EPA 600/R-10/177 | January 2011 | www.epa.gov/ord

Biological Sample Preparation 

Collaboration Project: 

Detection of Bacillus 

anthracis Spores in Soil

Final Study RepoRt

Office of Research and Development

National Homeland Security Research Center

Biological Sample Preparation 

Collaboration Project: 

Detection of Bacillus 

anthracis Spores in Soil

Final Study RepoRt

Meranda D. Bradley, Ph.D. 

Laura Rose, M.S.

Judith Noble-Wang, Ph.D.

Matthew Arduino, M.S., Dr.P.H. 

United States Environmental Protection Agency

Office of Research and Development

National Homeland Security Research Center

Cincinnati, Ohio 45268

Centers for Disease Control and Prevention

National Center for Emerging and Zoonotic Infectious Diseases

Atlanta, Georgia 30329
ii
Disclaimer

U.S. Environmental Protection Agency (EPA), National Homeland Security Research Center and 

the Centers for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic 

Infectious Diseases (Proposed), under IA #DW-75-92259701 (CDC IA# Cl10-001), collaborated in 

the development of the analysis procedure described here. 

This report has been peer and administratively reviewed and has been approved for publication 

as a joint EPA and CDC document. Note that approval does not signify that the contents 

necessarily reflect the views of the Agency. CDC and EPA do not endorse the purchase or sale of 

any commercial products or services. The findings and conclusions in this report are those of the 

author(s) and do not necessarily represent CDC or EPA.

Questions concerning this document or its application should be addressed to: 

Erin Silvestri, MPH

Project Officer

U.S. Environmental Protection Agency

Office of Research and Development

National Homeland Security Research Center 

26 W. Martin Luther King Drive, MS NG16 

Cincinnati, OH 45268 

513-569-7619 

Silvestri.Erin@epa.gov 

Laura Rose, MS

Centers for Disease Control and Prevention

National Center for Emerging and Zoonotic Infectious Diseases (Proposed)

Division of Healthcare Quality Promotion

Clinical and Environmental Microbiology Branch

1600 Clifton Avenue

Atlanta GA, 30329

404-639-2161

Lmr8@cdc.gov

If you have difficulty accessing these PDF documents, please contact Nickel.Kathy@epa.gov or 

McCall.Amelia@epa.gov for assistance. 

iii
iv
Foreword

Following the terrorist events of 2001, the U.S. Environmental Protection Agency’s (EPA) 

mission was expanded to meet critical needs related to homeland security. Presidential Directives 

identified EPA as the primary federal agency responsible for the country’s water supplies and 

for decontamination following a chemical, biological, and/or radiological attack. To provide 

scientific and technical support for EPA to meet this expanded role, EPA’s National Homeland 

Security Research Center (NHSRC) was established. The NHSRC research program is focused on 

conducting research and delivering products that improve the capability of the Agency to carry out 

its homeland security responsibilities. 

As a part of its long-term goals, NHSRC has been charged with delivery of detection techniques 

that will enable the rapid characterization of threats, identification of specific contaminants to 

protect workers, and development of plans for recovery operations. Substantial effort and resources 

have been invested in the development of molecular assays and culture techniques for pathogens; 

however, initial sample collection and preparation methodologies lag behind in development. To 

bridge this critical data gap, EPA collaborated with the Centers for Disease Control and Prevention 

to develop and optimize an immunomagnetic separation (IMS) method for isolation of Bacillus 

anthracis (BA) spores from soil. The developed BA IMS method will support environmental 

remediation and recovery activities. 

This report summarizes the experimental development of the method and the corresponding study 

results that move EPA one step closer to achieving our homeland security mission, and our overall 

mission of protecting human health and the environment. 

Jonathan Herrmann, Director

National Homeland Security Research

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vi
Acknowledgements

T

vii
viii
Contents

D

ix
4.0 Conclusions ................................................................................................................................... 15

5.0 References ..................................................................................................................................... 17

appendix a: Conjugation of paramagnetic Beads ............................................................................. 19

appendix B: time Resolved Fluorescence assay Summary ............................................................. 21

appendix C: Final protocols/Method for Real-World Sample analysis ............................................ 23

appendix d: Quality assurance and Quality Control (Qa/QC) ....................................................... 27

x
List of Figures

Figure 1. BeadRetriverTM automated immunomagnetic Separation Benchtop System........................ 6

Figure 2. average % Recovered Ba spores from sterile arizona test dust (atd), 

Minnesota loam (Ml), potting soil (pS), and sand (S) using HSGS extraction .................................. 9

Figrure 3. percentage recovery of Ba spores from soils using two different antibody 

to bead ratios ....................................................................................................................................... 10

Figure 4. effects of preprocessing on mean percent recovery of Ba spores (103 /g) 

from sterile arizona test dust .............................................................................................................. 11

Figure 5. SeM image of arizona test dust and sand processed through aiMS ................................. 12

xi
List of Tables

T

xii
List of Acronyms

%R percent Recovery

aBi applied Biosystems by life technologies 

aiMS automated immunomagnetic Separation

atCC american type Culture Collection

atd arizona test dust

Ba Bacillus anthracis

BSa Bovine Serum albumin

CFu Colony Forming units

CFu/g Colony Forming units per gram of soil

CdC Centers for disease Control and prevention

Coa Certificate of Analysis

dHHS department of Health and Human Services

edta ethylenediaminetetraacetic acid

epa 

h 

environmental protection agency

Hour

HSGS High Specific Gravity Sucrose

igG immunoglobulin G

iMS immunomagnetic Separation

lRn Laboratory Response Network

min Minute

Ml Minnesota loam

nHSRC national Homeland Security Research Center

nMRC naval Medical Research Center

pBS phosphate Buffered Saline

pBSt phosphate Buffered Saline with tween® 20

pCR polymerase Chain Reaction

pS potting Soil

Qa Quality assurance

Qapp Quality assurance project plan

QC Quality Control

RCF Relative Centrifugal Force 

Ro Reverse osmosis 

RpM Revolutions per Minute

RV-pCR Rapid Viability-polymerase Chain Reaction 

plet 

 

polymyxin B, lysozyme, ethylenediaminetetraacetic acid (edta), 

thallous acetate

xiii
s Seconds

Sd Standard deviation

SeM 

SNF 

SNFS 

SnS 

tRF 

Scanning electron Microscopy

Sonicating and filtering

Sonicating, filtering, and settling 

Sonicating and settling 

time Resolved Fluorescence

tSaii BBlTM trypticaseTM Soy agar with 5% Sheep Blood

xiv
Executive Summary

The objective of this work was to develop and optimize 

an automated immunomagnetic separation (AIMS) 

method for isolation of Bacillus anthracis (BA) 

spores from soil. In this study, the AIMS method for 

separating BA spores from various soil matrices was 

developed using the Dynabeads® technology (Invitrogen 

Corporation, Carlsbad, CA). This involved optimizing 

paramagnetic beads conjugation with antibodies 

specific to BA spores, designing an automated program 

(BeadRetrieverTM, Invitrogen) and developing standard 

procedures for separating BA spores from four soil types 

using AIMS. 

IMS technology utilizes paramagnetic beads conjugated 

with BA spore antibodies (polyclonal) to separate 

BA spores from spiked soil samples. Specifically, 

conjugated Dynabeads® MyOneTM beads were used 

for immunomagnetic separation of BA spores (Sterne 

34F2; Colorado Serum Company, Denver, CO) from 

four soil types; Arizona test dust (Powder Technology 

Inc, Burnsville, MN), Minnesota loam, potting soil, 

and sand (sources described below). Both sterile and 

non-sterile soils were evaluated. Spores were recovered 

from one gram of each of the soil type spiked with 

101-104colony forming units (CFU). Positive controls 

consisted of buffer (10X phosphate buffered saline with 

0.05%Tween® 20, PBST) spiked with the same inocula, 

negative controls consisted of soils without added spores. 

The percent of spores recovered (%R) was determined 

using the ratio of colony forming units per gram (CFU/g) 

recovered from the soil to the number of CFU of the 

spore stock inoculated into the gram of soil. The spore 

stock was cultured onto trypticase soy agar with 5% 

sheep blood plates (TSAII; BD, Franklin Lakes, NJ) and 

polymyxin B, lysozyme, ethylenediaminetetraacetic acid 

(EDTA)-thallous acetate (PLET) agar (non-sterile soils 

only). 

Although the soils were spiked with spore inocula 

ranging from 101-104, the lowest limit of reliable 

quantitation by AIMS varied. The limit of detection 

for all sterile soil types, as well as non-sterile sand and 

Arizona test dust was at least 102 spores/g. The limit 

of reliable quantitation (CFU ≥25 on each plate) for 

sand and Arizona test dust (sterile and non-sterile) was 

also 102 spores/g. The limit of reliable quantitation 

for Minnesota loam and potting soil, however, was 

103spores/g (sterile) and 104spores/g (non-sterile), 

respectively. The mean %R (±SD) of BA spores per 

gram of soil from inoculum levels 102 -104 varied by soil 

type, with 61.39% (±9.67) from sand, 38.08% (±4.38)

from arizona test dust, 29.09% (±5.79) from potting 

soil and 15.39% (±3.44) from Minnesota loam. the %R 

from the positive control (pBSt) was 59.50% (±7.89). 

Several published articles have evaluated various 

techniques for isolating Ba spore dna from soil (Ryu et 

al. 2003; Herzog et al. 2009; Gulledge et al. 2010). one 

frequently cited culture based method for recovering 

spores from soil is the high specific gravity sucrose 

(HSGS) plus non-ionic detergent method (dragon and 

Rennie 2001). When the HSGS method was compared 

to the iMS method, the %R using HSGS was lower and 

the limit of reliable quantitation was 1 to 2 orders of 

magnitude higher, depending upon the soil type. With 

the HSGS method, the %R ranged from 0.75% - 9.0 % 

with soils inoculated with 104-106 spores/g. the aiMS 

method was demonstrated to improve the efficiency of 

separating Ba spores from soil as compared to the HSGS 

extraction method. 

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1.0

Introduction

The U.S. Environmental Protection Agency (EPA) has 

identified detection of pathogenic microorganisms in 

environmental samples following a terrorist attack as a 

critical component of an effective response. Detection 

of such pathogens would require development and 

validation of sampling techniques that could be used 

by multiple laboratories following a homeland security 

event. To meet this requirement, EPA’s National 

Homeland Security Research Center (NHRSC), along 

with other EPA divisions and sister agencies, published 

a compendium of standard analytical methods, the 6th

revision in 2010 (EPA 2010). The compendium contains 

suggested assays for use by laboratories tasked with 

performing confirmatory analysis of environmental 

samples following a homeland security event. 

Sample preparation, however, remains the limiting step 

in the detection techniques available today whether 

for non-culture methods like PCR (polymerase chain 

reaction), semi-quantitative methods, or quantitative 

culture-based methods. Sample preparation includes 

extraction of the analyte from sample matrix for 

molecular (e.g., nucleic acid extraction) or culture-based 

techniques. Extracting pathogens from environmental 

matrices is challenging because the matrices are 

composed of non-target biological and chemical 

analytes. These non-target analytes may interfere 

(compete) with the extraction of the target analyte, and 

if present in the extracted product, they can inhibit PCR 

detection of the target

The Centers for Disease Control and Prevention (CDC), 

part of the U.S. Department of Health and Human 

Services (DHHS), has extensive knowledge of potential 

biological hazards. In this project, CDC, in collaboration 

with EPA, developed and improved methods for 

extraction of Bacillus anthracis (BA) spores from 

soil. Immunomagnetic separation (IMS) technology 

utilized tosylactivated beads conjugated with BA spore 

antibodies (polyclonal) to separate BA spores from 

spiked soil samples. IMS enables both concentration 

and purification of spores from soil samples. The 

IMS method for separating BA spores from various 

soil matrices in this study was developed using the 

Dynabeads® technology (Invitrogen Corporation, 

Carlsbad, CA). Due to the size of BA spores ( 1-2 

μm), Dynabeads®MyOneTM beads (1 μm in diameter) 

were conjugated with antibodies specific for BA spores 

(obtained from the Naval Medical Research Center 

(NMRC, Silver Spring, MD)). Conjugated Dynabeads 

MyOne beads are added to a slurry of BA spores and soil 

to allow binding of the spore antigens to the antibodies 

coating the beads Spore separation is performed in a 

BeadRetriverTM Automated Immunomagnetic Separation 

Benchtop System (Invitrogen). The magnetic beads, 

along with the bound target, are collected using a 

magnet. The excess solution and most of the soil is 

left behind, leaving only the beads with spores attached 

to the magnet. The beads are then washed to remove 

inhibitors and any remaining soil. The optimum bead 

conjugation parameters for the IMS protocol were 

determined in this investigation. The percent recovery 

(%R) of IMS method was compared to that of the 

published method for extraction of spores from soil, the 

high specific gravity sucrose plus non-ionic detergent 

method (HSGS, Dragon and Rennie 2001). 

1
2
2.0

Materials and Methods

2.1 Spore preparation

Bacillus anthracis Sterne 34F2 spores (BA) (Colorado 

Serum) were grown on soil extract peptone beef extract 

agar (Atlas 1996) at 35°C for seven days, harvested with 

sterile reverse osmosis (RO) water, and concentrated by 

centrifugation at 5000 RCF (relative centrifugal force) 

for 15 min. Centrifugation and washes were performed 

three times. The pelleted spores were placed in 50% 

ethanol (25 mL) at room temperature on a shaker table 

at 100 RPM (revolutions per minute) for 1 h. The 

suspension was centrifuged and washed with sterile 

RO water an additional 5 times and the spores were 

suspended in 5mL sterile RO water for storage at -70 

°C. Spore preparations were validated by culture and 

hemocytometry. The purified spore suspension density 

was determined by optical density and culture. 

2.2 Inoculum preparation 

The inoculum was initially prepared by diluting 

the frozen spore stock in sterile RO water to attain a 

concentration of 108 spores/mL. This standard was 

diluted in series with RO water, to make four spore 

concentrations; 104, 103, 102 and 101 spores/mL. These 

spore suspensions were quantified by culturing on TSAII 

(BBLTM TrypticaseTM Soy Agar with 5% Sheep Blood, 

Becton Dickinson Microbiology Systems, Franklin 

Lakes, NJ) at 35°C for 18 h. Colony forming units 

(CFU) were counted and used to determine the number 

of spores for inoculation of soils. A pilot experiment 

comparing spore suspensions diluted in sterile RO

water and in phosphate buffered saline, pH 7.4 with 

0.05% Tween® 20 surfactant (PBST) showed that the 

suspension in RO water yielded fewer CFU than the 

spores suspended in PBST. The PBST most likely 

assisted in disaggregating spores. Thereafter, the spore 

inoculum consisted of spores suspended in and diluted in 

series in PBST. 

2.3 High specific gravity sucrose 

extraction (HSGS)

In a 15 mL conical tube, autoclaved soil (2.5 g) was 

spiked with 104-106 BA spores. Sucrose extraction 

solution (12 mL of 1.22 g/mL sucrose/Triton®X-100 

surfactant) was added to each soil sample. The mixture 

was shaken by hand for 1 min, placed on a shaker at 75 

RPM for 15 min, and centrifuged at 850 RCF for 45 

s. The supernatant (3mL) was transferred to a new 15 

mL conical tube containing 6 mL filtered 1% bovine 

serum albumin in 0.01 mol/L phosphate buffered saline 

(BSA/PBS) and centrifuged in a swing bucket rotor 

at 5100 RCF for 10 min. The pellet was resuspended 

in 1 mL filtered 50% ethanol, gently agitated at room 

temperature for 60 min, and centrifuged at 5100 RCF 

for 10 min. The pelleted spores were resuspended in 1 

mL 1% BSA/PBS, 0.10 mL of purified samples were 

spread onto TSAII or PLET (Polymyxin B, Lysozyme, 

Ethylenediaminetetraacetic Acid thallous Acetate) agar 

plates, and incubated at 35°C for 24 - 48 h (Dragon and 

Rennie 2001). 

2.4 Soil inoculation

Four soil types were obtained for this project; Arizona 

test dust ( Powder Technologies, Inc.), potting soil (Home 

Depot, Atlanta, GA), Minnesota loam (Eagan, MN), sterile 

sand (Fisher Scientific, Sewanee, GA, cat# AC61235-

5000), and non-sterile sand (Destin, FL). 

The soil physical and chemical characterizations were 

performed by the Plant, and Water Laboratory at the 

University of Georgia, Athens, GA (Table 1).

3
Table 1. Physical and Chemical Properties of Soils

Soil Type Lime buffer 

capacity1 Soil pH2 Equivalent. water pH Base saturation3

 (Percent) Cation exchange capacity4

Arizona test dust NA5 8.08 8.68 100.00 29.87

Minnesota loam 371 4.51 5.11 61.94 4.27

Sand NA 7.87 8.47 100.00 0.22

Potting soil 618 7.46 8.06 100.00 26.99

Soil sample

Mineral Composition of Each Soil, ppm 

Calcium Cadmium Chromium Copper Iron Potassium Magnesium

Arizona test dust 5423 <0.01 <0.02 0.02 0.58 117.90 201.90

Minnesota loam 377 0.07 0.05 0.21 34.30 33.46 75.94

Sand 28 0.02 <0.02 0.04 7.21 4.89 5.56

Potting soil 4020 <0.01 0.06 0.11 3.47 95.44 897.10

Soil sample

Mineral Composition of Each Soil, ppm

Molybdenum Sodium Nickel Phosphorus Lead Zinc

Arizona test dust 0.05 176.30 0.06 6.69 <0.10 0.79

Minnesota loam <0.02 9.53 0.42 14.16 0.10 2.20

Sand <0.02 4.15 0.07 0.71 <0.10 0.62

Potting soil <0.02 24.99 0.24 30.92 <0.10 3.68

Soil sample

Percent Composition of Each Soil

Sand Silt Clay Total carbon

Arizona test dust 0.00 86.00 14.00 0.65

Minnesota loam 46.00 50.00 4.00 1.02

Sand 36.00 62.00 2.00 0.02

Potting soil ND6 ND ND ND

1

 ppm CaCO3/pH 2 Measurement of pH in dilute salt (http://www.caes.uga.edu/publications/caespubs/pubcd/C875.html)

3 Percent of soil exchange sites occupied by basic cations 4 Measures the soils ability to retain nutrients; unit of measurement millequivalent/100g of soil

5 Not applicable 6 Not done

4
Soils were inoculated as is, or sterilized (autoclaved at 

121oC, 32.5 psi for 30 min). One mL of spore suspension 

was added to 1 g of soil, and 2 mL 10X buffer (positive 

control). Two to 3.5 mL of 10X PBST was added to 

the soils to form a slurry. An evaluation of two buffer 

concentrations (1X and 10X PBST) for use in making the 

slurry was conducted and the 10X was found to provide 

the optimum spore recovery. The soil slurry and spiked 

buffer were rotated end over end for at least 10 min, 

allowed to settle for 10 min, and then filtered through a 

30 μm pre-separation filter (Miltenyi Biotec, Auburn, 

CA), before IMS processing (see Appendix C for final 

protocols/methods for real-world sample analysis). 

2.5 Immunomagnetic Separation

2.5.1 Conjugation of tosylactivated 

paramagnetic beads

BA anti-spore polyclonal antibodies (goat affinity 

anti-anthrax antibody (IgG, lot# 031104-01 and lot# 

2610006-01) were acquired from NMRC (Bethesda, 

MD), purified in-house (CDC Core Facility), and initially 

conjugated by Invitrogen to tosylactivated magnetic 

beads. Obtaining the conjugated beads was delayed by 

several months because of the merging of Invitrogen 

Corporation with Applied Biosystems Inc.TM (ABI) by 

Life Technologies, and subsequent reorganization of 

the company. In the reorganization, ABI eliminated the 

division that performs the conjugation, so arrangements 

were made for the CDC Core Facility to perform the 

conjugations. The conjugation method (see attached 

Appendix: Protocols/methods for real-world sample 

analysis) was adapted from Invitrogen’s “Dynabeads 

MyOneTM Tosylactivated Product Description and 

Instructions for Use” (Invitrogen 2006).” Two different 

temperatures and times of the reactions were evaluated 

(37°C for 24 h and 20°C for 48 h), as well as two 

different antibody to bead ratio (50 μg/mg and 40 μg/

mg). Optimum conditions for conjugation were found to 

be 37°C for 24 h with an antibody to bead ratio of 40μg/

mg. 

2.5.2 Exploratory Bead Retriever methods

Two programs were designed for the immunomagnetic 

BeadRetriever system (Invitrogen Corporation, Carlsbad, 

CA) to optimize recovery of spores from sand and 

Arizona test dust (the soils with the largest and smallest 

particle size, respectively). The alternative and the final 

programs were developed using a manual magnetic 

bead retriever. The parameters evaluated for optimum 

recovery were (a) the volume of slurry necessary, (b) 

the time needed for homogenous mixing, and (c) the 

wash times. The results of these evaluations were 

used to configure the KingFisherTM software (Thermo 

Fisher Scientific, Waltham MA) which operates the 

automated BeadRetriever system. Once the parameters 

were configured, a pilot run using the automated 

BeadRetriever to separate BA from spiked sand and 

Arizona test dust was performed. Approximately 3 mL

total of soil slurry was divided into 3 equal portions and 

placed into wells 1-3 (~1 mL per well) within a tube strip 

(Figure 1). Each of these 3 tubes contained 20 μL of 

antibody-conjugated MyOne beads. The automated Bead 

RetrieverTM was started and the beads and soil slurries 

were mixed vigorously. The instrument then collected 

the beads, along with the bound target BA spores, from 

tubes one through three, into a fourth tube in the tube 

strip where they were washed with PBST. The washed 

automated immunomagnetic separation (AIMS) product, 

i.e. the magnetic beads with bound target BA spores, was 

then transferred to the fifth and final tube within the tube 

strip and resuspended in 400 μL of PBST. The software 

program is available at Life TechnologiesTM.

5
Figure 1. BeadRetreiverTM Automated immunomagnetic seperation bench top system

The BeadRetriever system is capable of processing up 

to 15 samples at a time. Soil and spore slurry samples 

are pipetted into tube strips (A) which consists of 5 wells 

linked together and held on a stationary platform. The 

samples are processed by a moving platform consisting 

of plastic tip comb holders (B) and magnetic rods (C) 

which slide down into the plastic comb holders during 

AIMS processing. Antibody conjugated paramagnetic 

beads along with bound spores adhere to the plastic 

enclosed magnetic rods in the first three wells and are 

mixed by an up and down motion of the magnetic rods. 

The platform moves the magnetic rods with captured 

beads and spores up, over, and releases the collected 

beads into the fourth well in the tube strip. In the fourth 

well the beads with bound spores are washed in PBST

by a similar mixing action. The final washed bead with 

bound spores are then collected by the magnetic rod 

and placed into the fifth well of the tube strip containing 

PBST (see http://tools.invitrogen.com/content/sfs/

manuals/1189_BeadRetriever_Manual.pdf). 

2.5.3 Exploratory Method: Miltenyi 

Biotec Immunomagnetic Separation (IMS)

Miltenyi Biotec (Auburn, CA) super-paramagnetic 

particles, approximately 50nm in diameter, were also 

evaluated as an alternative to the MyOne beads. It was 

thought that the nano-sized beads utilized in this IMS 

system would increase magnetic retrieval and spore 

recovery by allowing more beads to bind to each spore. 

We evaluated the manual IMS system OctoMACSTM

(Miltenyi Biotec) along with their anti- mouse IgG 

bead for its ability to recover 104 spores/g of soil. The 

mouse IgG beads were pre-complexed with monoclonal 

(mouse) BA spore antibody and used for manual 

immunomagnetic separation of BA spores from sand. 

Recovery using this manual system was well below the 

recoveries observed using the manual Invitrogen® IMS 

system and the HSGS. A preliminary study comparing 

MyOne beads conjugated with polyclonal antibodies and 

MyOne beads conjugated with monoclonal antibodies 

revealed that the recovery was not sufficient using the 

6
BA monoclonal (mouse) antibody. The BA polyclonal 

(goat) antibody could give better results, however, the 

Miltenyi cell separation technology does not have an 

appropriate microbead strategy available for goat derived 

antibodies. Nonetheless, we did incorporate the Miltenyi 

Biotec pre-processing step (filtering the soils with a 30 

μm pre-separation filter) into the final AIMS method. 

2.5.4 Final AIMS method: Applied Biosystems 

Inc., IMS

Three mL of the spiked soil slurry was dispensed into 

three tube strips containing 100μL of 10X PBST and 

20 μL of MyOne conjugated beads. Three or more 

replicates of each soil were performed. The mixture was 

then gently agitated for approximately 30-35 min. The 

beads, along with bound spores, were collected into one 

tube, washed in 1 mL of 1X PBST for approximately 

10 min, and concentrated in 300 μL of 1X PBST. This 

magnetic bead retrieving process was accomplished with 

the BeadRetriever. This AIMS system utilizes inverse 

magnetic bead processing technology, which rather 

than transferring liquids, transfers paramagnetic beads 

through a series of tubes containing specific reagents 

with the aid of internal rather than external magnetic 

rods. The spores and beads captured by AIMS were 

diluted in series and using the spread plate technique 

cultured onto TSAII or PLET plates (non-sterile soils only) 

in triplicate and incubated at 35°C for 24 h. Colonies were 

counted after 24 h and the %R was determined using the 

ratio of CFU/g of soil to the CFU of the inoculum (see 

final protocols/methods for real-world sample analysis). 

The eluent was stored at -20°C for detection by real-time 

PCR.

2.6 Spore recovery by culture

The AIMS product was diluted in series and spread 

plated (100 μL each) onto TSAII and PLET agar, 

incubated at 35°C overnight and the colonies were then 

counted. The CFU/g of soil was determined and the %R 

calculated, relative to the inoculum. In order to confirm 

that colony counts on both media were comparable, a 

growth challenge test was conducted. TSAII and PLET

agar were inoculated with BA spores with a low level 

of challenge spore concentration (10-100 CFU). The 

plates were incubated at 35°C overnight and counted the 

next day. Media acceptance requires that the average 

number of CFU on the PLET agar must fall within ±70% 

of the average number of CFU found on the TSAII plates 

(adapted from USP 32, NF 27, Chapter 1227 in U.S. 

Pharmacopeia 2009).

2.7 Spore detection by real-time PCR 

In addition to culture, real-time PCR was evaluated for 

the ability to detect the spores present in the product 

of the AIMS. The DNA was extracted from the 

BeadRetriever product initially using a simple boil prep 

procedure (100°C for 5 min). The real-time PCR was 

performed using an ABI 7500 Fast DX platform. The 

cycling parameters were programmed according to the 

LRNTM protocol “Detection of Bacillus anthracis DNA

by fluorogenic 5’ nuclease assay using the Applied 

Biosystems® 7500 Fast DX Real-Time PCR System.” 

The primers and probes were obtained from the LRNTM

and are described in Hoffmaster et al. (2002). The only 

modification made was that the PCR was performed 

using 1X PCR master mix [Light Cycler FastStart PLUS 

DNA Master Hybprobe (Roche Molecular Biochemicals, 

Indianapolis, IN)]. We evaluated two DNA extraction 

kits, the MO BIO UltraClean® Soil DNA Isolation Kit 

(MO BIO Laboratories, Inc., Carlsbad, CA) and the 

QIAamp DNA Blood Mini Kit (Qiagen Inc., Valencia, 

CA), for DNA extraction from the AIMS product. Both 

kits are supported by the LRNTM. The MO BIO soil 

kit was used by collaborators at Lawrence Livermore 

National Labs. Both kits were evaluated for their 

efficiency of DNA purification from AIMS products 

(from spiked potting soil and Arizona test dust with 103 

spores/g of soil). 

7
8
3.0 

Results and Discussions

3.1 High specific gravity sucrose 

(HSGS) extraction

This study was designed to improve existing methods 

to extract BA spores from soil samples. The HSGS 

method exploits the difference in specific gravities 

between BA spores and spores from other organisms 

and from background materials in order to isolate the 

spores. The HSGS method is inexpensive relative to 

immunomagnetic separation; however, we found the 

technique to be highly variable. Dragon and Rennie 

(2001) found that using HSGS, the %R of BA (ATCC 

4229) spores (2 x105 to 8x105) seeded into 2.5 g of 

sterile field soil was 4.5%. Using this sucrose flotation 

method, we recovered 0.75 - 9% Ba spores on tSa ii 

from 1 g of spiked sterile soils (Figure 2). As seen in 

Figure 2, the best average %R for each soil were 5% (106

spores/g) from arizona test dust, 3.7% (104 spores/g) 

from Minnesota loam, 9% (104 spores/g) from potting 

soil, and 5.8% (105 spores/g) from sand. the standard 

deviations decreased slightly as the inoculum level 

increased. We also observed that this method yielded the 

highest %R from potting soil, regardless of the inoculum 

level tested. dragon and Rennie (2001) also found 

potting soil to yield the highest %R when compared to 

spiked field soils.

Figure 2. The mean percent of spores recovered using HSGS plus non-ionic detergent extraction (Y-axis) (n≥3) 

is shown for each spiked BA spore inoculum level (shown on the X-axis) and for each sterile soil type: Arizona 

test dust, Minnesota loam, potting soil, and sand. Error bars shown in graph represent standard deviations. 

9
3.2 Immunomagnetic Separation 

3.2.1 Exploratory methods and challenges

3.2.1.1 Alternate IMS system

Soon after obtaining the BeadRetrieversystem from 

Invitrogen, a merger between Invitrogen and Applied 

Biosystems led to reconstruction of the division 

responsible for programming the BeadRetriever system 

and conjugating the MyOne beads with our antibody. 

This restructuring impeded the programming of the 

BeadRetriever system and delayed obtaining conjugated 

beads; therefore, an alternative IMS method was 

explored. The Miltenyi Biotec IMS system was selected 

as an alternative because it uses nanoscale paramagnetic 

beads. Theoretically, smaller beads should allow for 

multiple beads to bind to each BA spore. This is a 

different approach than the Invitrogen system’s 1μm 

MyOne beads in which fewer beads bind per spore 

simply because the spore and the bead are of equivalent 

size. Unfortunately, all spore recovery attempts with 

the Miltenyi system failed, most likely due to the use 

of monoclonal, rather than polyclonal, antibodies. 

Fortunately, we were able to get the BeadRetriever King 

FisherTM Software programmed by Applied Biosystems 

technicians, and were able to continue with the 

BeadRetriever instrument as originally planned. 

3.2.1.2 Conjugation of tosylactivated paramagnetic 

beads

The performance of beads conjugated at 40 μg IgG/

mg beads and 50 μg IgG/mg beads were evaluated 

using the three soil types with lower spore recovery 

rates. Results showed that the %R of spores from soil 

was greater when the beads were conjugated with an 

antibody to bead ratio of 40 μg IgG/mg (Figure 3). The 

one unexplained exception was potting soil spiked with 

102 BA spores. The %R of spores from positive controls 

(buffer) was greater when using beads conjugated with 

an antibody to bead ratio of 50 μg IgG/mg. We found 

that conjugation at the different times and temperatures 

evaluated (20°C for 48 h and 37°C for 24 h) did not 

affect the %R (data not shown). We therefore chose to 

conjugate the antibody to the beads at 37°C because of 

the shorter incubation time.

Figure 3. Comparison of the mean percent of BA spores recovered (n≥3) from Arizona test dust (ATD); 

Minnesota loam (ML); potting soil (PS), and PBST (buffer positive control) spiked with 102-104 spores/g of soil 

using conjugated bead concentrations of 50 μg IgG/mg and 40 μg IgG/mg.

During the course of the experiments, we received more 

antibodies from NMRC with a different lot number 

than previously used. In order to confirm comparable 

performance, we evaluated the two antibodies, 

conjugated to the beads in the same manner (37°C for 

24 h), for their ability to recover spores from soil and 

buffer. The results indicated little difference in the %R 

when using beads conjugated with either of the two lot 

numbers of antibody.

10
3.2.2 Final method and challenges 

Our optimization of the AIMS protocol revealed 

that separation of the BA spores from soil was best 

accomplished by preprocessing the soil slurry samples, 

sonicating and vortexing (3 min each) the spore slurry to 

disrupt clumps, filtering the slurry through a 30 μm pore 

size filter, allowing the slurry to settle, and removing the 

liquid from the top of the sediment and placing it in the 

Bead Retriever tube tray. The preprocessing procedures 

improved the recovery of BA spores in all soil types and 

decreased the amount of unrecovered spores in the wash. 

These improvements were most noticeable with Arizona 

test dust (Figure 4). 

Figure 4. Effects of Preprocessing on the mean recovery of BA spores from sterile Arizona test dust. 

(Experiment done in duplicate.) Unrecovered spores = percent of spores recovered in IMS wash tube. 

Preprocessing abbreviations: SNFS – sonicating, filtering, and settling, and; SNF-sonicating and filtering; 

SNS- sonicating and settling and, No- no preprocessing. 

The AIMS system program was designed for the 

immunomagnetic separation of spores from Arizona 

test dust and sand. Each program specifies the amount 

of time the beads are allowed contact with the soil, the 

amount of vigor used in agitation of the slurry and beads, 

and the length of time for the wash step. The entire 

AIMS procedure consists of two steps: soil extraction 

which includes all of the preprocessing of samples 

and automated immunomagnetic separation by the 

BeadRetriever system.

When the conjugated beads are mixed with the soil, the 

critical step is the binding of the bead to the target BA

spore. This binding depends upon the specificity of the 

conjugated antibody to the spore and the contact time 

between the beads and the soil slurry. The antibody 

specificity was tested previously, by CDC’s Division of 

Bioterrorism and Preparedness Response, using time 

resolve fluorescence (TRF). The results showed that the 

antibody can differentiate between closely related and 

nonrelated bacterial strains (see appendix B). In the 

TRF study, only BA spores, not vegetative cells, were 

screened.

Our data show that the AIMS method recovered other 

organisms in addition to BA. It is possible that the soil 

aggregates contain metallic or magnetic particles that are 

attracted to the magnetic rod during AIMS processing. 

These soil aggregates may have other bacterial species 

adhering to them. In order to determine if this were 

true, we looked at Arizona test dust, which contains iron 

oxide, and sand using scanning electron microscopy 

(SEM). Observation by SEM of non-sterile Arizona 

test dust processed AIMS without any BA spores 

revealed possible magnetic particle aggregating with 

MyOnebeads and other particles (Figure 5). 

11
Figure 5. Scanning electron microscopy image of AIMS (automated immunomagnetic separation) processed 

non-sterile Arizona test dust (A) and sand (B) (Both without BA spores and at 10,000× and 3,500× respectively). 

(A) In Arizona test dust, clumps or aggregates containing beads and other particles are seen. (B). In sand, only 

beads are seen. 

Very few other organisms were cultured when recovering 

spores from sand and Arizona test dust, but the numbers 

were significant when recovering from potting soil and 

Minnesota loam. For this reason, the use of the selective 

agar (PLET) for recovery of BA from unknown soil 

samples is recommended. A comparison of recovery on 

both media (PLET and TSAII) was conducted, and the 

results demonstrated that recovery of BA on PLET agar 

was comparable to growth on TSAII agar. Specifically 

the PLET CFUs were within 72-77% of the number of 

CFUs found on TSAII agar after overnight growth at 

35°C for 48 h.

We also noted that the concentration of PBST buffer 

used in making the soil slurry influenced the %R. Using 

10X PBST in culturing methods reduced clumping and 

increased the %R when compared to 1X PBST. Results 

from a preliminary study showed the %R from Arizona 

test dust spiked with 10 3/g BA spores were 9.23% and 

0.52% when 10X PBST and 1X PBST, respectively, 

were used to make the slurries. 

3.2.2.1 Soil and Inoculum level evaluations

The average %R for each inoculum level and 

preprocessed soil type is shown in Table 2. The number 

of spores recovered from all soils inoculated with 101

spores and some soil types inoculated with 102 spores 

were below the limit of reliable quantitation (<25 CFUs 

recovered by plate count). Therefore, the average %R is 

only discussed for inoculum levels 102-104 in this report. 

The %R was found to be optimal within the 102-104

range of inoculum. 

The number of spores recovered does not necessarily 

increase with increasing inoculum (Table 2). According 

to the MyOne Tosylactivated product description and 

instructions for use (Invitrogen 2006), the amount 

of ligand per mg of beads is optimal at 40 μg ligand 

per mg beads. Thus it seems at higher spore (ligand) 

concentrations, the target sites on the beads become 

saturated and can no longer bind additional spores.

12
Table 2. AIMS Mean %R (SD) of BA Spores Recovered From All Four Sterile and Non-Sterile Preprocessed 

Soil Types and PBST. Percent recovery determined from culture on TSAII and PLET Agar. 

Soil Types

Arizona test dust Minnesota loam Potting soil Sand

Phosphate Buffered 

Saline with Tween®

20

Avg. % Recovered BA spores/g (Std. Deviation)

Inoculum level Sterile TSAII

104 39.44 (4.57) 14.46 (2.18) 31.28 (2.75) 61.95 (10.02) 56.12 (8.98)

103 35.32 (1.64) 16.89 (3.33) 23.64 (5.53) 55.67 (3.96) 57.22 (18.42)

102 25.05 (5.29) *14.81 (4.92) *31.24 (6.45) 68.01 (11.25) 67.82 (11.70)

101 *25.25 (5.05) *24.23 (24.24) *50.51 (0.00) ‡25.67 (2.14) *173.86 (117.27)

Overall** 38.08 (4.38) 15.39 (3.44) 29.09 (5.79) 61.39 (9.67) 59.50 (14.88)

Nonsterile TSAII

104 51.07 (7.39) 10.09 (1.76) 19.52 (4.80) 54.99 (9.02) 61.54 (19.03)

103 28.84 (5.03) *10.86 (1.94) 18.52 (2.20) 56.23 (5.87) 62.96 (3.56)

102 48.82 (9.55) *10.1 (3.64) *16.84 (3.55) 53.53 (11.25) 70.38 (7.59)

101 *131.3 (113.83) *116.16 (7.14) *50.51 (0.00) *202 (0.00) *148.2 (123.8)

Overall** 41.22 (12.74) 10.35 (2.06) 18.71 (3.64) 55.32 (7.74) 64.48 (16.36)

Nonsterile PLET

104 37.60 (6.62) 13.31 (3.40) 15.57 (6.42) 53.87 (14.42) 70.99 (19.53)

103 24.35 (12.25) 15.49 (5.79) *20.09 (16.96) 48.60 (18.90) 53.94 (19.40)

102 20.2 (9.55) *29.63 (10.22) *17.51 (5.74) 51.18 (4.98) 60.00 (8.64)

101 *45.45 (28.56) *121.21 (14.28) 16.84 (0.00) *117.85 (119.04) *173.9 (133.01)

Overall** 29.43 (11.75) 17.07 (10.48) 17.78 (11.81) 51.23 (15.32) 62.64 (19.13)

‡ no preprocessing

below limit of reliable quantitation (<25 CFU)

**overall % recovered excluding 10^1 data

Sand 10^1 sterile is from experiment 10-14-2008 no preprocessing was done

3.3 Spore extraction and rapid viability 

PCR detection

In evaluating the DNA extraction kits, we found that 

neither kit was able to detect the chromosomal target, 

most likely because the inoculum was at or below the 

limit of detection. The extraction efficiencies of the 

two kits were affected by the soil type. The Qiagen 

Blood kit extracted DNA from Arizona test dust more 

efficiently than the MO BIO soil kit. The MO BIO soil 

kit extracted DNA from potting soil more efficiently than 

the Qiagen Blood kit. 

Both DNA extraction kits were able to extract DNA 

from the AIMS products, although the results were not 

consistent between replicates, and the level of detection 

was still >103 spores/g of soil. Additionally, because 

of the chemistry of the MO BIO soil kit (personal 

communication with the MO BIO technical support 

department), a consistent DNA extraction of spores from 

a suspension of spores in PBS, necessary for a positive 

control, was not possible.

Considering all of these challenges with the DNA

extraction kits, we decided that the best way to reduce 

the level of detection and to get consistent DNA

extraction was by using the rapid viability PCR (RVPCR) method combined with an enzymatic lysis 

protocol developed by scientists at Lawrence Livermore 

Laboratories. The RV-PCR method includes an 

incubation and replication step to increase the DNA copy 

number. The enzymatic lysis uses Bacillus cereus Zebra 

Killer lysin, currently in use within another laboratory at 

CDC, and has been extensively evaluated and found to 

consistently lyse vegetative BA cells. 

Because of the challenges with the DNA extraction 

from spores, the timeline for the RV-PCR portion of the 

project was extended to December 2010. The protocol 

has been finalized and work is ongoing. A separate report 

will be submitted in 2011 that will include the RV-PCR 

results and recommendations. 

13
14
4.0 

Conclusion

An optimized AIMS method was successfully 

developed. The optimized method improved the %R of 

BA spores from four soil types and lowered the limit 

of quantitation, as compared to the extraction method, 

HSGS. Percent recovery of spores from soils inoculated 

with 102 to 103 spores/g with the AIMS method ranged 

from 15% to 68% as compared to <9% with the HSGS 

(range 104 – 106 spores/g) method. When the number of 

spores in the sample was greater than 103, the amount 

of target sites on the beads could have become saturated 

resulting in an inability to bind additional spores. The 

addition of pre-processing steps improved spore recovery 

from soil. The pre-processing steps were vortexing, 

sonication, filtration and allowing for soil to settle before 

AIMS processing. The %R of spores from soils with 

more organic matter and background organisms (i.e., 

Minnesota loam and potting soil) was lower than soil 

types with less organic matter and fewer background 

organisms (i.e., Arizona test dust and sand). In cases 

where soil types contain high levels of background 

organisms, PLET agar was helpful as a selective agar, 

but an additional day of incubation was required before 

countable colonies were visible. The limit of detection 

of the AIMS may be enhanced with the addition of the 

RV-PCR method. 

15
16
5.0 

References

Atlas R M. 1996. Handbook of Microbiological Media. 

2nd edition. New York, NY:CRC Press.

Dragon DC, Rennie RP. 2001. Evaluation of spore 

extraction and purification methods for selective 

recovery of viable Bacillus anthracis spores. Lett Appl 

Microbiol. 33(2):100-5.

Gulledge J S, V A Luna, Luna, AJ, Zartman R, Cannons 

AC. 2010. Detection of low numbers of Bacillus 

anthracis spores in three soils using five commercial 

DNA extraction methods with and without an enrichment 

step. J Appl Microbiol.109(5):1509-20. 

Herzog A B, McLennan SD, Pandey, Gerba C P, Haas 

CN, Rose JB, Hashsham SA. 2009. Implications of 

limits of detection of various methods for Bacillus 

anthracis in computing risks to human health. Appl 

Environ Microbiol. 75(19): 6331-9.

Hoffmaster AR, Meyer RF, Bowen MD, Marston CK, 

Weyant RS, Thurman K, Messenger SL, Minor EE, 

Winchell JM, Rassmussen MV, Newton BR, Parker 

JT, Morrill WE, McKinney N, Barnett GA, Sejvar JJ, 

Jernigan JA, Perkins BA, Popovic T. 2002. Evaluation 

and validation of a real-time polymerase chain reaction 

assay for rapid identification of Bacillus anthracis. 

Emerg Infect Dis. 8:1178-82

Invitrogen. 2006. Dyabeads® MyOneTM Tosylactivated. 

[product description and instructions for use] Oslo, 

Norway: Invitrogen Dynal AS. http://www.siercheng.

com/UploadFile/200961193927447.pdf 

Ryu C, Lee K, Yoo C, Seong W K, Oh H-B. 2003. 

Sensitive and rapid quantitative detection of anthrax 

spores isolated from soil samples by real-time PCR. 

Microbiol Immunol 47(10): 693-9.

U.S. Department of Health and Human Services. 

Public Health Service, Centers for Disease Control, 

and National Institutes for Health. 2009. Biosafety in 

Microbiological and Biomedical Laboratories. 5th ed.

HHS Publication No (CDC ) 21-1112. Atlanta, GA: 

Centers for Disease Control and Prevention. http://

www.cdc.gov/biosafety/publications/bmbl5/index.htm

U.S. Environmental Protection Agency. Office of 

Research and Development. National Homeland 

Security Research Center. 2010. Standardized analytical 

methods for environmental restoration following 

homeland security events. Revision 6.0. EPA/600/R10/122. October, 2010. http://www.epa.gov/nhsrc/

pubs/600r10122.pdf 

U.S. Pharmacopeia 32. National Formulary 27. 2009. 

Validation of Microbial Recovery. Vol 1 of The Official 

Compendia of Standards. Baltimore, Maryland: United 

Book Press.

17
18
Appendix A

Conjugation of Paramagnetic Beads

http://tools.invitrogen.com/content/sfs/manuals/655_01_02_rev003.pdf

19
20
Appendix B

Time Resolved Fluorescence 

Assay Summary

TRF assay Bacillus anthracis (Spore) 

Antibody Summary Information: 

Agent: B. anthracis (spore assay) 

Strains Tested: B. anthracis Pasteur vaccine strain, 

Sterne vaccine strain, B. anthracis strains AO34, AO39, 

AO62, AO102, AO149, AO158, AO174, AO188, 

AO193, AO248, AO256, AO264, AO267, AO297, 

AO328, AO367, AO379, AO419, AO442, AO462, 

AO463, AO465, AO488, AO489, M36 (vollum), ASC-1, 

ASC-3, ASC-32, ASC-38, ASC-45, ASC-58, ASC-68, 

ASC-69, ASC-78, Z-1, Z-6, PB-292, PB-293 

Near-neighbor Screen: Bacillus thuringiensis ssp.

kurstaki, B. thuringiensis ssp. israelensis 

Unrelated Screen: Paenibacillus macerans, Bacillus 

mycoides, Bacillus cereus (Laboratory Response 

Network (LRN) control strain), Brevibacillus 

laterosporus, Bacillus licheniformis, B. cereus, (Fri42), Bacillus epiphytus, Bacillus alvei, Bacillus 

firmus, Bacillus amyloliquefaciens, Bacillus badius, 

Brevibacillus brevis, Bacillus circulans, Bacillus 

subtilis, Bacillus subtilis (globigii), Paenibacillus 

polymyxa, Geobacillus stearothermophilus, Bacillus 

sphaericus, Bacillus lentus, Thuricide (pesticide 0.8% B. 

thuringiensis ssp. kurstaki (A.G Organics, Prosper, TX), 

Dipel dust.

Negative Control Antigen: B. cereus (LRN control 

strain) 

Markers Targeted (if known): B. anthracis spore coat 

Ag

Sensitivity: Assay was optimized to detect 100 B. 

anthracis spores total. 

Optimization: Antibodies were optimized using a 

range of 0.1 μg/mL to 10.0 μg/mL for both capture and 

detection. The assay utilizes 0.75 μg/mL for both goat 

anti-B. anthracis capture and anti-B. anthracis mouse 

monoclonal detector antibodies. Assay requires 90-min 

antigen incubation. 

Limitations: Assay also reacts with B. anthracis 

vegetative cells. 

21
22
Appendix C

Final Protocols/Methods for 

Real World Sample Analysis

Procedure Title: Automated Immunomagnetic Separation of Bacillus anthracis

spores from soil

Purpose: Immunomagnetic Separation (AIMS) of Bacillus anthracis spores from soil. 

Reagents: Custom coated Dynabeads® (Invitrogen Dynal MyOneTM beads, Carlsbad, CA) 

Sample buffer (10X Phosphate buffered Saline pH7.4 + 0.05% Tween 20) 

Equipment: Dynal® BeadRetrieversystem (Invitrogen Dynal cat# 159-50)

End over end rotator (such as VWR cat # 13916-822)

Supplies: 

• 50 mL conical tubes

• 1.5 mL Eppendorf tubes

• 30 μm Pre-Separation filter (Miltenyi Biotec cat. No. 130-041-407)

• BeadRetrievertubes and strips (Invitrogen Cat. No. 159-51)

• Polymyxin lysozyme EDTA thallous acetate select agar (PLET) 

• Tryptic Soy Agar II with 5% sheep blood (TSA II, BD Diagnostic Systems)

• Cell spreaders

• Pipettors (100 mL, and 1000 mL)

• Pipette tips (100 mL, and 1000 mL)

Specimen: 1 gram of soil sample, potentially contaminated with B. anthracis spores

Special safety precautions: Follow all safety precautions as outlined in Biosafety in Microbiological and Biomedical 

Laboratories, 5th edition (U.S. Department of Health and Human Services 2009)

Quality control: Check sterility of PLET agar and TSA II by incubating 10% of media at 35°C for 7 days and check for 

growth.

Procedure:

1. Place 1 g of soil sample in a 15 mL conical tube. 

2. Add enough 10X PBST to enable a 3 mL volume to be retrieved for AIMS. This can be estimated by subtracting the volume occupied by the soil 

from the total volume as observed on the side of the tube. 

3. Place the 15 mL conical tube (s) on the rotator and allow the tube(s) to rotate end over end for 10 min.

4. Remove tube from rotator, place in a tube rack and allow the soil to settle for 10 min.

5. Withdraw as much of the liquid from the top of the settled soil and filter it through a 30 μm pore size nylon filter (Miltenyi) that is set on top 

of a second 15 mL conical tube.

6. Prepare the BeadRetriever tube strips for AIMS by placing the tube trays which consists of 5 wells into BeadRetrieverrack.

Add 100μL of 10X PBST to well 1, 2 and 3 of a BeadRetrievertube strip.

Add 20μL of beads to well 1, 2 and 3.

Add 1 mL of 1X PBST to well 4.

Add 300μL of 1X PBST to well 5.

7. Mix filtered sample eluent by pipetting up and down a few times and place 1 mL of filtered sample to each of tubes 1, 2, and 3.

8. Insert BeadRetriever tips into slot.

9. Choose BA sand program and press start. The sample will be ready in ~ 50 min.

10. After the cycle is finished, remove the cleaned eluent (in the final tube containing 300 μL of buffer) and place in an eppendorf tube.

11. Mix sample by pipetting up and down a few times. If large numbers of spores are expected, serial dilutions of the AIMS product may be 

necessary. 

12. Spread 0.1mL from well 5 onto TSAII and/or PLET plates in duplicate. Incubate plates at 35o

C for 24 h

13. Count and record colonies on plates. 

14. Calculate the number of CFU/g soil: 

mean number of colonies per plate x total dilution factor

 g of soil

23
Flowchart of AIMS method

24
Limitations of AIMS

(1) False negative results may occur due to high 

background contamination and may prevent 

identification of growth of BA on TSAII plates. 

(2) The limit of detection as determined by culture is 

dependent upon the soil type. Based on the four soil 

types evaluated, the limit of detection for soils with 

more organic material and background microorganisms 

was approximately 103 spores/g. The limit of detection 

for soils with less organic material and background 

organisms was approximately 102 spores/g. 

(3) Growth on PLET agar requires an additional 24 h 

of incubation (as compared to growth on TSAII) and 

the resulting BA colonies are very small with slight 

differences in colony size and morphology.

(4) During the pre-processing step, filtration of some soil 

types may reduce recovery of BA spores, due to clogging 

of the 30μm mesh filter.

(5) Metallic or magnetic particles in soil can bind beads 

and cause aggregation of particles and other organisms 

to be recovered. 

Interpretation/examination

Examine the serial dilution plates for suspect B. anthracis 

colonies. 

To calculate spore recovery per gram of soil from serial 

dilution plates

containing between 25 and 250 B. anthracis colonies:

CFU/g = 

mean number of colonies per plate x total dilution factor

 g of soil

25
26
Appendix D

Quality Assurance and Quality Control 

(QA/QC)

QA/QC Categories QA/QC implemented and QAPP* deviations

Equipment, media and supplies 

As described, the certificate of analysis (COA) for all commercially available media were obtained and kept; 

sterility was also assessed with no deviations. Quality control of each lot of selective PLET agar media was 

performed as described. 

Bacillus spp. and soil sources

The storage and propagation conditions for the Bacillus anthracis Sterne 34F2 veterinary vaccine strain 

were as outlined in the QAPP with no deviations. Cross reactivity of the obtained antibodies was assessed 

by CDC’s Division of Bioterrorism and Preparedness Response and the findings are included in the report. 

Physical and chemical properties of each soil type were determined and the results are found in the report. 

Immunomagnetic bead separation

AIMS parameters such as buffer types, buffer strength and pH were optimized for the final method as 

described. For experimental trials positive and negative controls were always included. The negative control 

consisted of soil slurries made with soil and buffer only. We found this to be a more relevant negative 

control considering the nature of the beads and soil types. Appropriate interpretation of results was based on 

the use of these controls.

DNA extraction

Experimental trials of the DNA extraction method included BA spiked sterile soil slurries as a positive 

control and buffer only as a negative control. Appropriate interpretation of the performance of the DNA

isolation kit included considered findings from the controls. 

High density spore flotation Experimental trials assessed spore recovery from spiked sterile soils and positive and negative controls as 

described. Appropriate interpretation of results was based on the use of these controls.

Real time PCR and RV-PCR

All trials included the use of the 16S Ribosomal Ribonucleic acid (rRNA), and a plasmid containing all 

select agent primer and probe sets in the detection assays as positive controls and a no template control 

(water only). No deviations were observed. Findings will be covered in a another report

Training Standard laboratory procedures were followed and all personnel were trained on using real time PCR and 

other critical equipment. No deviations were observed.

Data management All documentation and records of testing and results were kept in a secured laboratory notebook and in 

project-specific electronic files as described in the QAPP. No deviations were observed. 

Data reporting Monthly and quarterly progress reports were prepared and submitted to EPA’s project assistant and project 

manager.

*QAPP, Quality Assurance Project Plan

27
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