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.

4. Ames, Iowa

6. Basics of Flow Cytometry

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

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

14. MicroPRO Standard Labeling Protocol

18. MicroPRO System

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

22. MicroPRO Various Results/ Applications

23. Purified Water Monitoring

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

31. Product Testing Presence/Absence

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

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

45. Negative Control Positive C. albicans

46. Negative Control Positive A. niger

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

53. Environmental Monitoring (swabs)

55. 1 4 3 2

59. Environmental Swab Protocol – specific pathogens ( Salmonella ) Negative control Positive ( 22 isolates tested)

60. Fermentation / Pure Culture Enumeration

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)

67. Challenge Tests

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

74. Antimicrobial Effectiveness Test

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

84. Applications Summary

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)

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