Quantitation of Growth of Shigella Dysenteriae, Salmonella choleraesuis, Listeria

monocytogenes, Esherichia coli on Cardboard and ABS Plastic Surfaces

Final Report Submitted to:

Mr. Ken Krall, President
Kut Products, Inc.
P.O. Box 704
San Anselmo, CA 94979
PH: 415-454-2311
FAX: 415-459-2311

Submitted by:

Tania L. Weiss, Ph.D.
Marin Biologic
3152 Paradise Drive
Tiburon, CA 94920
PHONE: 415-435-6636 FAX: 415-435-9299

DATE: June 10, 1998


Five infectious bacteria commonly found as kitchen/food contamination were tested to determine whether growth on ABS plastic was inhibited compared to cardboard and whether washing the plastic with kitchen antibacterial soap inhibited bacterial growth. The experimental design was to transfer an array of bacteria as discrete drops on to the plastic or cardboard. The plastic and cardboard materials were incubated overnight then blotted onto a sterile agar plate that would support bacterial growth. Data show that although the bacteria grew little on plastic, if at all, they were still present. Even though the bacteria were dried on the plastic, when the plastic was blotted onto sterile agar growth medium, the inoculate was able to sustain growth. However, data show that the antibacterial soap reduced/eliminated the bacterial contamination on the plastic, so that when plastic was blotted onto sterile agar growth medium, there was no bacterial growth. Data also show that moist/wet cardboard is a rich medium for bacterial growth. The results demonstrate that a "lawn" of bacteria grew on the sterile agar growth medium when the cardboard was blotted on the surface.

   View digital images of bacteria growth

1 . Organisms: Shigella dysenteriae ATCC #13313

      Escherichia coli ATCC #15922

      Salmonella choleraesuis ATCC #13311

      Listeria monocytogenes ATCC #51414

     Campylobacter jejuni ATCC #49943

2. Lyophilized bacteria were resuspended in LB broth and incubated at 37C

    overnight to achieve a growing culture. The Campylobacter did not grow.

    To determine the concentration of bacteria which would grow as discrete,

    individual colonies, serial dilutions of the E. coli were made (1 /10, 1 /100,

    1 /1000, 1 /10,000, 1 /100,000, 1 /1,000,000) and 1 00ul of culture were

    plated onto 100CM agar plates. The plates were incubated overnight at

    37C. The two concentrations resulting in individual colonies were

    1/100,000 and 1/1,000,000. The optimum concentration was

    approximately 1/500,000.

3. All the bacteria were diluted to 1/500,000 and 1 /1,000,000 and 100ul of

    culture were transferred to the agar plates and incubated overnight at

    37C. Depending upon the bacterial strain, the concentration was

    appropriate, but there was uneven distribution of colonies. Some colonies

    were very close together.


     A blot of the bacteria colonies was made by laying the plastic or cardboard
     upon the agar plate in order to transfer the bacteria to the test material. The
     cardboard was pretreated by applying water to all surfaces including between
     the two faces in the corrugated portion.

    The plastic and cardboard were grown in a moist atmosphere, at 37C
    overnight. The bacteria were present on the plastic but did not grow.
    The cardboard contained a lawn of bacteria. These plates were kept
    at 4C until they were digitally imaged.


4. A different approach was used to minimize the visual bacterial detection

on the plastic. Using a multichannel pipettor, less than I ul was applied as

individual spots directly onto the plastic and dried cardboard. The plastic

was incubated overnight at 37C, in a moist atmosphere that was allowed

to dry out. The cardboard was "floated" on water, and incubated in a moist

atmosphere overnight at 37C. The bacteria appeared as dried spots on

the plastic. On some of the cardboard pieces, the bacteria could not be

visualized. On others, the drops of bacteria were remained as drops rather

than soak into the cardboard. One replicate set of plastic samples was

washed with a dilute antibacterial soap. The washing process included

squirting the samples five times with soap then squirting with purified

water to rinse off the soap. The samples were air dried before blotting onto

the agar plates.

The plastic and cardboard pieces were blotted onto a sterile agar plate.

The plates were incubated at 37C overnight and imaged using a digital

camera imaging system. The photos were touched-up in Adobe Photoshop

and replicated in floppy discs and hard copy. The agar plates blotted with the

unwashed plastic materials resulted in the growth of small individual spots of

bacteria replicating the pattern of the bacterial application to the plastic. The

agar plates blotted with the washed plastic materials supported no growth of

bacteria. The agar plates blotted with some of the cardboard showed a "lawn"

of bacteria. In agar plates blotted with other cardboard samples a replicate pattern

of the bacterial application to the plastic was observed, however, the colonies

were larger than that on plastic indicating growth.


CONCLUSIONS                          View digital images of bacteria growth

The ABS plastic did not support the growth of the various bacteria, however, the bacteria remained viable after 24 hours even after the medium in the drop dried out. The wet cardboard is a rich medium supporting vigorous growth of the bacteria. Washing the ABS plastic with antibacterial soap was an effective means to reduce the organisms. In this case, all the bacteria were eliminated. In the cardboard samples where the bacteria were applied to wet cardboard rather than dry cardboard, and incubated in a moist atmosphere overnight at 37C, a thicker "lawn" of bacteria was observed. This suggests that either wet cardboard supports better growth than moist cardboard or the results may be due to the fact that the bacteria spread out during the application process, rather than soak into the cardboard as discrete spots.

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proprietary to KENKUT Products Inc. Any reproduction,

disclosure or use of its contents or any part thereof is

expressly prohibited without prior written permission from

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