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MicroPRO, A Rapid Microbiology Method Based on Flow Cytometry

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This document provides information on disruptive technologies and rapid microbiology products represented by the company. It summarizes their product range including instruments for biological sample preparation, dissolution/formulation, physico-chemistry analysis, and rapid microbiology detection. It also discusses their markets in pharmaceutical, personal care, fermentation, and more. The MicroPRO instrument allows detection of bacteria, yeast and mold from various samples within 24 hours.

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Disruptive Technologies Product Range and 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? 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 Presentation 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 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)
Fluorescence Detector Laser Beam – shaped and focused; 635 nm laser excitation Labeled microorganism Fluorescence signal High performance optical filters Scatter Detector Scatter signal Fluorescence plus Scatter = One Count Optic System
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 Fluorescence Threshold Level Time = 1 count = 0 count Detector output
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 Scatter Intensity vs. Counts Intensity Plot 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) Ideal for enumerating Live & Dead microbes in: Fermentation cultures Spore preparations RBD Total Cell Count / mL
TVO Labeling Protocol (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 Detection Labeling Protocol Mixed Sample Ideal for enumerating microbes for/in: Specific pathogen detection Mixed cultures High background matrices
MicroPRO System
 
Auto-Prep Adds reagents Mixes sample On-board automated cleaning and bubble removal Auto-Sampler Holds up to 42 samples
Analysis on the Micro 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 SetUp 1 2 3 4
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
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: MicroPRO TVO & R2A Plate Counts warning action
Purified Water System From: Hasher-Homesley, P .1, 2006. R&D Applications for the RBD3000. 1 Johnson & Johnson Vision Care. Rapid Microbial Methods User’s Meeting, Chicago, IL
Product Research Lab Water Testing Kozak, K .1 , 2006. Rapid Microbiological Testing in Support of Product Development. 1 Procter and Gamble. Rapid Microbial Methods User’s Meeting. Chigaco, IL
 
 
Product Testing Presence/Absence
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 Bacteria, Yeast & Mold 1:10 dilution of product 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 35  m filter Micro PRO ™ Tube A - GEM Add product, enrich Transfer swab from Tube A to B Tube B - PB Add swab, mix Tube C - PB Add 0.1mL from Tube B mold
Experimental Procedure Prepare 1:10 product suspension in buffer or neutralizing growth enhancement media Add 1mL 1:10 product suspension to Tube A & neutralize 30 min 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 Prepare non-spiked product controls Enrich at 30 o C for 24-48 hours Transfer substrate from Tube A to Tube B and vortex Transfer 0.1mL from Tube B to Tube C and load in the Micro PRO™ Plate samples post-enrichment
Analysis on MicroPRO Load sample vials and syringes Reagent additions and sample injection performed automatically as defined in the Method Select Method 1 2 3 4 Results
Micro PRO™ Output
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 Results 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
Micro PRO ™ output shows many counts within the area definition ( > 3X product baseline) Fail result indicates that the sample contains microbial contamination Results Bacteria Mold counts/mL Bacteria counts/mL Yeast counts/mL Mold Result 1 Bacteria Result 2 Yeast Result 3 Overall Result 212002 648372 28379 Fail Fail Fail Fail
Mold Yeast Mold counts/mL Bacteria counts/mL Yeast counts/mL Mold Result 1 Bacteria Result 2 Yeast Result 3 Overall Result 43 1913 1770 Pass Pass Fail Fail Mold counts/mL Bacteria counts/mL Yeast counts/mL Mold Result 1 Bacteria Result 2 Yeast Result 3 Overall Result 2452 2144 65 Fail Pass Pass 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 Approximately 300 samples were run parallel to standard cultural methods. Results from RBD are equivalent to the plate method. 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 + +
Solid Perfume 1 Micro PRO™ Counts/mL listed are for the appropriate box; Mold (Area 1), Bacteria (Area 2) or Yeast (Area 3). Negative Product Control samples contain all 3 areas and are listed as Area 1 / Area 2 / Area 3. 2 A (+) Plate result indicates growth on the agar plate, while a (-) Plate Result indicates no growth on the agar plate. Results Interpretation for Solid Perfume PA Pass/Fail Cutoff Value = (46713 + 24066)*3 = 212337 Therefore Micro PRO™ results that are < 212337 Pass and ≥ 212337 Fail CA Pass/Fail Cutoff Value = (30217 + 21218)*3 = 154305 Therefore Micro PRO™ results that are < 154305 Pass and ≥ 154305 Fail AN Pass/Fail Cutoff value = (1749+ 698)*3 = 7341 Therefore Micro PRO™ results that are < 7341 Pass and ≥ 7341 Fail Sample Enrichment Time Micro PRO™ Counts/mL 1 mold / bacteria / yeast Micro PRO™ Result Plate Result 2 Negative Product Control-1 48h 1969 / 63888 / 50952 ---- - Negative Product Control-2 48h 2310 / 57046 / 31152 ---- - Negative Product Control-3 48h 968 / 19206 / 8547 ---- - Average Negative Product Control 48h 1749 / 46713 / 30217 ---- ---- PA -1 spiked in Solid Perfume 48h 847528 Fail + PA -2 spiked in Solid Perfume 48h 654423 Fail + CA -1 spiked in Solid Perfume 48h 258819 Fail + CA -2 spiked in Solid Perfume 48h 543521 Fail + AN -1 spiked in Solid Perfume 48h 12342 Fail + AN -2 spiked in Solid Perfume 48h 2772 Pass +
PSA1 PSA2 CA1 CA2 AN1 NC1 AN2 NC2 Intensity Plots Solid Perfume @ 48 hours
Sun Protection Eye Cream 1 Micro PRO™ Counts/mL listed are for the appropriate box; Mold (Area 1), Bacteria (Area 2) or Yeast (Area 3). Negative Product Control samples contain all 3 areas and are listed as Area 1 / Area 2 / Area 3. 2 A (+) Plate result indicates growth on the agar plate, while a (-) Plate Result indicates no growth on the agar plate . Results Interpretation for Sun Protection Eye Crème PA Pass/Fail Cutoff Value = (165 + 29)*3 = 582 Therefore Micro PRO™ results that are < 582 Pass and ≥ 582 Fail CA Pass/Fail Cutoff Value = (238 + 67)*3 = 915 Therefore Micro PRO™ results that are < 915 Pass and ≥ 915 Fail AN Pass/Fail Cutoff Value* = (88 + 31)*3 = 357 Therefore Micro PRO™ results that are < 357 Pass and ≥ 357 Fail *48 hour negative control Mold Area counts/mL = 66, 110 average = 88 Sample Enrichment Time Micro PRO™ Counts/mL 1 mold / bacteria / yeast Micro PRO™ Result Plate Result 2 Negative Product Control-1 24h 66 / 143 / 297 ---- - Negative Product Control-2 24h 33 / 154 / 253 ---- - Negative Product Control-3 24h 44 / 198 / 165 ---- - Average Negative Product Control 24h 48 / 165 / 238 ---- ---- PA- 1 spiked in Eye crème 24h 723635 Fail + PA -2 spiked in Eye crème 24h 898810 Fail + CA -1 spiked in Eye crème 24h 15433 Fail + CA -2 spiked in Eye crème 24h 20757 Fail + AN -1 spiked in Eye crème 24h 66 Pass - AN -2 spiked in Eye crème 24h 44 Pass - AN- 3 spiked in Eye crème 48h 1012 Fail + AN- 4 spiked in Eye crème 48h 2453 Fail +
Intensity Plots Sun Eye Protection Cream @ 24hours PSA1 PSA2 CA1 CA2 AN1 NC1 AN2 NC2
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 Place a swab sample in a 5mL snap-cap tube containing 900 µ L 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 4 3 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 42 o C ( + 2 o C) 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 < 10 6 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 *RBD 3000 counts/mL are background corrected and have been adjusted for reagent additions. ~10 4 cfu/mL PB Background ~10 1 cfu/mL ~10 2 cfu/mL ~10 3 cfu/mL Box = 3 count/0.25 mL Box = 36 counts/0.25 mL Box = 401 counts/0.25 mL Box = 3725 counts/0.25mL Box = 1 count/0.25 mL Sample RBD 3000 Counts/0.25mL RBD 3000 Counts/mL* Plate Counts/mL RBD 3000 log 10 Counts/mL Plate Counts log 10 cfu/mL PB Background 1 4 ~10 1 E. coli in PB 3 9 19 0.95 1.28 ~10 2 E. coli in PB 36 150 280 2.18 2.45 ~10 3 E. coli in PB 401 1707 1665 3.23 3.22 ~10 4 E. coli in PB 3725 15894 18050 4.20 4.26
Correlation of RBD 3000 TVO Counts vs. Plate Counts (Poster presented at SIM 2006) Candida albicans (n = 14, R 2 = 0.9982), Escherichia coli (n = 17, R 2 = 0.9959), Mycoplasma bovis (n = 15, R 2 = 0.9891) and Salmonella typhimurium (n = 15, R 2 = 0.9952).
Bacillus atrophaeus (n = 15, R 2 = 0.9839), Clostridium perfringens Type A (n = 12, R 2 = 0.9981), Staphylococcus aureus (n = 18, R 2 = 0.9857) and Streptococcu bovis (n = 13, R 2 = 0.9832) Correlation of RBD 3000 TVO Counts vs. Plate Counts (Poster presented at SIM 2006)
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 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 Enumerated 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 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
CLT – Results Company A In-process Product: Specification <1000cfu/g Spiked Below Specification Spiked Above Specification Product contains >100cfu/g and <1000cfu/g; product is within specification Actual spike was 559 cfu Ps. aeruginosa /g product Product contains >1000cfu/g; product fails Actual spike was 3920 cfu Ps. aeruginosa /g product Sample Dilution Theoretical Result Detection by RBD 3000 Growth on YM Petrifilm TM 1:100 + + + 1:1,000 + + + 1:10,000 - - - 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 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 3/0 3/0 >1,000
CLT – Results Company B 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 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 specification Actual spike was 35 cfu C. albicans /g body butter Sample Dilution Theoretical Result Detection by RBD 3000 Growth on YM Petrifilm TM 1:100 + + + 1:1,000 + + + 1:10,000 - - - Sample Dilution RBD 3000 Result (# pos./# neg.) Growth on YM Petrifilm TM (# 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 Sample Dilution RBD 3000 Result (# pos./# neg.) Growth on YM Petrifilm TM (# 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
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 10 5 – 10 6 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 post-inoculation 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
0.01% Benzoic Acid Challenged with Ps. aeruginosa – Test 2
0.01% Benzoic Acid Challenged with S. aureus
0.01% Benzoic Acid Challenged with C. albicans
Antimicrobial Effectiveness Test – Action Items Test other preservative systems Determine utility in various liquid product formulations Demonstrate feasibility with A. niger spores
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
Benefits 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 sur l’eau 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
Benefits of MicroPRO 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.
Benefits of MicroPRO 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.
Benefits of MicroPRO 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.
Benefits of MicroPRO 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.
Benefits of MicroPRO Le système permet d’analyser jusqu’à 20 échantillons à l’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 et en conforme/non conforme/ambigu à condition d'avoir préalablement indiqué les limites pour accepter un lot, le refuser ou dans certain cas de préciser si la mesure est ambigue 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 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 Disruptive Technologies 3 allée des Camélias 94440 Villecresnes France Contact: William Amoyal Tel. +33 (0)6 98 64 98 81 Email : [email_address]
Thank You ! www.aati-us.com www.disruptechno.com

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MicroPRO, A Rapid Microbiology Method Based on Flow Cytometry

  • 1.  
  • 2. Disruptive Technologies Product Range and 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)
  • 3. 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.
  • 5. Overview of Microbial Detection Presentation 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
  • 6. Basics of Flow Cytometry
  • 7. 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
  • 8. Fluidic 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)
  • 9. Fluorescence Detector Laser Beam – shaped and focused; 635 nm laser excitation Labeled microorganism Fluorescence signal High performance optical filters Scatter Detector Scatter signal Fluorescence plus Scatter = One Count Optic System
  • 10. 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 Fluorescence Threshold Level Time = 1 count = 0 count Detector output
  • 11. Signal Processing cumulative Microbe A Microbe A + B Microbe A + B +C
  • 12. Example of Micro PRO Intensity Plot Cell size Amount of label
  • 13. Results Fluorescence Intensity vs. Counts Scatter Intensity vs. Counts Intensity Plot Box = 9907 counts/0.25mL 97.4% of the counts are in the box
  • 15. Biomass Labeling Protocol (Membrane Permeable – all Cells are Stained) Ideal for enumerating Live & Dead microbes in: Fermentation cultures Spore preparations RBD Total Cell Count / mL
  • 16. TVO Labeling Protocol (Only Viable Cells are Stained) Ideal for enumerating Viable microbes in: Process water monitoring Presence/Absence testing Fermentation cultures Final product testing Pure cultures
  • 17. Antibody-Specific Detection Labeling Protocol Mixed Sample Ideal for enumerating microbes for/in: Specific pathogen detection Mixed cultures High background matrices
  • 19.  
  • 20. Auto-Prep Adds reagents Mixes sample On-board automated cleaning and bubble removal Auto-Sampler Holds up to 42 samples
  • 21. Analysis on the Micro 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 SetUp 1 2 3 4
  • 24. 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
  • 25. 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
  • 26. Purified Water Monitoring: MicroPRO TVO & R2A Plate Counts warning action
  • 27. Purified Water System From: Hasher-Homesley, P .1, 2006. R&D Applications for the RBD3000. 1 Johnson & Johnson Vision Care. Rapid Microbial Methods User’s Meeting, Chicago, IL
  • 28. Product Research Lab Water Testing Kozak, K .1 , 2006. Rapid Microbiological Testing in Support of Product Development. 1 Procter and Gamble. Rapid Microbial Methods User’s Meeting. Chigaco, IL
  • 29.  
  • 30.  
  • 32. 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
  • 33. Advanced Analytical Method Bacteria, Yeast & Mold 1:10 dilution of product 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
  • 34. Product Test Kit 35  m filter Micro PRO ™ Tube A - GEM Add product, enrich Transfer swab from Tube A to B Tube B - PB Add swab, mix Tube C - PB Add 0.1mL from Tube B mold
  • 35. Experimental Procedure Prepare 1:10 product suspension in buffer or neutralizing growth enhancement media Add 1mL 1:10 product suspension to Tube A & neutralize 30 min 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 Prepare non-spiked product controls Enrich at 30 o C for 24-48 hours Transfer substrate from Tube A to Tube B and vortex Transfer 0.1mL from Tube B to Tube C and load in the Micro PRO™ Plate samples post-enrichment
  • 36. Analysis on MicroPRO Load sample vials and syringes Reagent additions and sample injection performed automatically as defined in the Method Select Method 1 2 3 4 Results
  • 37. Micro PRO™ Output
  • 38. 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 Results 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
  • 39. Micro PRO ™ output shows many counts within the area definition ( > 3X product baseline) Fail result indicates that the sample contains microbial contamination Results Bacteria Mold counts/mL Bacteria counts/mL Yeast counts/mL Mold Result 1 Bacteria Result 2 Yeast Result 3 Overall Result 212002 648372 28379 Fail Fail Fail Fail
  • 40. Mold Yeast Mold counts/mL Bacteria counts/mL Yeast counts/mL Mold Result 1 Bacteria Result 2 Yeast Result 3 Overall Result 43 1913 1770 Pass Pass Fail Fail Mold counts/mL Bacteria counts/mL Yeast counts/mL Mold Result 1 Bacteria Result 2 Yeast Result 3 Overall Result 2452 2144 65 Fail Pass Pass Fail
  • 41. 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
  • 42. 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
  • 43. 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
  • 44. Negative Control Positive Ps. aeruginosa
  • 47. Results Approximately 300 samples were run parallel to standard cultural methods. Results from RBD are equivalent to the plate method. 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 + +
  • 48. Solid Perfume 1 Micro PRO™ Counts/mL listed are for the appropriate box; Mold (Area 1), Bacteria (Area 2) or Yeast (Area 3). Negative Product Control samples contain all 3 areas and are listed as Area 1 / Area 2 / Area 3. 2 A (+) Plate result indicates growth on the agar plate, while a (-) Plate Result indicates no growth on the agar plate. Results Interpretation for Solid Perfume PA Pass/Fail Cutoff Value = (46713 + 24066)*3 = 212337 Therefore Micro PRO™ results that are < 212337 Pass and ≥ 212337 Fail CA Pass/Fail Cutoff Value = (30217 + 21218)*3 = 154305 Therefore Micro PRO™ results that are < 154305 Pass and ≥ 154305 Fail AN Pass/Fail Cutoff value = (1749+ 698)*3 = 7341 Therefore Micro PRO™ results that are < 7341 Pass and ≥ 7341 Fail Sample Enrichment Time Micro PRO™ Counts/mL 1 mold / bacteria / yeast Micro PRO™ Result Plate Result 2 Negative Product Control-1 48h 1969 / 63888 / 50952 ---- - Negative Product Control-2 48h 2310 / 57046 / 31152 ---- - Negative Product Control-3 48h 968 / 19206 / 8547 ---- - Average Negative Product Control 48h 1749 / 46713 / 30217 ---- ---- PA -1 spiked in Solid Perfume 48h 847528 Fail + PA -2 spiked in Solid Perfume 48h 654423 Fail + CA -1 spiked in Solid Perfume 48h 258819 Fail + CA -2 spiked in Solid Perfume 48h 543521 Fail + AN -1 spiked in Solid Perfume 48h 12342 Fail + AN -2 spiked in Solid Perfume 48h 2772 Pass +
  • 49. PSA1 PSA2 CA1 CA2 AN1 NC1 AN2 NC2 Intensity Plots Solid Perfume @ 48 hours
  • 50. Sun Protection Eye Cream 1 Micro PRO™ Counts/mL listed are for the appropriate box; Mold (Area 1), Bacteria (Area 2) or Yeast (Area 3). Negative Product Control samples contain all 3 areas and are listed as Area 1 / Area 2 / Area 3. 2 A (+) Plate result indicates growth on the agar plate, while a (-) Plate Result indicates no growth on the agar plate . Results Interpretation for Sun Protection Eye Crème PA Pass/Fail Cutoff Value = (165 + 29)*3 = 582 Therefore Micro PRO™ results that are < 582 Pass and ≥ 582 Fail CA Pass/Fail Cutoff Value = (238 + 67)*3 = 915 Therefore Micro PRO™ results that are < 915 Pass and ≥ 915 Fail AN Pass/Fail Cutoff Value* = (88 + 31)*3 = 357 Therefore Micro PRO™ results that are < 357 Pass and ≥ 357 Fail *48 hour negative control Mold Area counts/mL = 66, 110 average = 88 Sample Enrichment Time Micro PRO™ Counts/mL 1 mold / bacteria / yeast Micro PRO™ Result Plate Result 2 Negative Product Control-1 24h 66 / 143 / 297 ---- - Negative Product Control-2 24h 33 / 154 / 253 ---- - Negative Product Control-3 24h 44 / 198 / 165 ---- - Average Negative Product Control 24h 48 / 165 / 238 ---- ---- PA- 1 spiked in Eye crème 24h 723635 Fail + PA -2 spiked in Eye crème 24h 898810 Fail + CA -1 spiked in Eye crème 24h 15433 Fail + CA -2 spiked in Eye crème 24h 20757 Fail + AN -1 spiked in Eye crème 24h 66 Pass - AN -2 spiked in Eye crème 24h 44 Pass - AN- 3 spiked in Eye crème 48h 1012 Fail + AN- 4 spiked in Eye crème 48h 2453 Fail +
  • 51. Intensity Plots Sun Eye Protection Cream @ 24hours PSA1 PSA2 CA1 CA2 AN1 NC1 AN2 NC2
  • 52. 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
  • 54. Environmental Swab Protocol Place a swab sample in a 5mL snap-cap tube containing 900 µ L 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
  • 55. 1 4 3 2
  • 56. 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
  • 57. 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
  • 58. Environmental Swab Protocol – specific pathogens ( Salmonella ) Place a swab sample in a 5mL snap-cap tube containing 900µL Buffered Peptone Water incubate at 42 o C ( + 2 o C) 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 .
  • 59. Environmental Swab Protocol – specific pathogens ( Salmonella ) Negative control Positive ( 22 isolates tested)
  • 60. Fermentation / Pure Culture Enumeration
  • 61. Enumeration of Fermentation/Pure cultures Collect sample ; if necessary dilute to < 10 6 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
  • 62. TVO - Escherichia coli *RBD 3000 counts/mL are background corrected and have been adjusted for reagent additions. ~10 4 cfu/mL PB Background ~10 1 cfu/mL ~10 2 cfu/mL ~10 3 cfu/mL Box = 3 count/0.25 mL Box = 36 counts/0.25 mL Box = 401 counts/0.25 mL Box = 3725 counts/0.25mL Box = 1 count/0.25 mL Sample RBD 3000 Counts/0.25mL RBD 3000 Counts/mL* Plate Counts/mL RBD 3000 log 10 Counts/mL Plate Counts log 10 cfu/mL PB Background 1 4 ~10 1 E. coli in PB 3 9 19 0.95 1.28 ~10 2 E. coli in PB 36 150 280 2.18 2.45 ~10 3 E. coli in PB 401 1707 1665 3.23 3.22 ~10 4 E. coli in PB 3725 15894 18050 4.20 4.26
  • 63. Correlation of RBD 3000 TVO Counts vs. Plate Counts (Poster presented at SIM 2006) Candida albicans (n = 14, R 2 = 0.9982), Escherichia coli (n = 17, R 2 = 0.9959), Mycoplasma bovis (n = 15, R 2 = 0.9891) and Salmonella typhimurium (n = 15, R 2 = 0.9952).
  • 64. Bacillus atrophaeus (n = 15, R 2 = 0.9839), Clostridium perfringens Type A (n = 12, R 2 = 0.9981), Staphylococcus aureus (n = 18, R 2 = 0.9857) and Streptococcu bovis (n = 13, R 2 = 0.9832) Correlation of RBD 3000 TVO Counts vs. Plate Counts (Poster presented at SIM 2006)
  • 65. 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 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
  • 66. 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 Enumerated 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
  • 68. 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
  • 69. 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
  • 70. CLT – Protocol for <100cfu/g specification
  • 71. CLT – Results Company A In-process Product: Specification <1000cfu/g Spiked Below Specification Spiked Above Specification Product contains >100cfu/g and <1000cfu/g; product is within specification Actual spike was 559 cfu Ps. aeruginosa /g product Product contains >1000cfu/g; product fails Actual spike was 3920 cfu Ps. aeruginosa /g product Sample Dilution Theoretical Result Detection by RBD 3000 Growth on YM Petrifilm TM 1:100 + + + 1:1,000 + + + 1:10,000 - - - 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 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 3/0 3/0 >1,000
  • 72. CLT – Results Company B 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 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 specification Actual spike was 35 cfu C. albicans /g body butter Sample Dilution Theoretical Result Detection by RBD 3000 Growth on YM Petrifilm TM 1:100 + + + 1:1,000 + + + 1:10,000 - - - Sample Dilution RBD 3000 Result (# pos./# neg.) Growth on YM Petrifilm TM (# 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 Sample Dilution RBD 3000 Result (# pos./# neg.) Growth on YM Petrifilm TM (# 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
  • 73. 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
  • 75. 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
  • 76. 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
  • 77. Antimicrobial Effectiveness Test – Protocol Challenge 0.01% (w/v) Benzoic Acid with 10 5 – 10 6 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 post-inoculation 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
  • 78. 0.01% Benzoic Acid Challenged with Ps. aeruginosa – Test 1
  • 79. 0.01% Benzoic Acid Challenged with Ps. aeruginosa – Test 2
  • 80. 0.01% Benzoic Acid Challenged with S. aureus
  • 81. 0.01% Benzoic Acid Challenged with C. albicans
  • 82. Antimicrobial Effectiveness Test – Action Items Test other preservative systems Determine utility in various liquid product formulations Demonstrate feasibility with A. niger spores
  • 83. 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
  • 85. 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
  • 86. Benefits 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 sur l’eau 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
  • 87. Benefits of MicroPRO 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.
  • 88. Benefits of MicroPRO 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.
  • 89. Benefits of MicroPRO 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.
  • 90. Benefits of MicroPRO 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.
  • 91. Benefits of MicroPRO Le système permet d’analyser jusqu’à 20 échantillons à l’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 et en conforme/non conforme/ambigu à condition d'avoir préalablement indiqué les limites pour accepter un lot, le refuser ou dans certain cas de préciser si la mesure est ambigue 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.
  • 92. 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)
  • 93. For More Information Disruptive Technologies 3 allée des Camélias 94440 Villecresnes France Contact: William Amoyal Tel. +33 (0)6 98 64 98 81 Email : [email_address]
  • 94. Thank You ! www.aati-us.com www.disruptechno.com

Editor's Notes

  • #10: In the animation, notice the labeled microbial cell coming down the flow. When hit with the laser, it emits a fluorescence single because it is tagged with dye and also the scatter detector because it is larger then the minimum size. Both detectors have to be activated for the cell to be counted.
  • #34: This slide gives an outline of the United States Pharmacopeia Chapter 61 which addresses microbial contamination in both raw materials and non-sterile finished products In the first line concerning Bacteria, notice that the incubation time is 48 to 72 hours Also, notice that the 1:10 dilution of the product means that if there is no counts, the results are recorded as &lt;10 cfu/mL. In the Personal Care market, this is the situation well over 90% of the time. In the second line, the incubation time for yeast &amp; mold is 5 to 7 days. The same reporting logic is followed.
  • #38: This information looks even simpler to the technician on the software On this screen the results are shown for the first sample in the tray Both mold and bacteria failed (red color) and the yeast passed (green color)