Advanced Analytical
Micro PRO™
Overview of Today’s Presentation
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History of Advanced Analytical Technologies, Inc.
How the Technology Works
The Micro PRO™ System
Micro PRO™ Applications and Results
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Qualitative Analysis – Presence/Absence
Quantitative Analysis – Enumeration
Customer Presentations
Advanced Analytical
Improving Process
Empowering Progress
OUR MISSION
Through innovative technology and collaborative
relationships we will:
> Deliver superior customer support, services, and solutions
> Create sustainable growth
> Foster a dynamic and fulfilling work environment
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
Basics of Flow
Cytometry
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
Flow Cytometry: Fluidics
Sample delivery
Sample
delivery
Labeled
bacteria
Sheath delivery
Sheath
flow
Core
flow
• Quantitative cell delivery
• Hydrodynamic Focusing
• Single File Passage
through detection region
Flow Cytometry: Optics
Labeled
microbial cell
Scatter
Detector
Fluorescence
Detector
Scatter
signal
Fluorescence
signal
High performance
optical filters
Fluorescence plus Scatter
= One Count
Laser Beam –
shaped and
focused;
635 nm
laser excitation
Flow Cytometry: Optics
Unlabeled
Particle
Scatter
Detector
Fluorescence
Detector
Scatter
signal
High performance
optical filters
Scatter without
Fluorescence = No Count
Laser Beam –
shaped and
focused;
635 nm
laser excitation
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
Time
Fluorescence
Threshold Level
Signal Processing
cumulative
Microbe A
Microbe A + B
Microbe A + B +C
Interpretation of intensity plots
Cell
size
Amount of label
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
Micro PRO™
INSTRUMENTATION
Introducing the Micro PRO™
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
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
Analysis on the Micro PRO™
1
Load sample vials and syringes
3
Reagent additions and
sample injection
performed automatically
as defined in the Method
2
Select Tray Setup
Micro PRO™ Output:
Pass/Fail
&
Counts/mL
4
The Micro PRO™
Hardware upgrades
• High sample throughput
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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
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
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View/download results during sample analysis
Reagent level alarm
Context-sensitive Help files
21 CFR part 11 compliant
Micro PRO™ Qualitative
Analysis
Product Screening
Traditional methods USP <61>
Bacteria
1:10
dilution of
product
1ml in each of two Petri
dishes with Soybean Casein
Digest medium melted <45 C
Incubate 48 to
72 hours at 30° C
Count
If zero counts, results are expressed as less than 10 cfu/ml
Yeast and Mold
1:10
dilution of
product
1 ml in each of two Petri
dishes Saboraud Dextrose
Agar
Incubate 5 to 7
days at 20-25° C
If zero counts, results are expressed as less than 10 cfu/ml
Count
Micro PRO™ Product Screening
Protocol
1) Dilute 10g product in 90mL phosphate buffer or media (1:10
product suspension)
2) Mix to achieve a homogeneous solution
3) Add 1mL of the 1:10 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
Micro PRO™ Media Kit
Microbes
35µm filter
Transfer swab
from Tube A to B
Tube A - GEM
Add product, enrich
Tube B - PB
Add swab, mix
Micro PRO™
Tube C - PB
Add 0.1mL from Tube B
Micro PRO™ Reagent Kit
Micro PRO™
Pass/Fail
Result
Test Sample
w/ Microbes
All Microbes
Stained
Viable Microbes
Stained
Ideal for detecting microorganisms post-enrichment in:
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Finished Products
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Raw Materials
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In-Process Samples
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
Interpretation of results
• Micro PRO™ picture shows many counts within the
analysis box (>4X 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
Negative
Control
Positive
A. niger
Negative
Control
Positive
P. aeruginosa
Negative
Control
Positive
C. albicans
Micro PRO™ Product Screening Results
Example of Signal:Noise ratios in various finished products
Average Signal:Noise
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
>50000
1150.5
25
1869
4933
8.5
Toothpaste
>50000
13
41
Body wash
1280.5
10569
88
Hand Soap
269
400
23.5
Laundry detergent
Fabric freshener
Micro PRO™ Product Screening Results
Household Cleaner – 24hr
Neg Ctrl
Box cnts/mL: 0/87/0
S. aureus spike
S:N 11779
C. albicans spike
S:N 439
A. niger spike
S:N 406
Hand soap – 24hr
Neg Ctrl
Box cnts/mL: 21/549/21
S. aureus spike
S:N 268.8
C. albicans spike
A. niger spike
S:N 399.6
S:N 23.5
Examples of Products Tested
Bacteria-Yeast-Mold detection in 1 assay
Personal Care Products
OTC Pharma Products
Hand soap
Shave Gel
Antacid Tablets
Body wash
Shampoo
Liquid Antacid
Face Scrub
Conditioner
Anti-itch cream
Toothpaste
Lotion
Cold Syrup
Mouthwash
Multi-vitamin (chewable)
Multi-vitamin (adult)
Household Products
Nose Drops
Dish soap
Stomach Relief
Laundry detergent
Fabric Freshener
All-purpose household cleaner
Product Testing Results
Bacteria
Yeast
Negative
Control
Mold
Micro
PRO™
Agar
Plate
Micro
PRO™
Agar
Plate
Micro
PRO™
Agar
Plate
Micro
PRO™
Agar
Plate
Dish Soap
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Toothpaste
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Shave Gel
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Multivitamin
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Results from Micro PRO™ are equivalent to the plate method.
Micro PRO™ Quantitative
Analysis
Total Viable Organisms (TVO)
Labeling Kit
Viable Count/mL
Test Sample
w/ Microbes
All Microbes
Stained
Ideal for enumerating viable microorganisms in:
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Process/Purified Water
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Surface Swabs
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Pure cultures
Viable Microbes
Stained
Purified Water Monitoring
•Results in less then
4 minutes
•Quantitative output in
counts/mL
•Significant savings in
technician labor
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.
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
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
Results – Defining the Analysis Box
Micro PRO™ Intensity Plots
E. coli
Ps. aeruginosa
Staph. aureus
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
R. pickettii
Faucet Isolate Mix
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
Clean Water TVO: Ps. aeruginosa Spike
Filtered DI H2O
Box = 2 counts/0.25mL
~101 cfu/mL
~102 cfu/mL
Box = 17 counts/0.25mL
Box = 106 counts/0.25mL
~103 cfu/mL
Box = 1241 counts/0.25mL
~104 cfu/mL
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
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~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
Purified Water Monitoring
TVO & R2A Plate Counts
Micro PRO
R2A Plates 5-day
R2A Plates 10-day
Action Limit
100
90
80
Counts/mL
70
60
Alert Limit
50
40
30
20
10
0
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Sample Number
11
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17
Customer Studies
Purified Water System
counts/mL (CFU/mL)
MQ system CPL2 Micro qual
2.50
2.00
1.50
1.00
0.50
0.00
MQ MQ MQ MQ MQ MQ MQ MQ MQ MQ MQ MQ MQ MQ MQ MQ MQ MQ
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9 10 11 12 13 14 15 16 17 18
RBD
Plate
From: Hasher-Homesley, P.1, 2006. R&D Applications for the RBD3000.
1Johnson & Johnson Vision Care. Rapid Microbial Methods User’s Meeting,
Chicago, IL
Ecoli diluted in SPW RBD 3000 Counts vs. Standard Plate Counts over 10 Samples--Reproducibility
RBD 3000 (counts/mL)
5.00
Plate Counts (cfu/mL)
4.50
4.00
3.50
Log10 Value
3.00
2.50
2.00
4.11 4.05
4.10 4.06
4.12 4.03
4.09 4.00
4.11 4.03
4.11 4.06
4.13 4.04
4.09 3.97
4.10 4.06
4.10 4.02
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10
1.50
1.00
0.50
0.00
Sample
Surface Swab Analysis/
Environmental Monitoring
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
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
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
Environmental Swab Protocol
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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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2
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4
Environmental Sample Testing
• Swab samples are directly analyzed on the Micro PRO™
• Result is obtained in minutes rather than overnight
Negative Control Swab
E.coli Spiked Swab
Environmental Swab
Box counts/0.25mL: 2
Box counts/0.25mL: 1,195
Indicative of residual product
Microbial population
within a predefined box
Residual product; an additional
box may be created to capture
this data
Pure Culture Enumeration
Enumeration of Fermentation/Pure
Cultures
• Collect sample; if necessary dilute to <106 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
Fermentation Applications
Upstream
1. Media Contamination
2. Starter Culture Viability
and/or Titer
Downstream
1. Number of cells post
inactivation
2. Number of cells after
processing
3. Cell Concentrations
Fermentation
1. Process Monitoring
2. Biomass and/or Viable
cell enumerations
3. Determination of when
to harvest or induce
Spores +
Vegetative
Vegetative
Spores
Bacillus atrophaeus
Spores vs. Vegetative
TVO – Candida albicans in PB
PB Background
Box = 1 count/0.25mL
~101 cfu/mL
~102 cfu/mL
~103 cfu/mL
~104 cfu/mL
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
Correlation of Micro PRO™ TVO
Counts vs. Plate Counts
E. coli 8739
E. coli 25922
S. aureus 6538
Ps. aeruginosa 9027
Micro PRO Counts (log10/mL)
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
0.0
1.0
2.0
3.0
4.0
5.0
Plate Counts (log 10/mL)
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).
6.0
7.0
Correlation of Micro PRO™ TVO
Counts vs. Plate Counts
A. niger spores 16404
C. albicans 10231
Micro PRO Counts (log 10/mL)
6.0
5.0
4.0
3.0
2.0
1.0
0.0
0.0
1.0
2.0
3.0
4.0
Plate Count (log 10/mL)
Aspegillus niger spores 16404 (R2 = 0.9959) and Candida albicans 10231 (R2 = 0.9971)
5.0
6.0
Microorganisms Enumerated
with AATI Technology
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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
Enterobacter aerogenes
Enterobacter cloacae
Enterococcus casseliflavus
Enterococcus durans
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Enterococcus faecium
Enterococcus faecalis
Enterococcus gallinarum
Enterococcus hirae
Enterococcus mundtii
Erysipelothrix rhusiopathiae
Escherichia coli
Escherichia coli O157:H7
Escherichia coli O25:HN
Escherichia coli O15:NM
Escherichia coli O1:NM
Escherichia coli O7:NM
Escherichia coli O78:NM
Escherichia coli ON:H8
Escherichia coli ON:NM
Escherichia coli O8:HN
Geobacillus stearothermophilus
Geobacillus stearothermophilus
spores
Giardia lamblia cysts
Haemophilus parasuis
Haemophilus somnus
Halobacterium salinarum
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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
Microorganisms Enumerated
with AATI Technology
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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 enteriditis
Salmonella hadar
Salmonella heidelberg
Salmonella iverness
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Salmonella schalwijk
Salmonella typhimurium
Salmonella worthington
Serratia marcescens
Shigella boydii
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus saprophyticus
Stenotrophomonas maltophila
Streptococcus bovis
Streptococcus equinus
Streptococcus pyogenes
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 client’s
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
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 Analytical’s
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
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
THANK YOU
www.aati-us.com
RBD3000 Customer
Case Studies
VISTAKON (J&J)
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
incubate
use of AATI RBD 3000
From conducting test to
data output for 24 samples = 6
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
hours
QC PA ATCC 9027
RBD 3000 counts/ml (log)
RBD 3000 ID 1508 vs. Pour Plate
7.00
6.00
R2 = 0.9903
5.00
4.00
3.00
2.00
1.00
0.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
Pour Plate CFU/ml (log)
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.
7.00
cfu/ml: counts/ml
RBD 3000 PQ RUN 1: method equivalency
8
6
4
2
0
1
2
3
4
5
6
7
8
9
10
11
12
13
samples
Plate Count
RBD 3000
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.
14
Equipment equivalency ID 1508 vs 1506
RBD 3000 # 1508 VS. 1506
R2 = 0.9914
RBD 3000 # 1508
6.00
5.00
4.00
3.00
2.00
1.00
0.00
0.00
1.00
2.00
3.00
4.00
5.00
RBD 3000 # 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.
6.00
Annual Cost for Efficacy studies
Current method vs. RBD 3000 system
• Current method
Estimated annual expenditure for Efficacy
studies
• RBD 3000
Estimated 1st year expenditure for
Efficacy studies using the RBD 3000
system
$219,960
$126,640
Estimated 1st year
savings of $93,320
After 1 year warranty maintenance
contract
$9600
Estimated thereafter
savings of $172,320
Estimated annual expenditure for
Efficacy studies
$47,640
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.
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.
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.
Personal Care Products
Company
Evaluation performed :
- Presence/Absence of bacteria in the final product
- Bacterial water testing
- Environmental surface monitoring on the production line
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
Results
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
+
+
Sample
Approximately 300 samples were run parallel to
standard cultural methods. Results from RBD
are equivalent to the plate method.
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)
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.
Negative control
Microbial population
Product residue
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
Procter & Gamble
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.
Typical Scatterplots of Adsorbent Study
Control
• Microbial removal over time
• Quickly know if adsorbent is
efficacious
After 60 minutes
After 120 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.
E. Coli with Adsorbent
1.00E+07
Counts/mL
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
RBD counts/mL
1.00E+01
Colilert cfu/mL
1.00E+00
0
10
20
30
40
50
Time (minutes)
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.
60
Counts/mL
R. terrigena with Adsorbent
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
RBD counts/mL
Colilert cfu/mL
0
10
20
30
40
50
60
Time (minutes)
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.
Counts/mL
P. aeruginosa with Adsorbent
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
RBD counts/mL
Plates cfu/mL
0
10
20
30
40
50
Time (minutes)
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.
60
Loose Adsorbent Screening
•
How is this useful to R&D?
• 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.
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 Analytical’s 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
(R&D) Applications.” Encyclopedia of Rapid Microbiological Methods, Volume
2. Ed. M. J. Miller. River Grove, IL, USA: DHI Publishing, LLC, 2005. (P&G)
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. (J&J)