of remediation 

activities: 

•	 Clorox® HealthcareTM bleach germicidal wipe; 

•	 Hype-WipeTM bleach towelette; and 

•	 pAB wipe prepared in house by soaking FisherbrandTM dry wipes in 4 mL pAB 

The surface tested was glass (to represent nonporous indoor building materials). The coupons measured 

3.5 by 3.5 feet (12.25 ft2). The test coupons were first subjected to a specific decontamination wipe. The 

coupons were not inoculated before decontamination. Table B-1 summarizes the test matrix. 

The testing approach consisted of the following steps: 

1.	 After an exposure time of 30 minutes, the coupons were wipe sampled as follows: 

a) Sections a, b, and c were sampled together and labeled as the Day 1 sample; 

b) Sections d, e, and f were sampled together and labeled as the Day 4 sample; and 

c) Sections g, h, and i were sampled together and labeled as the Day 7 sample. 

2.	 Each sampled wipe container was inoculated on Day 1 (1 day after sampling) with 0.1 mL of 2 x 108 

CFU/mL microbial suspension (test organism solution) to result in a final suspension containing 

approximately 2 x 107 CFU of the microorganism. 

3.	 The samples were analyzed by adding 20 mL of PBST to each container on the scheduled day (Day 1, 

4, or 7), followed immediately by extraction and plating. 

Table B-1. Holding Time Testing Sequence Test Matrix 

Test ID Decontaminant Coupon 

Section 

Analyzed and 

Plated (Day) 

No. of Wipe 

Samples 

73-(C/H/P)-0-G-1-Ta-1 a 

Day 1 

C test samples (3) 

H test samples (3) 

P test samples (3) 

73-(C/H/P)-0-G-1-Tb-1 b 

73-(C/H/P)-0-G-1-Tc-1 C = Clorox® HealthcareTM c 

73-(C/H/P)-1-G-1-Td-1 bleach germicidal wipe d 

Day 4 

C test samples (3) 

H test samples (3) 

P test samples (3) 

73-(C/H/P)-1-G-1-Te-1 H = Hype-WipeTM bleach e 

73-(C/H/P)-1-G-1-Tf-1 towelettes f 

73-(C/H/P)-7-G-1-Tg-1 P = pAB wipe g 

Day 7 

C test samples (3) 

H test samples (3) 

P test samples (3) 

73-(C/H/P)-7-G-1-Th-1 h 

73-(C/H/P)-7-G-1-Ti-1 i 

B-1 
 

 

 

 

 

 

The positive control and negative control coupons were not subjected to the decontamination wipe. The 

negative control coupon and each of the positive control coupon sections were directly wipe-sampled with 

a PRB wipe. While the positive coupons were inoculated with spores, the negative control coupons were 

not. The procedural blank coupon was decontaminated with the hypochlorite wipe and then sampled but 

not inoculated. Table B-2 shows the control sample test matrix for this sample holding time study. 

Table B-2. Holding Time Testing Sequence Control Sample Test Matrix 

Test ID Type Decontaminant Coupon 

Section 

Analyzed 

and Plated 

(Day) 

No. of Wipe 

Samples 

73-(C/H/P)-0-G-1-Pa-1 

Positive 

control 

C = Clorox® 

HealthcareTM 

bleach 

germicidal wipe 

H = HypeWipeTM bleach 

towelette 

P = pAB 

a 

Day 1 

C test samples (3) 

H test samples (3) 

P test samples (3) 

73-(C/H/P)-0-G-1-Pb-1 b 

73-(C/H/P)-0-G-1-Pc-1 c 

73-(C/H/P)-1-G-1-Pd-1 d 

Day 4 

C test samples (3) 

H test samples (3) 

P test samples (3) 

73-(C/H/P)-1-G-1-Pe-1 e 

73-(C/H/P)-1-G-1-Pf-1 f 

73-(C/H/P)-7-G-1-Pg-1 g 

Day 7 

C test samples (3) 

H test samples (3) 

P test samples (3) 

73-(C/H/P)-7-G-1-Ph-1 h 

73-(C/H/P)-7-G-1-Pi-1 i 

73-(C/H/P)-(0/1/7)-G-1-XT-1 Procedural 

blank NA 

0 = Day 1 

1 = Day 4 

7 = Day 7 

1 per 

decontamination 

wipe per day (9) 

73-(C/H/P)-(0/1/7)-G-1-NT-1 Negative 

control NA 

1 per 

decontamination 

wipe per day (9) 

The results from the hold tests are summarized in Table B-3 and indicate that the samples were not 

affected by the longer holding time before analysis. There was no reduction of bacterial spores in the 

sample containers as a function of time. 

Table B-3. Sample Holding Test Results for Decontamination Efficacy Tests 

Wipe Cloth Day 

Positive Control 

Average Recovery 

(CFU) 

Test Sample Average 

Recovery 

(CFU) 

LR 

Average STD Average STD Average STD 

Clorox® 

HealthcareTM 

bleach germicidal 

wipe 

1 1.42 x 107 9.33 x 105 1.34 x 107 9.34 x 105 0.02 0.03 

4 1.40 x 107 1.04 x 106 1.35 x 107 1.83 x 106 0.08 0.06 

7 1.26 x 107 4.82 x 105 1.55 x 107 1.08 x 106 -0.04 0.03 

Hype-WipeTM 

bleach towelette 

1 1.37 x 107 1.48 x 106 1.46 x 107 7.57 x 105 -0.03 0.02 

4 1.44 x 107 5.18 x 105 1.40 x 107 4.54 x 105 -0.01 0.01 

7 1.21 x 107 4.73 x 105 1.36 x 107 3.85 x 105 0.00 0.01 

pAB wipe 

1 1.42 x 107 1.34 x 106 1.47 x 107 1.77 x 106 -0.01 0.05 

4 1.35 x 107 1.03 x 106 1.38 x 107 7.49 x 105 0.01 0.02 

7 1.29 x 107 9.05 x 105 1.27 x 107 4.23 x 105 0.05 0.01 

B-2 
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