Disruptive Technologies Product Range and Manufacturers Represented

Disruptive Technologies Product Range Disruptive Technologies Product Range and Manufacturers Representedand Manufacturers Represented

• Biolgical Sample Preparation– Nucleic acides, protein and small

molecules extraction from hard-to-lyse tissue samples (Pressure Biosciences)

• Dissolution/Formulation– Dissolution baths, friability and

disintegration instruments (Distek)

• Physico Chemistry– HT Log P and pKa analyzer (AATI)– Kinetic solubility instruments

(Analiza)

• Rapid microbiology with MicroPRO (AATI)

• Service– Agilent Channel Partner to service

their range of HPLC, UV spectrophotometers and CE systems

• Preparative– 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 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?Who are we?Our History: Founded 1997, 4 Scientific Co-founders,

Privately Funded

Our Business: Rapid Microbial Detection Technology

Capillary Electrophoresis (January 2007)

Our Markets: Pharmaceutical, Personal Care Products, Fermentation, Environmental, Drug

Discovery, Oligonucleotides Production.

Our Solution: 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.

Our Products: MicroPRO Instrument, Custom Kits. OligoPRO and pKa PRO analyzers.

Ames, Iowa

Overview of Microbial Detection Overview of Microbial Detection PresentationPresentation

• 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 Basics of Flow CytometryCytometry

Basics of Flow CytometryBasics 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 SystemFluidic System

• Quantitative cell delivery

• Hydrodynamic Focusing

• Single File Passage through detection region

Sheath flow

Labeled bacteria

Core flow

Sample delivery(0.05mL/min)

Sheath delivery(16-18mL/min)

FluorescenceDetector

Laser Beam – shaped and

focused; 635 nm

laser excitation

Labeled microorganism

Fluorescencesignal

High performance optical filters

ScatterDetector

Scatter signal

Fluorescence plus Scatter = One Count

Optic SystemOptic System

Electronic SystemElectronic 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

Fluorescence Threshold Level

Time

= 1 count

= 0 count

Detectoroutput

Signal ProcessingSignal Processing

cumulative

Microbe A

Microbe A + B

Microbe A + B +C

Example of Micro PRO Intensity PlotExample of Micro PRO Intensity Plot

Cellsize

Amount of label

ResultsResultsFluorescence Intensity vs. Counts

Scatter Intensity vs. Counts

Intensity Plot

Box = 9907 counts/0.25mL97.4% of the counts are in the box

MicroPRO Standard MicroPRO Standard Labeling ProtocolLabeling Protocol

Biomass Labeling ProtocolBiomass Labeling Protocol

RBD TotalCell

Count / mL

(Membrane Permeable – all Cells are Stained)(Membrane Permeable – all Cells are Stained)

Ideal for enumerating Live & Dead microbes in: Fermentation cultures Spore preparations

TVO Labeling ProtocolTVO Labeling Protocol(Only Viable Cells are Stained)(Only Viable Cells are Stained)

Ideal for enumerating Viable microbes in: Process water monitoring Presence/Absence testing Fermentation cultures

Final product testing Pure cultures

Antibody-Specific DetectionAntibody-Specific DetectionLabeling ProtocolLabeling Protocol

Mixed Sample

Ideal for enumerating microbes for/in: Specific pathogen detection Mixed cultures High background matrices

MicroPRO SystemMicroPRO System

Auto-PrepAuto-Prep

• Adds reagents• Mixes sample• On-board automated cleaning

and bubble removal

Auto-SamplerAuto-Sampler

• Holds up to 42 samples

Analysis on the Analysis on the Micro PROMicro PRO™™

Load sample vials and syringes

Reagent additions and sample injection

performed automatically as defined in the Method

Micro PRO™ Output:Pass/Fail

& Counts/mL

Select Tray SetUp1 2

3 4

MicroPROMicroPROVarious Results/ Various Results/

ApplicationsApplications

Purified Water Purified Water MonitoringMonitoring

Sample PreparationSample 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

Box = 0 counts/0.25mL Box = 2 counts/0.25mL Box = 22 counts/0.25mL

Background / Neg.Ctrl Sample 3 Sample 7

1 2 3

Purified Water Monitoring: Purified Water Monitoring: MicroPRO TVO & R2A Plate CountsMicroPRO TVO & R2A Plate Counts

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Sample Number

Co

un

ts/m

L

RBD 3000 R2A Plates 5-day R2A Plates 10-day

warning

action

0,00

0,50

1,00

1,50

2,00

2,50

MQ 1

MQ 2

MQ 3

MQ 4

MQ 5

MQ 6

MQ 7

MQ 8

MQ 9

MQ 10

MQ 11

MQ 12

MQ 13

MQ 14

MQ 15

MQ 16

MQ 17

MQ 18

Lo

g1

0 c

ou

nt/

ml

or

cfu

/ml

MQ system CPL2 Micro qual

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

Purified Water SystemPurified Water System

Product Research Lab Water TestingProduct Research Lab Water TestingComparison of RBD vs. manual plate count for PRL water

0

100

200

300

400

500

600

700

800

900

1000

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

Sample port

cfu

/ml RBD ct/ml

TSA plate

Alert level

Kozak, K.1, 2006. Rapid Microbiological Testing in Support of Product Development.

1Procter and Gamble. Rapid Microbial Methods User’s Meeting. Chigaco, IL

E. coli , ATCC 25922 Diluted in SPW--Reproducibility of RBD 3000 #150804/08/04 JNT

Plate CountsSample Counts/0.25mL Counts/mL* CFU/mL

SPW 1 4 --------10-5 Ecoli in SPW--1 2957 13006 11300

10-5 Ecoli in SPW--2 2861 12584 11500

10-5 Ecoli in SPW--3 2979 13103 10700

10-5 Ecoli in SPW--4 2815 12382 9900

10-5 Ecoli in SPW--5 2927 12874 10700

10-5 Ecoli in SPW--6 2933 12901 11500

10-5 Ecoli in SPW--7 3062 13468 10900

10-5 Ecoli in SPW--8 2810 12360 9300

10-5 Ecoli in SPW--9 2866 12606 11500

10-5 Ecoli in SPW--10 2841 12496 10400Average 2905.10 12778.04 10770.00Average Deviation 66.50 292.60 570.00Percent Deviation 2.29 2.29 5.29

RBD 3000

Ecoli diluted in SPW RBD 3000 Counts vs. Standard Plate Counts over 10 Samples--Reproducibility

4.11 4.10 4.12 4.09 4.11 4.11 4.13 4.09 4.10 4.104.05 4.06 4.03 4.00 4.03 4.06 4.04 3.97 4.06 4.02

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

1 2 3 4 5 6 7 8 9 10

Sample

Log 1

0 Va

lue

RBD 3000 (counts/mL) Plate Counts (cfu/mL)

Product Testing Product Testing Presence/AbsencePresence/Absence

Traditional methods USP <61>Traditional methods USP <61>

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

Bacteria

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 Count

If zero counts, results are expressed as less than 10 cfu/ml

Advanced Analytical Method Advanced Analytical Method

Bacteria, Yeast & Mold

1:10dilution ofproduct

1ml in Tube A

Incubate 24 to 48 hours at

30°C

Micro PRO™

Transfer substrate tube A to

Tube B; vortex; 0.1mL to Tube C

Product Test Kit

35m filter

Micro PRO™

Tube A - GEM Add product, enrich

Transfer swabfrom Tube A to B

Tube B - PBAdd swab, mix

Tube C - PBAdd 0.1mL from Tube B

mold

Experimental ProcedureExperimental 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 min3. 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 vortex7. Transfer 0.1mL from Tube B to Tube C and load in the Micro PRO™8. Plate samples post-enrichment

Analysis on MicroPROAnalysis on MicroPRO

Load sample vials and syringes

Reagent additions and sample injection

performed automatically as defined in the Method

Select Method

12

3 4

Results

Micro PRO™Micro PRO™ Output Output

Mold counts/mL

Bacteriacounts/mL

Yeast counts/mL

MoldResult 1

BacteriaResult 2

YeastResult 3

Overall Result

54 1550 10 Pass Pass Pass Pass

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

ResultsResults

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

Bacteriacounts/mL

Yeast counts/mL

MoldResult 1

BacteriaResult 2

YeastResult 3

Overall Result

212002 648372 28379 Fail Fail Fail Fail

ResultsResults

Bacteria

Mold counts/mL

Bacteriacounts/mL

Yeast counts/mL

MoldResult 1

BacteriaResult 2

YeastResult 3

Overall Result

43 1913 1770 Pass Pass Fail Fail

Mold counts/mL

Bacteriacounts/mL

Yeast counts/mL

MoldResult 1

BacteriaResult 2

YeastResult 3

Overall Result

2452 2144 65 Fail Pass Pass Fail

Mold

Yeast

Results SummaryResults Summary

Personal Care ProductsEnrichment 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


OTC PharmaceuticalsEnrichment 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


OthersEnrichment 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

ResultsResults

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 The Advanced Analytical Solution ProvidesSolution 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 Environmental Monitoring (swabs)Monitoring (swabs)

Environmental Swab Protocol• 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

43

2

Environmental Sample Testing• Swab samples are directly analyzed on the MicroPRO• Result is obtained in minutes rather than overnight

Swab Control

Box counts/0.25mL: 2

E.coli Swab

Box counts/0.25mL: 1,195

Swab Sample: Population

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)

• 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 Fermentation / Pure Culture EnumerationCulture 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 RBD 3000 Sample Tray with capped syringe

• Select pre-defined (or create new) Analysis/Tray Sequence

• MicroPRO count result in 5 minutes

TVOTVO - Escherichia coli

Sample

RBD 3000

Counts/0.25mL

RBD 3000

Counts/mL*

Plate

Counts/mL

RBD 3000

log10 Counts/mL

Plate Counts

log10 cfu/mL

PB Background 1 4

~101 E. coli in PB 3 9 19 0.95 1.28

~102 E. coli in PB 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.

~104 cfu/mLPB Background ~101 cfu/mL ~102 cfu/mL ~103 cfu/mL

Box = 3 count/0.25 mL Box = 36 counts/0.25 mL Box = 401 counts/0.25 mL Box = 3725 counts/0.25mLBox = 1 count/0.25 mL

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

6.50

7.00

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 6.50 7.00

Plate Counts (log10 cfu/mL)

RB

D 3

000

(log

10

coun

ts/m

L)

C. albicans E. coli M. bovis S. typhimurium

Correlation of RBD 3000 TVO Counts vs. Plate CountsCorrelation of RBD 3000 TVO Counts vs. Plate Counts

(Poster presented at SIM 2006)

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).

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)

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

6.50

7.00

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 6.50 7.00

Plate Counts (log10 cfu/mL)

RB

D 3

000

(log

10

coun

ts/m

L)

B. atrophaeus C. perfringens Type A S. aureus S. bovis

Correlation of RBD 3000 TVO Counts vs. Plate CountsCorrelation of RBD 3000 TVO Counts vs. Plate Counts

(Poster presented at SIM 2006)

Some of the Microorganisms Some of the Microorganisms Enumerated with the MicroPROEnumerated 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

• 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

• 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

• 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

Some of the Microorganisms Some of the Microorganisms Enumerated with the MicroPROEnumerated with the MicroPRO

• 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 TestsChallenge Tests

OverviewOverview

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 – Contamination Level Test –

Feasible in a Variety of Complex MatricesFeasible 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 specificationCLT – Protocol for <100cfu/g specification

CLT – Results Company ACLT – Results Company AIn-process Product: Specification <1000cfu/g

Sample DilutionRBD 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


(# 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 3/0 3/0 >1,000

Spiked Below Specification

Spiked Above Specification

Product contains >100cfu/g and <1000cfu/g; product is within specificationActual spike was 559 cfu Ps. aeruginosa/g product

Product contains >1000cfu/g; product fails Actual spike was 3920 cfu Ps. aeruginosa/g product

CLT – Results Company BCLT – Results Company B

Product contains >100cfu/g; product failsActual 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


(# 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 <1000


(# 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

Body Butter & Juice Products: Specification <100cfu/g or mL

Juice: Spiked Above Specification

Body Butter: Spiked Below Specification

Product contains >10cfu/g and <100cfu/g; product is within specificationActual spike was 35 cfu C. albicans/g body butter

Contamination Level Test – BenefitsContamination 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 TestAntimicrobial Effectiveness Test

Antimicrobial Effectiveness TestAntimicrobial 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 – Antimicrobial Effectiveness Test – SolutionSolution

• Use the RBD 3000 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 – Antimicrobial Effectiveness Test – ProtocolProtocol

• Challenge 0.01% (w/v) Benzoic Acid with 105 – 106

cfu/mL

• Perform challenge studies with Ps. aeruginosa, S.

aureus, and C. albicans

• Use RBD 3000 to determine titer of inoculum

• Sample at 0, 2hr, 4hr, 6hr, 8hr & 24hr post-

inoculation– Dilute samples 1:10 in phosphate buffer for RBD 3000 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 with0.01% Benzoic Acid Challenged withPs. aeruginosaPs. aeruginosa – Test 1 – Test 1

5.76 5.83 5.83 5.84 5.83 5.81

4.14

4.684.93

5.14 5.22

5.735.785.64

5.345.12

5.29

3.98

3.0

3.5

4.0

4.5

5.0

5.5

6.0

0 2 4 6 8 24

Time post-inoculation (hours)

RB

D 3

000

log

10 C

ou

nts

/mL

Ps .aeruginosa-Biomass Count Ps. aeruginosa-Dead Cell Count Ps. aeruginosa-Plate Count

0.01% Benzoic Acid Challenged with0.01% Benzoic Acid Challenged with Ps. aeruginosaPs. aeruginosa – Test 2 – Test 2

5.72 5.76 5.75 5.75 5.77 5.77

4.46

4.865.10

5.27 5.36

5.715.59

5.465.25

5.10 5.18

3.0

3.5

4.0

4.5

5.0

5.5

6.0

0 2 4 6 8 24

Time post-inoculation (hours)

RB

D 3

00

0 lo

g 10 C

ou

nts

/mL

Ps. aeruginosa-Biomass Count Ps. aeruginosa-Dead Cell Count Ps. aeruginosa-Plate Count

2.4

0.01% Benzoic Acid Challenged 0.01% Benzoic Acid Challenged withwith S. aureus S. aureus

5.59 5.61 5.62 5.61 5.60 5.58

5.145.37 5.47 5.52 5.53 5.59

5.965.81

5.50

5.174.94

3.0

3.5

4.0

4.5

5.0

5.5

6.0

0 2 4 6 8 24Time Post-Inoculation (hours)

RB

D 3

000

log

10 C

ou

nts

/mL

S. aureus-Biomass Count S. aureus-Dead Cell Count S. aureus-Plate Count

1.78

0.01% Benzoic Acid Challenged 0.01% Benzoic Acid Challenged withwith C. albicans C. albicans

5.115.24 5.31 5.31 5.25

5.37

3.93 4.03 4.00 3.983.79

4.36

5.55 5.45 5.45 5.44 5.53 5.56

3.0

3.5

4.0

4.5

5.0

5.5

6.0

0 2 4 6 8 24

Time Post-Inoculation (hours)

RB

D 3

00

0 lo

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Co

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C. albicans-Biomass Count C. albicans-Dead Cell Count C. albicans-Plate Count

SummarySummary

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 SummaryApplications Summary

Advanced Analytical Provides Advanced Analytical Provides Solutions forSolutions 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

Benefits of MicroPROBenefits of MicroPRO

• Détection simultanée des bactéries, levures et moisissures en 24 heures dans plus de 95% des produits testés à ce jour et pratiquement 100% en 48 heures

• Détection simultanée des bactéries, levures et moisissures en 5 minutes dans l’eau purifiée et l’environnement– Cela vous permet de prendre des actions correctives

immédiatement afin de ne pas contaminer tout un lot. • Limite de quantification égale à 20 ufc/mL

– Soit 5 fois mieux que notre concurrent le plus proche grâce à l’utilisation d’un laser émettant dans le rouge (moins de bruit généré par les produits et l’eau à cette longueur d’onde) et à une meilleure conception de notre cellule

• Plus grande stabilité, plus longue durée de vie de notre laser qui est en fait une diode laser émettant dans le rouge par rapport à un laser émettant dans le bleu.

• Cellule en quartz dont la conception évite les bouchages et contamination internes


• La technologie de nos réactifs permet de marquer 100% des organismes morts et vivants ou vivants seulement ou mort seulement– La technique de marquage consiste à fixer une sonde fluorescente

sur les acides nucléiques des micro-organismes. Il ne s’agit pas d’une réaction enzymatique qui est souvent incomplète ou qui ne génère pas suffisamment de fluorescence pour être détectée. Nous pouvons aussi concevoir des kits pour marquer des micro-organismes spécifiques sur demande.

• Les réactifs peuvent se conserver pendant 10 jours à température ambiante dans les réservoirs de l’instrument– Par rapport aux technologies concurrentes, vous évite beaucoup de

perte et donc limite les coûts. Cette technologie permet d’utiliser le système même pour un seul échantillon alors que dans la plupart des autres techniques il est préférable d’attendre d’avoir suffisamment d’échantillons à analyser avant d’ouvrir un kit qui ne se conservera que quelques heures après l’ouverture.


• Très grande reproductibilité– Le MicroPRO ajoute les réactifs lui-même contrairement à d’autres

techniques qui nécessitent de les ajouter manuellement avant introduction dans l’automate. Les temps d’incubation sont donc les mêmes pour des échantillons identiques ce qui garantie une très grande reproductibilité des résultats.

• La quantité de réactifs nécessaire à l'analyse de quantités inférieures à 100 ou 200 échantillons par semaine peut être dosée, ce qui permet de remettre le reste au frais pour un usage ultérieur plusieurs semaines ou mois après l'ouverture de l'emballage– Ceci est également une source d'économie considérable par

rapport à d'autres techniques.


• La capacité du MicroPRO est de 42 échantillons sur 3 racks de 12 échantillons et 1 rack prioritaire de 6 échantillons. Lorsque le MicroPRO a terminé l’analyse d’un rack il est possible de le sortir et de le recharger sans interrompre la séquence d’analyse– Par conséquent le système peut fonctionner en continu toute la

journée.– Si des échantillons sont positionnés sur le rack prioritaire, le

système les détectera et interrompra la séquence d’analyse sur un des racks standard après l’analyse des échantillons en cours. Il analysera les échantillons du rack prioritaire et reviendra à l’endroit où il s’était arrêté sur le rack standard pour poursuivre la séquence.

– Ceci vous permet d’analyser des échantillons en urgence sans devoir arrêter manuellement la séquence d’analyse.

– Les racks n’ont pas besoin d’être totalement remplis pour démarrer une analyse. A partir du moment où un seul échantillon se trouve sur un rack, l’analyse peut démarrer.


• Pas de risque de contamination croisée– Les racks sont conçus pour recevoir 12 échantillons ou 6

échantillons pour le rack prioritaire ainsi que 12 ou 6 seringues à usage unique d’un millilitre

– Ces seringues sont utilisées par l’automate pour injecter chaque échantillon une seule fois dans le cytomètre.

– Cela permet d’éliminer un risque de contamination important lorsque la même seringue est utilisée pour injecter tous les échantillons.

– Par ailleurs le port d’injection du cytomètre est lavé systématiquement et automatiquement entre deux injections avec une solution de détergent qui a été qualifiée par le fabriquant. Cette fonction n’est pas débrayable par l’utilisateur ni les managers qui entrent les méthodes dans le système. Cela garantit une absence totale de risque de contamination croisée.

– Notre système à fait l’objet de tests très approfondis à la fois en interne et par nos clients pour nous assurer que le risque de contamination est totalement absent.


• Le système permet d’analyser jusqu’à 20 échantillons par heure.

• Le logiciel très convivial répond à la norme 21 CFR Part 11 exigée par l’industrie pharmaceutique. Ce logiciel permet de visualiser les résultats en ufc/ml ou en pass/ambiguous/fail à condition d'avoir préalablement indiqué les limites pour accepter un lot, le refuser ou dans certain cas de préciser si la mesure est ambiguë et ainsi relancer des analyses complémentaires. Ce logiciel comprend plusieurs niveaux. – Le premier est le niveau utilisateur qui ne permet pas de modifier

ou d’entrer des nouvelles méthodes. – Le deuxième niveau est le niveau manager qui permet d’entrer ou

modifier des méthodes. – Le troisième niveau est le niveau administrateur– Le quatrième niveau service permet à nos équipes de maintenance

d’intervenir sur le système en coupant toutes les sécurités.

RBD 3000/MicroPRO ReferencesRBD 3000/MicroPRO ReferencesMiller, 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)

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