Disruptive Technologies Product Range
and Manufacturers Represented
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Biolgical Sample Preparation
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Rapid microbiology with MicroPRO
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Service
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Agilent Channel Partner to service
their range of HPLC, UV
spectrophotometers and CE
systems
OPLC chromatography solutions for semi
preparative applications (OPLC systems,
pumps, sample applicator, video imaging
and densitometry instruments, reagent
sprayer (OPLC-NIT)
Flash chromatography (Gyan)
Automated SPE system (HTA)
Analytical
HT Log P and pKa analyzer (AATI)
Kinetic solubility instruments
(Analiza)
(AATI)
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Physico Chemistry
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Dissolution baths, friability and
disintegration instruments (Distek)
Preparative
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Nucleic acides, protein and small
molecules extraction from hard-tolyse tissue samples (Pressure
Biosciences)
Dissolution/Formulation
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HT oligonucleotides purity analyzer (AATI)
HT proteins analyzer (AATI)
HT DNA analyzer (AATI)
HT Chiral analyzer (AATI)
Spotter for MALDI and tissue MALDI
imaging (SunChrom)
Type-C silica hydride HPLC columns Flat
sorbent beds for OPLC (MicroSolv)
Accessories and consumables for CE and
HPLC (MicroSolv)
Validation kits for HPLC systems
(MicroSolv)
Who are we?
Our History:
Our Business:
Our Markets:
Our Solution:
Our Products:
Founded 1997, 4 Scientific Co-founders,
Privately Funded
Rapid Microbial Detection Technology
Capillary Electrophoresis (January 2007)
Pharmaceutical, Personal Care Products,
Fermentation, Environmental, Drug Discovery,
Oligonucleotides Production.
Replace current microbial detection methods
(requiring 24 – 72 hours). High throughput
quality analysis of oligonucleotides and fast pKa
and Log P determinations of chemical
compounds.
MicroPRO Instrument, Custom Kits. OligoPRO
and pKa PRO analyzers.
Ames, Iowa
Overview of Microbial Detection
Presentation
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How the Technology Works
Standard Labeling Protocol
The MicroPRO Instrument
Water Monitoring
Bacteria, Yeast and Mold in 24 hours in Products
Surface/Environment Monitoring Using Swabs
Fermentation Products
Challenge Tests in Products
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
Fluidic System
Sample delivery
Sheath delivery
(0.05mL/min)
(16-18mL/min)
Sheath
flow
Labeled
bacteria
Core
flow
• Quantitative cell delivery
• Hydrodynamic Focusing
• Single File Passage
through detection region
Optic System
Labeled
microorganism
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
Electronic System
• 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
Example of Micro PRO Intensity Plot
Cell
size
Amount of label
Results
Fluorescence Intensity vs. Counts
Intensity Plot
Scatter Intensity vs. Counts
Box = 9907 counts/0.25mL
97.4% of the counts are in the box
MicroPRO Standard
Labeling Protocol
Biomass Labeling Protocol
(Membrane Permeable – all Cells are Stained)
RBD Total
Cell Count / mL
Ideal for enumerating Live & Dead microbes in:

Fermentation cultures

Spore preparations
TVO Labeling Protocol
(Only Viable Cells are Stained)
Ideal for enumerating Viable microbes in:
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Process water monitoring
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Final product testing
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Presence/Absence testing
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Pure cultures
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Fermentation cultures
Antibody-Specific Detection
Labeling Protocol
Mixed Sample
Ideal for enumerating microbes for/in:
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Specific pathogen detection
Mixed cultures
High background matrices
MicroPRO System
Auto-Sampler
• Holds up to 42 samples
Auto-Prep
• Adds reagents
• Mixes sample
• On-board automated cleaning
and bubble removal
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
4
Select Tray SetUp
Micro PRO™ Output:
Pass/Fail
&
Counts/mL
MicroPRO
Various Results/
Applications
Purified Water
Monitoring
Sample Preparation
• Collect sample;
• Dispense 3mL sample into 5mL snap-cap tube
• Load sample into MicroPRO Sample Tray with
capped syringe
• Select pre-defined (or create new) Analysis/Tray
Sequence
• MicroPRO count result in 5 minutes
1
2
Background / Neg.Ctrl
Sample 3
Box = 0 counts/0.25mL
Box = 2 counts/0.25mL
3
Sample 7
Box = 22 counts/0.25mL
Purified Water Monitoring:
MicroPRO TVO & R2A Plate Counts
RBD 3000
R2A Plates 5-day
R2A Plates 10-day
action
100
90
80
Counts/mL
70
60
warning
50
40
30
20
10
0
1
2
3
4
5
6
7
8
9
10
Sample Number
11
12
13
14
15
16
17
Purified Water System
MQ system CPL2 Micro qual
Log10 count/ml or cfu/ml
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
1
2
3
4
5
6
7
8
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
Product Research Lab Water Testing
Comparison of RBD vs. manual plate count for PRL water
1000
900
800
700
cfu/ml
600
RBD ct/ml
TSA plate
Alert level
500
400
300
200
100
8U
31U
22U
4T
11U
19U
34U
30U
31U
8U
14U
33U
26U
13U
19U
16U
33U
34U
5T
14U
20U
5T
15U
21U
32U
31U
8U
10U
11U
33U
16U
4T
19U
30U
35T
22U
18U
33U
32U
11U
4T
21U
0
Sample port
Kozak, K.1, 2006. Rapid Microbiological Testing in Support of Product Development.
1Procter
and Gamble. Rapid Microbial Methods User’s Meeting. Chigaco, 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
1
2
3
4
5
6
7
8
9
10
1.50
1.00
0.50
0.00
Sample
Product Testing
Presence/Absence
Traditional methods USP <61>
Bacteria
1:10
dilution of
product
1 ml 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
Advanced Analytical Method
Bacteria, Yeast & Mold
1:10
dilution of
product
1ml in
Tube A
Incubate
24 to 48
hours at
30°C
Transfer substrate
tube A to
Tube B; vortex;
0.1mL to Tube C
Micro PRO™
Product Test Kit
35mm filter
mold
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
Experimental Procedure
1. Prepare 1:10 product suspension in buffer or neutralizing growth
enhancement media
2. Add 1mL 1:10 product suspension to Tube A & neutralize 30 min
3. Spike with <100 cfu Escherichia coli ATCC 8739 or 25922,
Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC
6538, Candida albicans ATCC 10231, Aspergillus niger ATCC 16404
4. Prepare non-spiked product controls
5. Enrich at 30oC for 24-48 hours
6. Transfer substrate from Tube A to Tube B and vortex
7. Transfer 0.1mL from Tube B to Tube C and load in the Micro PRO™
8. Plate samples post-enrichment
Analysis on MicroPRO
2
1
Select Method
Load sample vials and
syringes
Reagent additions and
sample injection
performed automatically
as defined in the Method
3
Results
4
Micro PRO™ Output
Results
Micro PRO™ output shows few to no counts within the area
definitions (product baseline)
Pass result indicates that the sample does not contain microbial
contamination
Mold
counts/mL
Bacteria
counts/mL
Yeast
counts/mL
Mold
Result 1
Bacteria
Result 2
Yeast
Result 3
Overall
Result
54
1550
10
Pass
Pass
Pass
Pass
Results
Micro PRO™ output shows many counts within the area definition
(>3X product baseline)
Fail result indicates that the sample contains microbial
contamination
Mold
counts/mL
Bacteria
counts/mL
212002
648372
Bacteria
Yeast
Mold
counts/mL Result 1
28379
Fail
Bacteria
Result 2
Yeast
Result 3
Overall
Result
Fail
Fail
Fail
Mold
counts/mL
Bacteria
counts/mL
43
1913
Yeast
Mold
counts/mL Result 1
1770
Pass
Bacteria
Result 2
Yeast
Result 3
Overall
Result
Pass
Fail
Fail
Bacteria
Result 2
Yeast
Result 3
Overall
Result
Pass
Pass
Fail
Yeast
Mold
counts/mL
Bacteria
counts/mL
2452
2144
Mold
Yeast
Mold
counts/mL Result 1
65
Fail
Results Summary
Personal Care Products
Enrichment Time – Positive
for Bacteria, Yeast & Mold
Face scrub
24 hrs
Hair gel
24 hrs
Hand soap, antibacterial
24 hrs
Lotion
24 hrs
Mouthwash
24 hrs
Shampoo
24 hrs
Shave gel
24 hrs
Sunscreen
24 hrs
Toothpaste
30 hrs
Results Summary
OTC Pharmaceuticals
Enrichment Time – Positive
for Bacteria, Yeast & Mold
Acetaminophen
24 hrs
Ibuprofen
24 hrs
Antacid tablets
24 hrs
Anti-itch cream
24 hrs
Nasal spray
24 hrs
Sleep aid capsules
24 hrs
Cold/flu liquid
24 hrs
Allergy liquid
24 hrs
Multivitamins
24 hrs
Fiber capsules & powder
48 hrs
Results Summary
Others
Enrichment Time – Positive
for Bacteria, Yeast & Mold
Excipients
24 hrs
Household cleaner
24 hrs
Industrial Emulsions
24 hrs
Antioxidant water
24 hrs
Cranberry juice
24 hrs
Lemonade
24 hrs
Vegetable juice
48 hrs
Negative
Control
Positive
Ps. aeruginosa
Negative
Control
Positive
C. albicans
Negative
Control
Positive
A. niger
Results
Sample
RBD 3000 Results
Plate Results
#1-Alberto Moisture Hold Gel
+
+
#2-TRESemme Instant Heat Tamer
-
-
#3-TRESemme Instant Heat Tamer
+
+
#4-VO5 Free Me Freesia Shampoo
-
-
#5-VO5 Tea Therapy Shampoo
-
-
#6-VO5 Free Me Freesia Conditioner
-
-
#7-St. Ives Apricot Radiance Cream Cleanser
-
-
#8-TRESemme Mega Sculpting Gel
-
-
#9-TRESemme Provitamin B5 & Aloe
Conditioner
-
-
#10- St. Ives Apricot Shower Cleanser/Mask
-
-
#11-VO5 Leave in Conditioner Anti-Frizz
+
+
Approximately 300 samples were run parallel to standard cultural methods.
Results from RBD are equivalent to the plate method.
The Advanced Analytical
Solution Provides
• One test for the detection of bacteria, yeast & mold in a
variety of matrices
• Rapid screening for microbial contamination with 24 hour
results for a majority of the products tested
• Objective Pass/Fail results requiring no additional
interpretation
• Pass/Fail criteria that generate no false positives or false
negatives
• Versatile system for quantification of microorganisms in
purified water, surface swabs, and pure cultures
Environmental
Monitoring (swabs)
Environmental Swab Protocol
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Place a swab sample in a 5mL snap-cap tube
containing 900µL 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 MicroPRO
1
2
3
4
Environmental Sample Testing
• Swab samples are directly analyzed on the MicroPRO
• Result is obtained in minutes rather than overnight
Swab Control
E.coli Swab
Swab Sample: Population
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
Environmental Sample Testing
• Surface swabs are analyzed on the
MicroPRO with results within 5 minutes
• Data generated by the MicroPRO not
only provides information about
microbial populations but also indicates
levels of residual product
Environmental Swab Protocol –specific
pathogens (Salmonella)
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Place a swab sample in a 5mL snap-cap tube
containing 900µL Buffered Peptone Water
incubate at 42oC (+2oC) with rocking* for 3 hours
Add 1mL 2X strength RV broth to each tube and
incubate with rocking for an additional 4 hours
Press the swab against the side of the tube to
express extra liquid
After enrichment, label samples with antibody (40
minutes)
Dilute enriched samples in phosphate buffer
Analyze samples and controls on the MicroPRO for
the presence/absence of Salmonella spp.
Environmental Swab Protocol –specific
pathogens (Salmonella)
Negative control
Positive (22 isolates tested)
Fermentation / 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 MicroPRO sample tray with capped syringe
• Select pre-defined (or create new) Analysis/Tray Sequence
• MicroPRO count result in 5 minutes
TVO - Escherichia coli
PB Background
~101 cfu/mL
Box = 1 count/0.25 mL
Box = 3 count/0.25 mL
~102 cfu/mL
~103 cfu/mL
Box = 36 counts/0.25 mL
Sample
RBD 3000
Counts/0.25mL
RBD 3000
Counts/mL*
PB Background
1
4
~101 E. coli in PB
3
~102 E. coli in PB
Box = 401 counts/0.25 mL
~104 cfu/mL
Box = 3725 counts/0.25mL
Plate
Counts/mL
RBD 3000
log10 Counts/mL
Plate Counts
log10 cfu/mL
9
19
0.95
1.28
36
150
280
2.18
2.45
~103 E. coli in PB
401
1707
1665
3.23
3.22
~104 E. coli in PB
3725
15894
18050
4.20
4.26
*RBD 3000 counts/mL are background corrected and have been adjusted for reagent additions.
Correlation of RBD 3000 TVO Counts vs. Plate Counts
(Poster presented at SIM 2006)
C. albicans
E. coli
M. bovis
S. typhimurium
7.00
6.50
RBD 3000 (log10 counts/mL)
6.00
5.50
5.00
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
Plate Counts (log10 cfu/mL)
Candida albicans (n = 14, R2 = 0.9982), Escherichia coli (n = 17, R2 = 0.9959), Mycoplasma
bovis (n = 15, R2 = 0.9891) and Salmonella typhimurium (n = 15, R2 = 0.9952).
6.50
7.00
Correlation of RBD 3000 TVO Counts vs. Plate Counts
(Poster presented at SIM 2006)
B. atrophaeus
C. perfringens Type A
S. aureus
S. bovis
7.00
6.50
6.00
RBD 3000 (log10 counts/mL)
5.50
5.00
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
Plate Counts (log10 cfu/mL)
Bacillus atrophaeus (n = 15, R2 = 0.9839), Clostridium perfringens Type A (n = 12, R2 = 0.9981),
Staphylococcus aureus (n = 18, R2 = 0.9857) and Streptococcu bovis (n = 13, R2 = 0.9832)
6.50
7.00
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Some of the Microorganisms
Enumerated with the MicroPRO
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
Some of the Microorganisms
Enumerated with the MicroPRO
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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
Challenge Tests
Overview
Contamination Level Test
–
–
–
–
For materials that contain low-level bio-burden
Determines that material meets microbial specification
Utilizes the Most Probable Number (MPN) protocol
Useful for complex matrices
Antimicrobial Effectiveness Test
– Rapidly screens candidate preservative systems
– Tracks an increase in dead cell count
Contamination Level Test –
Feasible in a Variety of Complex Matrices
Company A – In-process pharmaceutical product
Company B – Various nutraceutical finished products
– Juice, Body Butter, Fiber
Company C – Liquid nutraceutical finished product
Company D – Various alcoholic beverages
CLT – Protocol for <100cfu/g specification
1. Add 1mL of product to 9mL of Enrichment Media
Dilution A
<100 total cfu/10mL
2. Add 1mL of Dilution A to 9mL of Enrichment Media
Dilution B
<10 total cfu/10mL
3. Add 1mL of Dilution B to 9mL of Enrichment Media in
triplicate
Final Dilution
<1 total cfu/10mL
4. Enrich Final Dilution samples for 18 hours
5. Dilute enriched samples in phosphate buffer for a total
of 3mL per sample
6. Analyze Samples on the RBD 3000
CLT – Results Company A
In-process Product: Specification <1000cfu/g
Spiked Below Specification
Sample Dilution
RBD 3000 Result
(# pos./# neg.)
Plate Result
(# pos./# neg.)
Microbial level determined by
RBD 3000 (cfu/g)
1:10
3/0
3/0
>1
1:100
3/0
3/0
>10
1:1,000
3/0
3/0
>100
1:10,000
1/2
1/2
<1,000
Product contains >100cfu/g and <1000cfu/g; product is within specification
Actual spike was 559 cfu Ps. aeruginosa/g product
Spiked Above Specification
Sample Dilution
RBD 3000 Result
(# pos./# neg.)
Plate Result
(# pos./# neg.)
Microbial level determined by
RBD 3000 (cfu/g)
1:10
3/0
3/0
>1
1:100
3/0
3/0
>10
1:1,000
3/0
3/0
>100
3/0
>1,000
1:10,000
3/0
Product contains >1000cfu/g; product fails
Actual spike was 3920 cfu Ps. aeruginosa/g product
CLT – Results Company B
Body Butter & Juice Products: Specification <100cfu/g or mL
Body Butter: Spiked Below Specification
Sample Dilution
RBD 3000 Result
(# pos./# neg.)
Growth on YM
PetrifilmTM
(# pos./# neg.)
Microbial level determined by
RBD 3000 (cfu/g)
1:100
2/1
2/1
>10
1:1,000
0/3
0/3
<100
1:10,000
0/3
0/3
Product contains >10cfu/g and <100cfu/g; product is within specification
Actual spike was 35 cfu C. albicans/g body butter
<1000
Juice: Spiked Above Specification
Sample Dilution
RBD 3000 Result
(# pos./# neg.)
Growth on YM
PetrifilmTM
(# pos./# neg.)
Microbial level determined by
RBD 3000 (cfu/g)
1:100
3/0
3/0
>10
1:1,000
2/1
2/1
>100
1:10,000
1/2
1/2
<1000
Product contains >100cfu/g; product fails
Actual spike was 110cfu C. albicans/mL juice
Taken from poster presentation at ASM 2006 A Rapid Most Probable Number Test for Yeast
in Nutraceutical Products, K.A. Molitor and A.M. Steger, Advanced Analytical Technologies,
Inc., Ames, IA 50010 and D. Wright and M. Roblin, Morinda, Inc., Orem, UT 84604
Contamination Level Test – Benefits
• 18-24hr time-to-result
• Detects lower levels of potential microbial
contamination than the traditional plate count
method in complex/opaque matrices
• Results are confirmed by the “Most Probable Total
Count by Multiple-Tube Method” in USP Chapter
<61>, Microbial Limits Test
Antimicrobial Effectiveness Test
Antimicrobial Effectiveness Test
• Used to screen candidate preservatives for liquid
products
• Pharmaceutical and Cosmetic Industries routinely
perform
– New & reformulated products
• Problems with the current method:
– Compendial method takes 35 days
– Requires significant labor & materials
– Not all types &/or formulations of antimicrobials are screened due to time
& labor constraints
– Delays development of suitable preservative systems
– Delays product time to market
Antimicrobial Effectiveness Test –
Solution
• Use the MicroPRO to rapidly screen
candidate preservative systems
• Benefits:
– Shorter time to result
– Significant labor and materials savings
– Evaluate more types/formulations of antimicrobials
– Develop better preservative systems
– Speed product time to market
Antimicrobial Effectiveness Test –
Protocol
• Challenge 0.01% (w/v) Benzoic Acid with 105 – 106
cfu/mL
• Perform challenge studies with Ps. aeruginosa, S.
aureus, and C. albicans
• Use MicroPRO to determine titer of inoculum
• Sample at 0, 2hr, 4hr, 6hr, 8hr & 24hr postinoculation
– Dilute samples 1:10 in phosphate buffer for MicroPRO Biomass & Dead
Cell counts using the Biomass and Dead Cell Test Kits
– Dilute samples in phosphate buffer + 4% Tween 20 for TSA plate count
comparison
0.01% Benzoic Acid Challenged with
Ps. aeruginosa – Test 1
Ps .aeruginosa-Biomass Count
Ps. aeruginosa-Dead Cell Count
Ps. aeruginosa-Plate Count
6.0
RBD 3000 log 10 Counts/mL
5.5
5.0
4.5
5.78
5.76
5.83
5.64
5.84
5.83
5.34
4.0
4.93
5.83
5.14 5.12
5.81 5.73
5.22 5.29
4.68
3.5
4.14
3.98
3.0
0
2
4
6
Time post-inoculation (hours)
8
24
0.01% Benzoic Acid Challenged with
Ps. aeruginosa – Test 2
Ps. aeruginosa-Biomass Count
Ps. aeruginosa-Dead Cell Count
Ps. aeruginosa-Plate Count
6.0
RBD 3000 log10 Counts/mL
5.5
5.0
4.5
5.72
5.59
5.76
5.75
5.46
5.25
5.10
4.0
5.77
5.75
5.27
5.10
5.775.71
5.36
5.18
4.86
4.46
3.5
2.4
3.0
0
2
4
6
Time post-inoculation (hours)
8
24
0.01% Benzoic Acid Challenged
with S. aureus
S. aureus-Biomass Count
S. aureus-Dead Cell Count
S. aureus-Plate Count
6.0
RBD 3000 log10 Counts/mL
5.5
5.0
4.5
5.96
5.81
5.62
5.61
5.59
5.37
4.0
5.47 5.50
5.61 5.52
5.60 5.53
5.58 5.59
5.17
5.14
4.94
3.5
1.78
3.0
0
2
4
6
Time Post-Inoculation (hours)
8
24
0.01% Benzoic Acid Challenged
with C. albicans
C. albicans-Biomass Count
C. albicans-Dead Cell Count
C. albicans-Plate Count
6.0
RBD 3000 log10 Counts/mL
5.5
5.0
4.5
5.55
4.0
5.45
5.24
5.11
5.45
5.31
5.44
5.31
5.53
5.25
5.56
5.37
4.36
3.5
3.93
4.03
0
2
4.00
3.98
3.79
3.0
4
6
Time Post-Inoculation (hours)
8
24
Summary
Contamination Level Test
– Provides a rapid & sensitive detection method
for determining if materials meet particular
microbial specifications
Antimicrobial Effectiveness Test
– Provides a rapid method for screening candidate
preservative systems
Applications Summary
Advanced Analytical Provides
Solutions for
• Purified Water
• Final Product Testing/Raw Material Testing
• Fermentation/Animal Health/Vaccines
• Pure cultures/ Spore preparations (US Army)
• Residue testing/swab testing
• Specific Pathogen testing
• Challenge Tests
RBD 3000/MicroPRO References
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)
For More Information
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