Comparing the Growth Direct™ Environmental Monitoring Cassette to

Conventional Media

Presented by: Dawn McIver

President, MicroWorks, Inc.

Purpose

• The purpose of this protocol was to

compare Rapid Micro Biosystems

Environmental Monitoring cassette (RMB

EM Cassette) to Conventional media for

environmental monitoring.

MicroWorks Laboratory

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

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

Conventional Contact Plates

• Conventional contact plates containing

TSA with lecithin and polysorbate 80

utilized for surface sampling and for

airborne viable monitoring.

Side by Side Comparison

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

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.

GROWTH PROMOTION TESTING

Growth Promotion Testing

OrganismInoculum 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%

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%.

AIRBORNE VIABLE MONITORING

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.

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.

Results for Airborne Viable Monitoring

Sample Result for RMB EM Cassette (cfu/sample)Result for Conventional TSA w/LP80 plate

(cfu/sample)

Lab Bench100 90

Near Vitek72 88

Warehouse near sink81 102

Warehouse near door112 84

Warehouse back corner87 94

Utility Room70 72

Lunch Room76 89

Computer Room94 85

TOC Room74 82

Questioning Room70 53

Results for Airborne Viable Monitoring

Sample Result for RMB EM Cassette (cfu/sample)Result for Conventional TSA w/LP80 plate

(cfu/sample)

Vestibule125 TNTC

Office Area70 106

Hallway97 59

Lab 2 Near KTA16 11

Lab 2 Doorway54 17

Back of sink63 69

LFH right side8 4

LFH left side0 0

BSC right side0 5

BSC left side0 0

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.

SURFACE BIOBURDEN-NATURALLY CONTAMINATED

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.

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

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

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.

SURFACE BIOBURDEN-INOCULATED

Coupon Surfaces

• Epoxy wall

• Tile floor

• Glass

• Stainless Steel

• Polyvinyl chloride (PVC)

• Terrazzo flooring

• HEPA curtain (Plastic)

• Conveyor belt

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)

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.

Results-Inoculated Glass

Organism-Sample NumberResult 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-120 25

70%AB-2

19 31

CA-157 61

105%CA-2

46 37

BS-11 0

50%BS-2

0 0

PA-10 0

100%PA-2

0 0

Results-Inoculated Glass

Organism-Sample NumberResult 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-111 3

170%SA-2

6 7

EC-11 2

60%EC-2

2 3

ML-12 2

125%ML-2

3 2

DA-129 28

94%DA-2

17 21

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.

NEUTRALIZATION CAPABILITY OF RMB CASSETTES

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

Coupon Surfaces

• Epoxy wall

• Tile floor

• Glass

• Stainless Steel

Neutralization Results-Epoxy

Disinfectant % Recovery

LpH 64 – 148%

Vesphene 52 – 128%

SporKlenz 78 -133%

TBQ 55 – 123%

70% IPA 91 – 146%

Neutralization Results-Tile

Disinfectant % Recovery

LpH 55 – 172%

Vesphene 79 -117%

SporKlenz 84 - 145%

TBQ 63 – 124%

70% IPA 74 -152%

Neutralization Results-Glass

Disinfectant % Recovery

LpH 55 – 172%

Vesphene 79 -117%

SporKlenz 84 - 145%

TBQ 63 – 124%

70% IPA 74 -152%

Neutralization Results-SS

Disinfectant % Recovery

LpH 76 – 154%

Vesphene 57- 125%

SporKlenz 54 -130%

TBQ 60- 138%

70% IPA 72- 144%

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.

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.

RMB Cassettes

• RMB Cassettes are designed to be used in

conjunction with the GrowthDirect™

system so faster, automated EM results

are now a reality.

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