Advanced%20Analytical

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Advanced Analytical Micro PRO
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Overview of Todays Presentation

• History of Advanced Analytical Technologies, Inc.
• How the Technology Works
• The Micro PRO System
• Micro PRO Applications and Results
• Qualitative Analysis Presence/Absence
• Quantitative Analysis Enumeration
• Customer Presentations

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Advanced Analytical
Improving Process
Empowering Progress
OUR MISSION
Through innovative technology and collaborative
relationships we will
gt Deliver superior customer support, services,
and solutions
gt Create sustainable growth
gt Foster a dynamic and fulfilling work environment
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Advanced Analytical Technologies, Inc. Who are
We?

• History Founded 1997, 4 Scientific Co-founders
• Acquired CE Technology December, 2006
• Privately Funded
• Business Rapid Microbial Detection Technology
• Capillary Electrophoresis Technology
• Markets Pharmaceutical, Personal Care Products,
  Fermentation, Environmental
• Microbiology Solution Replace current microbial
  detection methods (requiring 24h 5 days) with
  a technician-friendly,
• rapid system capable of detection and
  enumeration
• Products Micro PRO Instrument, Micro PRO Media
  Kit, Micro PRO Reagent Kit, TVO Kit

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Basics of Flow Cytometry
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Basics of Flow Cytometry

• Laser-based irradiation of cells
• Fluorochromes bound to cells provide information
  on cell state (e.g., live, dead, spores,
  vegetative)
• Light scattering provides relative size
  information
• System composed of fluidic, optic and electronic
  components
• Advantages Rapid and quantitative analysis of
  individual cells

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Flow Cytometry Optics
Labeled microbial cell
Scatter Detector
Scatter signal
Laser Beam shaped and focused 635 nm laser
excitation
Fluorescence Detector
Fluorescence signal
High performance optical filters
Fluorescence plus Scatter One Count
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Flow Cytometry Optics
Unlabeled Particle
Scatter Detector
Scatter signal
Laser Beam shaped and focused 635 nm laser
excitation
Fluorescence Detector
High performance optical filters
Scatter without Fluorescence No Count
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Electronics

• Signal processing component
• The Micro PRO triggers on fluorescence
• Fluorescent event above the threshold is
  processed, along with the corresponding scatter
  event, and is plotted and recorded as a count

1 count
Detector output
0 count
Fluorescence Threshold Level
Time
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Signal Processing
Microbe A
cumulative
Microbe A B
Microbe A B C
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Interpretation of intensity plots
Cell size
Amount of label
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Setting Analysis Boxes
Fluorescence Intensity vs. Counts
Intensity Plot
Scatter Intensity vs. Counts
Box 166856 counts/mL 99.2 of the counts are in
the box
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Micro PRO INSTRUMENTATION
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Introducing the Micro PRO
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Automated, High-Throughput Analysis

• Qualitative analysis (Presence/Absence)
• 20 samples/hour
• Quantitative analysis (Enumeration)
• 15 samples/hour
• Holds up to 42 samples at once
• Automatically
• adds up to 3 reagents
• mixes samples
• cleaning and bubble removal

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The Micro PRO

• Additional Advantages
• Qualitative Analysis Presence/Absence
• Product Testing Finished, raw materials,
  in-process samples
• Screen products for bacteria, yeast mold in 1
  test
• Next day results for product release
• Quantitative Analysis Enumeration
• Purified/process water monitoring
• Surface swab analysis
• Pure culture enumeration
• Validation packages services available

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Analysis on the Micro PRO
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1
Load sample vials and syringes
Select Tray Setup
Reagent additions and sample injection performed
automatically as defined in the Method
Micro PRO Output Pass/Fail Counts/mL
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4
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The Micro PRO

• Hardware upgrades
• High sample throughput
• Qualitative analysis 20 samples/hour
  (3min/sample)
• Quantitative analysis 15 samples/hour
  (4min/sample)
• Preparation of multiple samples at a time
• Large capacity motors motor movements
  streamlined
• Sheath flow automatically stops after 30 minutes
  of inactivity
• Cover plate to protect reagent ports from
  incidental contamination
• Sample trays redesigned lighter, ergonomic,
  support cap-less syringes
• Re-designed vial de-capper
• Optical sensor eliminated

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The Micro PRO

• Software upgrades
• Windows-based, user-friendly interface
• Main page re-design
• Instant color-coded pass-fail results
• View sample and tray status
• View/download results during sample analysis
• Reagent level alarm
• Context-sensitive Help files
• 21 CFR part 11 compliant

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Micro PRO Qualitative AnalysisProduct Screening
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Traditional methods USP lt61gt
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Micro PRO Product Screening Protocol

1. Dilute 10g product in 90mL phosphate buffer or
   media (110 product suspension)
2. Mix to achieve a homogeneous solution
3. Add 1mL of the 110 product suspension to Tube A
   (Media Kit)
4. Incubate the samples at 30OC for 24-48 hours at
   200 rpm
5. After enrichment, prepare samples as directed in
   the Micro PRO Media Kit
6. Load samples on the Micro PRO for analysis
7. Pass/Fail results in 3 minutes/sample

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Micro PRO Media Kit
Microbes
35µm filter
Micro PRO
Transfer swab from Tube A to B
Tube A - GEM Add product, enrich
Tube B - PB Add swab, mix
Tube C - PB Add 0.1mL from Tube B
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Micro PRO Reagent Kit

• Ideal for detecting microorganisms
  post-enrichment in
• Finished Products
• Raw Materials
• In-Process Samples

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• Interpretation of results
• Micro PRO picture shows few to no counts within
  the analysis box (product baseline)
• Pass result indicates that the sample does not
  contain microbial contamination

Mold Area 1 counts/mL Bacteria Area 2 counts/mL Yeast Area 3 counts/mL Result 1 Result 2 Result 3 Overall Result
32 241 43 Pass Pass Pass Pass
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• Interpretation of results
• Micro PRO picture shows many counts within the
  analysis box (gt4X baseline)
• Fail result indicates that the sample contains
  microbial contamination

Mold Area 1 counts/mL Bacteria Area 2 counts/mL Yeast Area 3 counts/mL Result 1 Result 2 Result 3 Overall Result
2628 1066 54 Fail Ambiguous Pass Fail
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Negative Control
Positive A. niger
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Negative Control
Positive P. aeruginosa
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Negative Control
Positive C. albicans
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Micro PRO Product Screening Results
Example of SignalNoise ratios in various
finished products
Sample Average SignalNoise Average SignalNoise Average SignalNoise
Sample S. aureus C. albicans A. niger
Household cleaner 11779 241 384
Shave Gel 70369 686.5 16
Dish Soap 31172 1580 56.5
Mouthwash 6199 592 41.5
Laundry detergent gt50000 1150.5 25
Fabric freshener 1869 4933 8.5
Toothpaste gt50000 13 41
Body wash 1280.5 10569 88
Hand Soap 269 400 23.5
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Micro PRO Product Screening Results
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Examples of Products Tested
Bacteria-Yeast-Mold detection in 1 assay
Personal Care Products Hand soap Shave Gel Body
wash Shampoo Face Scrub Conditioner Toothpaste
Lotion Mouthwash
OTC Pharma Products Antacid Tablets Liquid
Antacid Anti-itch cream Cold Syrup Multi-vitamin
(chewable) Multi-vitamin (adult) Nose
Drops Stomach Relief
Household Products Dish soap Laundry
detergent Fabric Freshener All-purpose household
cleaner
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Product Testing Results
  Bacteria Bacteria Yeast Yeast Mold Mold Negative Control Negative Control
  Micro PRO Agar Plate Micro PRO Agar Plate Micro PRO Agar Plate Micro PRO Agar Plate
Dish Soap - -
Toothpaste - -
Shave Gel - -
Multivitamin - -
Results from Micro PRO are equivalent to the
plate method.
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Micro PRO Quantitative Analysis
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Total Viable Organisms (TVO) Labeling Kit

• Ideal for enumerating viable microorganisms in
• Process/Purified Water
• Surface Swabs
• Pure cultures

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Purified Water Monitoring

• Results in less then 4 minutes
• Quantitative output in counts/mL
• Significant savings in technician labor

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Implementation Procedure
1) Determine the background range of the Micro
PRO and TVO kit for accurate enumeration of TVO
in purified water. 2) Define the method for
purified water samples. Defining the analysis
box. 3) Demonstrate that the Micro PRO results
correlate well with the traditional plate
methods. 4) Provides a laboratory with the
necessary equipment to detect increases in
purified water TVO counts within minutes of
sample collection, enabling the scientist to
respond proactively.
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Determining Background Limits
1) Collect purified water sample 2) 0.2mm filter
sterilize 3) Background/negative control samples
are analyzed on the Micro PRO using the Water
Method 4) Background is typically less than 26
counts/ml
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Defining the Analysis Box
1) Microbes used a. Escherichia coli, ATCC 25922
b. Pseudomonas aeruginosa, ATCC 9027 c.
Staphylococcus aureus, ATCC 6538 d. Serratia
marcescens, ATCC 13880 e. Ralstonia pickettii,
ATCC 49129 f. A 6-isolate mix from in-house water
system (unknown species) 2) Grow pure cultures
and serially dilute in 0.2mm-filtered purified
water 3) Analyze on the Micro PRO with the TVO
kit 4) Set analysis box parameters in Water
Method so the box encompasses all microbial
populations 5) Use this standard analysis box in
the Water Method for all negative control and
test samples
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Results Defining the Analysis BoxMicro PRO
Intensity Plots
Staph. aureus
Ps. aeruginosa
Serratia marcescens
After the box parameters were set using ATCC
strains, a mix of isolates from Faucet A and
Faucet B were analyzed on the Micro PRO to
verify the placement of the analysis box
Faucet Isolate Mix
R. pickettii
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Purified Water Monitoring Protocol

1. Sanitize each sampling point if necessary.
2. Flush each sampling point for one minute.
3. Collect a minimum of 10mL per sampling point.
4. Dispense 2 x 3mL aliquots per sampling point into
   5mL sample tubes.
5. Prepare 0.2-micron filtered water sample
6. Load tray and analyze using Water Analysis Tray
   Setup in the Micro PRO software

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Clean Water TVO Ps. aeruginosa Spike
Filtered DI H2O 101 cfu/mL
102 cfu/mL 103 cfu/mL
104 cfu/mL
Box 2 counts/0.25mL Box 17
counts/0.25mL Box 106 counts/0.25mL
Box 1241 counts/0.25mL Box 16,069
counts/0.25mL
Sample Micro PRO Counts/mL Plate Counts/mL Micro PRO log10 Counts/mL Plate Counts log10 cfu/mL
Filtered DI H2O (Bkgd) -- 0 -- --
101 Ps. aeruginosa 63 68 1.80 1.83
102 Ps. aeruginosa 455 593 2.66 2.77
103 Ps. aeruginosa 5,449 5,875 3.74 3.77
104 Ps. aeruginosa 70,692 58,750 4.85 4.77
Micro PRO counts/mL are background corrected
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Purified Water Monitoring TVO R2A Plate Counts
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Customer Studies
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Purified Water System
From Hasher-Homesley, P.1, 2006. RD
Applications for the RBD3000. 1Johnson Johnson
Vision Care. Rapid Microbial Methods Users
Meeting, Chicago, IL
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Surface Swab Analysis/ Environmental Monitoring
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Environmental Sample Testing

• Surface swabs are analyzed on the Micro PRO with
  results within 4 minutes
• Data generated by the Micro PRO not only
  provides information about microbial populations
  but also indicates levels of residual product

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Microorganism Recovery

• Recovery was demonstrated by testing pure
  cultures of Pseudomonas aeruginosa,
  Staphylococcus aureus, and Candida albicans
• 100µL of the pure culture was pipetted onto a
  sterile Petri dish
• A dry swab was used to absorb the sample
• Additionally, 100µL of the final solution was
  spread plated onto Tryptic Soy Agar

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Microorganism Recovery
Sample Micro PRO counts Plate Counts/mL Micro PRO log10/mL Plate log10/mL Log10 Difference
Swab Control 17 NA NA NA NA
P. aeruginosa 7004 7700 3.85 3.89 0.04
S. aureus 1327 1420 3.12 3.15 0.03
C. albicans 1782 3280 3.25 3.52 0.26
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Environmental Swab Protocol

• Place a swab sample in a 5mL snap-cap tube
  containing 1mL filtered, sterile PB
• Break the swab handle over the rim of the tube
• Replace snap-cap and vortex swab and buffer for
  30 seconds
• Press the swab against the side of the tube to
  express extra liquid
• Bring the volume to 3mL with filtered, sterile PB
• Analyze samples and controls on the Micro PRO

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1
2
4
3
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Environmental Sample Testing

• Swab samples are directly analyzed on the Micro
  PRO
• Result is obtained in minutes rather than
  overnight

Negative Control Swab Box counts/0.25mL 2
E.coli Spiked Swab Box counts/0.25mL 1,195
Environmental Swab Indicative of residual product
Residual product an additional box may be
created to capture this data
Microbial population within a predefined box
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Pure Culture Enumeration
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Enumeration of Fermentation/Pure Cultures

• Collect sample if necessary dilute to lt106
  cfu/mL in PB
• Dispense 3mL sample into 5mL snap-cap tube
• Load sample into Micro PRO Sample Tray with
  capped syringe
• Select pre-defined (or create new) Analysis/Tray
  Sequence
• Micro PRO count result in 4 minutes

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Fermentation Applications

• Upstream
• Media Contamination
• Starter Culture Viability and/or Titer

• Fermentation
• Process Monitoring
• Biomass and/or Viable cell enumerations
• Determination of when to harvest or induce

• Downstream
• Number of cells post inactivation
• Number of cells after processing
• Cell Concentrations

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Bacillus atrophaeus Spores vs. Vegetative
Spores
Vegetative
Spores Vegetative
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TVO Candida albicans in PB
PB Background 101 cfu/mL
102 cfu/mL 103 cfu/mL
104 cfu/mL
Box 1 count/0.25mL Box 2 counts/0.25mL
Box 33 counts/0.25mL Box 216 counts/0.25mL
Box 2,027 counts/0.25mL
Sample Micro PRO Counts/mL Plate Counts/mL Micro PRO log10 Counts/mL Plate Counts log10 cfu/mL
PB Background -- 0 -- --
101 C. albicans in PB 8 12 0.90 1.08
102 C. albicans in PB 140 128 2.15 2.11
103 C. albicans in PB 921 1,175 2.96 3.07
104 C. albicans in PB 8,648 12,500 3.94 4.10
Micro PRO counts/mL are background corrected
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Correlation of Micro PRO TVO Counts vs. Plate
Counts
Escherichia coli 8739 (R2 0.9916), Escherichia
coli 25922 (R2 0.9992), Staphylococcus aureus
6538 (R2 0.9988) and Pseudomonas aeruginosa
9027 (R2 0.9984).
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Correlation of Micro PRO TVO Counts vs. Plate
Counts
Aspegillus niger spores 16404 (R2 0.9959) and
Candida albicans 10231 (R2 0.9971)
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Microorganisms Enumerated with AATI Technology

• Aeromonas caviae
• Aeromonas hydrophila
• Aspergillus niger spores
• Bacillus atrophaeus
• Bacillus atrophaeus spores
• Bacillus pumilus
• Bacillus pumilus spores
• Bacillus subtilis
• Bacillus subtilis spores
• Bordetella bronchisceptica
• Brachyspira (Serpulina) hyodysenteriae
• Burkholderia cepacia
• Campylobacter jejuni
• Candida albicans
• Candida glabrata
• Citrobacter freundii
• Clostridium perfringens
• Cryptococcus spp.
• Cryptosporidium parvum oocysts

• Enterococcus faecium
• Enterococcus faecalis
• Enterococcus gallinarum
• Enterococcus hirae
• Enterococcus mundtii
• Erysipelothrix rhusiopathiae
• Escherichia coli
• Escherichia coli O157H7
• Escherichia coli O25HN
• Escherichia coli O15NM
• Escherichia coli O1NM
• Escherichia coli O7NM
• Escherichia coli O78NM
• Escherichia coli ONH8
• Escherichia coli ONNM
• Escherichia coli O8HN
• Geobacillus stearothermophilus
• Geobacillus stearothermophilus spores
• Giardia lamblia cysts

• Klebsiella pneumoniae
• Lactobacillus acidophilus
• Lactobacillus casei
• Lactobacillus delbrueckii
• Lactobacillus lindneri
• Lactobacillus plantarum
• Lactococcus lactis
• Lawsonia intracellularis
• Leptospira pomona
• Listeria grayi
• Listeria innocua
• Listeria ivanovii
• Listeria monocytogenes
• Listeria seeligeri
• Listeria welshimeri
• Micrococcus candicans
• Micrococcus luteus
• Moraxella bovis

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Microorganisms Enumerated with AATI Technology

• Mycoplasma bovis
• Mycoplasma hyopneumoniae
• Nannocystis exedens
• Oxalobacter formigenes
• Pantoea agglomerans
• Pasteurella multocida
• Pediococcus acidilactici
• Pediococcus damnosus
• Proteus mirabilis
• Pseudomonas aeruginosa
• Pseudomonas fluorescens
• Pseudomonas putida
• Ralstonia pickettii
• Raoutella terrigena
• Saccharomyces cerevisiae
• Salmonella adelaide
• Salmonella anatum
• Salmonella choleraesuis
• Salmonella dublin

• Salmonella schalwijk
• Salmonella typhimurium
• Salmonella worthington
• Serratia marcescens
• Shigella boydii
• Staphylococcus aureus
• Staphylococcus epidermidis
• Staphylococcus saprophyticus
• Stenotrophomonas maltophila
• Streptococcus bovis
• Streptococcus equinus
• Streptococcus pyogenes

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Installation, Training, Validation Support for
the Micro PRO

• Pre-Installation
• Feasibility is demonstrated by providing
  information on similar applications or performing
  a feasibility study
• Potential clients can contact current users of
  the Micro PRO technology
• Potential clients are welcome to visit the Ames,
  IA facility for a hands-on demonstration
• Training
• Training for up to two employees at the Ames, IA
  facility is included in the purchase of the Micro
  PRO system
• Installation
• Advanced Analytical issues an Installation
  Checklist to ensure the clients lab is equipped
  with the materials and environment necessary to
  operate the Micro PRO
• Micro PRO is installed by Advanced Analytical
  personnel using Installation and Operation
  Qualification (IQ/OQ) documents

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Installation, Training, Validation Support for
the Micro PRO

• Validation
• Advanced Analytical provides a validation guide
  and protocols for several applications to assist
  in the implementation process
• Provide onsite validation support and testing
• Conduct customer validation testing at our Ames,
  IA facility
• On-going Support
• Comprehensive support in the US is provided by
  Advanced Analyticals team of microbiologists and
  service technicians
• International equipment and application support
  is provided by Advanced Analytical and our
  extensive network of distributors
• Preventive maintenance program
• 21 CFR Part 11 compliant software

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The Micro PRO Delivers on the Promise of Rapid
Microbiology

• Versatile system covering the many aspects of
  microbial testing (qualitative and quantitative
  modes of operation)
• Automated, high throughput processing (up to 20
  samples/hour)
• with unattended analysis
• Reagents stable at room temperature for 7 days
• Easy to use and maintain
• Media kit containing all necessary components
• Intuitive, 21 CFR compliant software
• Preventive maintenance agreements available
• Excellent technical and validation support

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THANK YOU
www.aati-us.com
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RBD3000 Customer Case Studies
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VISTAKON (JJ)
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Efficacy studies - Flow Diagram of current
methodology vs. use of RBD 3000
subculture organism 24 hours
set up testing 24-48 hours incubation
conduct testing
plate samples, 48 hour incubation
use of AATI RBD 3000
From conducting test to data output for 24
samples 6 hours
incubate
incubate
read plates
Data crunch
crunch data
From conducting test to data output for 24
samples 58 hours
From Hasher-Homesley, P.1, 2004. Validation of
the RBD3000 Flow Cytometer for Bacterial
Enumeration. 1Johnson Johnson Vision Care.
Rapid Microbial Methods Conference, San Diego, CA
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QC PA ATCC 9027
From Hasher-Homesley, P.1, 2004. Validation of
the RBD3000 Flow Cytometer for Bacterial
Enumeration. 1Johnson Johnson Vision Care.
Rapid Microbial Methods Conference, San Diego, CA.
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From Hasher-Homesley, P.1, 2004. Validation of
the RBD3000 Flow Cytometer for Bacterial
Enumeration. 1Johnson Johnson Vision Care.
Rapid Microbial Methods Conference, San Diego, CA.
75
Equipment equivalency ID 1508 vs 1506
From Hasher-Homesley, P.1, 2004. Validation of
the RBD3000 Flow Cytometer for Bacterial
Enumeration. 1Johnson Johnson Vision Care.
Rapid Microbial Methods Conference, San Diego, CA.
76
Annual Cost for Efficacy studies Current method
vs. RBD 3000 system

• Current method
• Estimated annual expenditure for Efficacy studies
• 219,960

• RBD 3000
• Estimated 1st year expenditure for Efficacy
  studies using the RBD 3000 system
• 126,640

Estimated 1st year savings of 93,320
After 1 year warranty maintenance contract
9600 Estimated annual expenditure for
Efficacy studies 47,640
Estimated thereafter savings of 172,320
From Hasher-Homesley, P.1, 2004. Validation of
the RBD3000 Flow Cytometer for Bacterial
Enumeration. 1Johnson Johnson Vision Care.
Rapid Microbial Methods Conference, San Diego, CA.
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Applications

• BI Enumerations
• In-Process and End-Use solution testing
• Label claim enumerations from outside vendor
  sources i.e. quanti-cults and spore preparations
• Routine finished goods Bioburden Testing
• Microbial adhesion assays
• Sample purity check

From Hasher-Homesley, P.1, 2004. Validation of
the RBD3000 Flow Cytometer for Bacterial
Enumeration. 1Johnson Johnson Vision Care.
Rapid Microbial Methods Conference, San Diego, CA.
78
Advantages of RBD 3000

• Rapid bacteria detection
• Counting organisms in real time within a defined
  volume
• Total bacterial testing or specific pathogen
  testing
• Non-specific labeling with fluorescent probes
• Organism specific labeling with selective
  fluorescent antibodies or gene probes
• Automated sample preparation and testing
• Real time validation
• 42-position auto sampler for high sample
  throughput
• Fast, accurate and easy to use
• Accurate detection 101-106 cfu/ml and
  reproducibility shown at 2-3 x 101
• Detection 0.1 micron and larger
• Red laser excitation for reduction in background
  signal of non-bacterial origin
• Standardized testing protocols
• In house method development in the form of
  application notes providing customers with
  working protocols
• On system reagents for unattended operation
• Reducing chances of cross contamination and human
  error
• 21 CFR Part 11 Compliant

From Hasher-Homesley, P.1, 2004. Validation of
the RBD3000 Flow Cytometer for Bacterial
Enumeration. 1Johnson Johnson Vision Care.
Rapid Microbial Methods Conference, San Diego, CA.
79
Personal Care Products Company
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- Presence/Absence of bacteria in the final
product- Bacterial water testing-
Environmental surface monitoring on the
production line
Evaluation performed
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Presence/Absence

1. Dilute 1mL product in 9mL Enrichment Broth
2. Place samples at 32oC and rock for 18-24 hours
3. Dilute enriched samples 10-3 in 10mM PB, 40µm
   filter if necessary
4. Dispense 3mL sample into a 5mL snap cap tube
5. Analyze on the RBD 3000 for TVO

Interpretation of Results Enriched samples are
considered positive if they are 10x the background
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Results
Sample RBD 3000 Results Plate Results
Sample 1
Sample 2 - -
Sample 3
Sample 4 - -
Sample 5 - -
Sample 6 - -
Sample 7 - -
Sample 8 - -
Sample 9 - -
Sample 10 - -
Sample 11
Approximately 300 samples were run parallel to
standard cultural methods. Results from RBD are
equivalent to the plate method.
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Bacterial water testing
- 5 minutes per water sample for microbial
results. - Samples can be analyzed directly on
the RBD with enumeration of viable cells per mL.
- Microbial situations can be evaluated and the
source determined in minutes. (i.e. city water
main break)
84
Environmental surface monitoring on the
production line

• RBD 3000 detected surface contamination of
  bacteria and residual product.
• RBD 3000 will help validate cleaning procedures
  and ensure all residual product is removed before
  beginning new batch.

Product residue
Negative control
Microbial population
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Conclusions

• Capable of detecting bacterial contamination
  using Presence/Absence test in finished goods
• Capable to enumerate bacterial contamination in
  DI water. Results were obtained within hours
  after pulling samples
• Environmental surface monitoring tests on the
  RBD 3000 detected microbial contaminants.
• Unexpected result showed residual product could
  also be seen after cleaning.
• Pass/Fail criteria were able to be set in RBD
  3000 to automatically tell user if samples are
  good/bad

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Procter Gamble
87
Loose Adsorbents study

• As a screener, loose adsorbents are placed in
  water to test their effectiveness on bacterial
  removal
• Enumerates the removal efficiency and potential
  capacity for microbial adsorption
• Prior to counting, the sample is passed through a
  5 µm syringe filter to remove adsorbent
• Background run to ensure filtered adsorbents had
  no adverse effect on RBD
• For correlation, the RBD counts were initially
  compared to other methods (Pour plating and
  ColilertTM)

From Kozak, K.1, Petersen, J. and Lasky,S.J.2,
2004. Rapid Microbiological Testing Using Flow
Cytometry in Support of Product Development.
1Procter and Gamble and 2Advanced Analytical.
European Microbiology Event of the Year (IVT),
Amsterdam, The Netherlands.
88
Typical Scatterplots of Adsorbent Study

• Microbial removal over time
• Quickly know if adsorbent is efficacious

Control
After 120 minutes
After 60 minutes
After 30 seconds
From Kozak, K.1, Petersen, J. and Lasky,S.J.2,
2004. Rapid Microbiological Testing Using Flow
Cytometry in Support of Product Development.
1Procter and Gamble and 2Advanced Analytical.
European Microbiology Event of the Year (IVT),
Amsterdam, The Netherlands.
89
E. Coli with Adsorbent
From Kozak, K.1, Petersen, J. and Lasky,S.J.2,
2004. Rapid Microbiological Testing Using Flow
Cytometry in Support of Product Development.
1Procter and Gamble and 2Advanced Analytical.
European Microbiology Event of the Year (IVT),
Amsterdam, The Netherlands.
90
R. terrigena with Adsorbent
From Kozak, K.1, Petersen, J. and Lasky,S.J.2,
2004. Rapid Microbiological Testing Using Flow
Cytometry in Support of Product Development.
1Procter and Gamble and 2Advanced Analytical.
European Microbiology Event of the Year (IVT),
Amsterdam, The Netherlands.
91
P. aeruginosa with Adsorbent
From Kozak, K.1, Petersen, J. and Lasky,S.J.2,
2004. Rapid Microbiological Testing Using Flow
Cytometry in Support of Product Development.
1Procter and Gamble and 2Advanced Analytical.
European Microbiology Event of the Year (IVT),
Amsterdam, The Netherlands.
92
Loose Adsorbent Screening

• How is this useful to RD?
• Good correlation between traditional methods and
  the RBD 3000
• Rapid screener for potential adsorbents
• Pretreatment (5um filter) allows for bacterial
  enumeration in a complex media
• Noticed differences in adsorption of microbes

From Kozak, K.1, Petersen, J. and Lasky,S.J.2,
2004. Rapid Microbiological Testing Using Flow
Cytometry in Support of Product Development.
1Procter and Gamble and 2Advanced Analytical.
European Microbiology Event of the Year (IVT),
Amsterdam, The Netherlands.
93
RBD 3000 Reference
Miller, M. J., Encyclopedia of Rapid
Microbiological Methods, Volume 2, DHI
Publishing, River Grove, IL, USA. 
2005.   Chapter 16 Steger, A. M. Rapid
enumeration of microorganisms using Advanced
Analyticals RBD 3000. Encyclopedia of Rapid
Microbiological Methods, Volume 2. Ed. M. J.
Miller. River Grove, IL, USA DHI Publishing,
LLC, 2005. (AATI)   Chapter 17 Kozak, K. C. and
D. E. Langworthy.  Rapid Microbial Counting by
Flow Cytometry Validation and Implementation for
Research and Development (RD) Applications.
Encyclopedia of Rapid Microbiological Methods,
Volume 2. Ed. M. J. Miller. River Grove, IL, USA
DHI Publishing, LLC, 2005. (PG) Chapter 18
Homesley, P. H.  The RBD 3000 Rapid Bacterial
Enumeration System as an Alternative to
Traditional Pour Plate Enumeration. Encyclopedia
of Rapid Microbiological Methods, Volume 2. Ed.
M. J. Miller. River Grove, IL, USA DHI
Publishing, LLC, 2005. (JJ)