A recent webinar discussed a protocol that was used to compare Rapid Micro Biosystem's Environmental Monitoring cassette (RMB EM Cassette) to Conventional media for environmental monitoring.

1. Comparing the Growth Direct™
Environmental Monitoring Cassette to
Conventional Media
Presented by: Dawn McIver
President, MicroWorks, Inc.

2. Purpose
• The purpose of this protocol was to
compare Rapid Micro Biosystems
Environmental Monitoring cassette (RMB
EM Cassette) to Conventional media for
environmental monitoring.

3. MicroWorks Laboratory

4. Scope of Study
• Growth promotion testing
• Airborne viable bioburden
• Surface bioburden
Naturally contaminated surfaces
Inoculated surfaces
• Demonstration of ability of cassettes to
neutralize common surface disinfectants

5. RMB Cassettes
• Novel contact plate developed to work with
the Growth Direct™ System
• 0.45 micron pore size black mixed
cellulose ester membrane as functional
surface rather than the traditional media
• TSA with lecithin and polysorbate 80
media

6. Conventional Contact Plates
• Conventional contact plates containing
TSA with lecithin and polysorbate 80
utilized for surface sampling and for
airborne viable monitoring.

7. Side by Side Comparison

8. Incubation
• 7 days using serial incubation
• A minimum of 4 days at 20-25°C
• Plus a minimum of 3 days at 30-35°C

9. Observations
• RMB Cassettes are slightly larger than the
conventional plates.
• RMB Cassettes have a black background
so many organisms were easier to see and
count.
• Colonies stayed much more compact on
the RMB Cassettes, especially spreaders
and molds, which made counting easier.

10. GROWTH PROMOTION TESTING

11. Growth Promotion Testing
Organism
Inoculum Level
(cfu/sample)
RMB EM Cassette Result
(cfu/sample)
Conventional TSA w/LP80
Result
(cfu/sample)
% Correlation between
Cassette and
Conventional Plate
Results
Bacillus subtilis 32 18 30 60%
Pseudomonas
aeruginosa 15 13 15 87%
Staphylococcus
aureus 17 28 18 156%
Escherichia coli 33 34 28 121%
Aspergillus
brasiliensis 30 32 33 97%
Candida albicans 55 45 54 83%

12. Conclusion- Growth Promotion Testing
• RMB EM Cassettes and TSA w/LP80
plates met the acceptance criteria
specified for growth promotion testing.
• Correlation between the two methods
was within the acceptance criteria of 50 to
200%.

13. AIRBORNE VIABLE MONITORING

14. SAS Air Sampler
• SAS air sampler was fitted to
accommodate RMB cassettes.
• SAS with RMB cassettes and SAS with
conventional contact plates were placed
side by side for sampling.

15. Airborne Viable Monitoring
• Samplers with each media type were
placed side by side and samples were
collected and incubated simultaneously.
• Samples were collected in lab, warehouse
and LFH and BSC locations to represent a
variety of contamination levels.
• 20 locations were sampled on 3 separate
days for a total of 60 test results.

16. Results for Airborne Viable Monitoring
Sample Result for RMB EM Cassette (cfu/sample)
Result for Conventional TSA w/LP80 plate
(cfu/sample)
Lab Bench
100 90
Near Vitek
72 88
Warehouse near sink
81 102
Warehouse near door
112 84
Warehouse back corner
87 94
Utility Room
70 72
Lunch Room
76 89
Computer Room
94 85
TOC Room
74 82
Questioning Room
70 53

17. Results for Airborne Viable Monitoring
Sample Result for RMB EM Cassette (cfu/sample)
Result for Conventional TSA w/LP80 plate
(cfu/sample)
Vestibule
125 TNTC
Office Area
70 106
Hallway
97 59
Lab 2 Near KTA
16 11
Lab 2 Doorway
54 17
Back of sink
63 69
LFH right side
8 4
LFH left side
0 0
BSC right side
0 5
BSC left side
0 0

18. Conclusion-Airborne Viable Monitoring
• Statistical analysis demonstrated there
was no significant difference between
methods when side by side airborne viable
monitoring was performed with the SAS air
samplers.

19. SURFACE BIOBURDEN-
NATURALLY CONTAMINATED

20. Surfaces
• A variety of surfaces were tested
throughout the office, lab and warehouse
and the surfaces of the LFH and BSC.
• Samples were collected in close proximity
to each other.
• 50 samples were collected.

21. Results of Sampling Naturally
Contaminated Surfaces
Site Description Result for RMB EM Cassette (cfu/sample)
Result for Conventional TSA w/LP80 plate
(cfu/sample)
Sink 35 34
Culture refrigerator 4 7
Microwave 0 0
Wall behind autoclave 2 4
Tile behind mop tub 6 8
Floor in front of refrigerator 26 40
Wall near light switch 4 spreader
TOC computer 54 TNTC
TOC computer chair back 31 96

22. Results of Sampling Naturally
Contaminated Surfaces
Site Description Result for RMB EM Cassette (cfu/sample)
Result for Conventional TSA w/LP80 plate
(cfu/sample)
Windowsill 19 TNTC
Door handle 30 TNTC
Floor 39 TNTC
Bench in front of pH meter 11 30
Inside wall of waterbath 70 41
Door near handle 0 9
LFH right side 6 29
LFH left side 6 19
BSC right side 2 2

23. Conclusion- Surface Sampling Naturally
Contaminated Surfaces
• Results for the RMB-Cassettes were lower than that
seen for the traditional media in some cases.
• For some plates results were recorded as TNTC or
spreader on conventional plates but were readable
with RMB-Cassettes.
• Statistical analysis demonstrated that there was a
significant difference between the methods.

24. SURFACE BIOBURDEN-
INOCULATED

25. Coupon Surfaces
• Epoxy wall
• Tile floor
• Glass
• Stainless Steel
• Polyvinyl chloride (PVC)
• Terrazzo flooring
• HEPA curtain (Plastic)
• Conveyor belt

26. Challenge Organisms
• Aspergillus brasiliensis, ATCC 16404
• Candida albicans, ATCC 10231
• Bacillus subtilis, ATCC 6633
• Pseudomonas aeruginosa, ATCC 9027
• Staphylococcus aureus, ATCC 6538
• Escherichia coli, ATCC 8739
• Delftia acidovorans (Environmental isolate)
• Micrococcus luteus (Environmental isolate)

27. Surface Bioburden-Inoculated
• Four replicates of each surface were
inoculated with each of the challenge
organisms. Two sampled with each
method.
• Target inoculum was 25-100 cfu/0.1 mL.
• 128 samples collected.

28. Results-Inoculated Glass
Organism-Sample Number
Result for RMB EM Cassette
(cfu/sample)
Result for Conventional TSA
w/LP80 plate (cfu/sample)
% Correlation between
Averages for Cassette and
Conventional Plate Results
AB-1
20 25
70%
AB-2
19 31
CA-1
57 61
105%
CA-2
46 37
BS-1
1 0
50%
BS-2
0 0
PA-1
0 0
100%
PA-2
0 0

29. Results-Inoculated Glass
Organism-Sample Number
Result for RMB EM Cassette
(cfu/sample)
Result for Conventional TSA
w/LP80 plate (cfu/sample)
% Correlation between
Averages for Cassette and
Conventional Plate Results
SA-1
11 3
170%
SA-2
6 7
EC-1
1 2
60%
EC-2
2 3
ML-1
2 2
125%
ML-2
3 2
DA-1
29 28
94%
DA-2
17 21

30. Conclusion-Inoculated Surfaces Testing
• Inoculated challenge organisms did not always
survive on the surface.
• This is most likely due to desiccation of the
organisms.
• There was a strong correlation between the two
methods.
• F-statistic showed that the difference between the 2
methods was not significant.

31. NEUTRALIZATION CAPABILITY
OF RMB CASSETTES

32. Disinfectants
• LpH- Acidic, phenolic cleaner/disinfectant
• Vesphene – alkaline phenolic cleaner/disinfectant
• SporKlenz – peracetic acid sterilant/disinfectant
• TBQ – ammonium based alkaline
cleaner/disinfectant
• 70% IPA – alcohol based sanitizer

33. Coupon Surfaces
• Epoxy wall
• Tile floor
• Glass
• Stainless Steel

34. Neutralization Results-Epoxy
Disinfectant % Recovery
LpH 64 – 148%
Vesphene 52 – 128%
SporKlenz 78 -133%
TBQ 55 – 123%
70% IPA 91 – 146%

35. Neutralization Results-Tile
Disinfectant % Recovery
LpH 55 – 172%
Vesphene 79 -117%
SporKlenz 84 - 145%
TBQ 63 – 124%
70% IPA 74 -152%

36. Neutralization Results-Glass
Disinfectant % Recovery
LpH 55 – 172%
Vesphene 79 -117%
SporKlenz 84 - 145%
TBQ 63 – 124%
70% IPA 74 -152%

37. Neutralization Results-SS
Disinfectant % Recovery
LpH 76 – 154%
Vesphene 57- 125%
SporKlenz 54 -130%
TBQ 60- 138%
70% IPA 72- 144%

38. Conclusion-Neutralization Study
• This study demonstrates neutralization of
common disinfectants has been achieved
with this novel contact plate on Epoxy,
Tile, Glass and Stainless Steel surfaces.

39. Summary
• This study demonstrates that RMB
Cassettes are suitable for use for
collecting surface samples and for
collecting airborne viable samples in place
of conventional media.
• Visible colonies can be read very easily on
the plate surface and can be sub-cultured
for further ID or retention.

40. RMB Cassettes
• RMB Cassettes are designed to be used in
conjunction with the GrowthDirect™
system so faster, automated EM results
are now a reality.

41. Contact Information
• Feel free to contact me with any future
questions.
Dawn McIver
MicroWorks, Inc.
2200 W. 97th Place
Crown Point, IN 46307
219-661-8620
www.mwiconsulting.com