Open-Cell Foam Biological Indicator for Bacteria Detection and Removal from Water or Air, and Methods of Use

Abstract

An open-cell foam biological indicator for bacteria detection and removal from water or air, and methods of use.

Claims

1. A method of detecting the presence of one or more biological contaminants, comprising; exposing to a biological contaminant a biological indicator comprising an open-cell polyolefin foam material or open-cell polyurethane foam material; after a time that is sufficient to accumulate a biological contaminant, removing some or all of the biological indicator; and testing the removed biological indicator for the presence of the biological contaminant.

2. The method of claim 1, wherein the foam comprises a cross-linked copolymer of ethylene and alkyl acrylate.

3. The method of claim 1, wherein the foam material comprises a cross-linked blend of a copolymer of ethylene and alkyl acrylate, and one or more other polymers.

4. The method claim 1, wherein the foam material comprises at least one of LDPE, EVA, LLDPE, HDPE, natural rubber, and EPDM.

5. The method of claim 1, wherein the biological indicator comprises a plurality of structures that are suspended at various levels through a water column.

6. The method of claim 1, wherein the contaminants are selected from the group of contaminants consisting of fungi and mold.

7. The method of claim 1, wherein the biological indicator comprises at least one structure that is selected from the group of structures consisting of strips, eelgrass, cubes, and small pieces.

8. The method of claim 1, wherein the open-cell foam material comprises a silane-grafted material or a silane-modified material.

9. The method of claim 1, wherein the foam is impregnated with a biocide or another chemical that is adapted to kill bacteria.

10. The method of claim 1, wherein testing the removed biological indicator for the presence of a biological contaminant comprises placing a portion of the removed biological indicator in a sterile container, adding a surfactant, blending to separate the biological contaminant from the removed biological indicator, and culturing the biological contaminant in a nutritive medium.

11. The method of claim 10, wherein culturing the biological contaminant comprises removing from the sterile container and plating some of a liquid portion of the blend.

12. The method of claim 1, wherein the open-cell foam material comprises one or more of ethylene methyl acrylate (EMA), ethylene vinyl acetate (EVA), ethylene-ethyl acrylate (EEA), ethylene-butyl acrylate (EBA), low density polyethylene (LDPE), linear low density polyethylene (LLDPE), very low density polyethylene (VLDPE), high density polyethylene (HDPE), polypropylene (PP), natural rubber, ethylene propylene diene monomer (EPDM), synthetic rubber, chlorinated polyethylene (CPE), olefin block copolymers, ethylene maleic anhydride copolymer, singe site initiated polyolefins, metallocene catalyzed polyolefins, grafted polymers including but not limited silane and maleic anhydride, styrene-butadiene-styrene copolymers, polyisoprene, and equivalents and blends thereof.

13. The method of claim 1, wherein the open-cell foam material comprises a polar component.

14. The method of claim 1, wherein the open-cell foam material is either crosslinked or not crosslinked, and is foamed with either a physical or chemical foaming agent.

15. The method of claim 1, wherein the exposing step comprises placing the biological indicator into water or air, or swabbing a surface with the biological indicator.

16. The method of claim 1, wherein the exposing step comprises placing the biological indicator and water into a container.

17. The method of claim 1, wherein the foam comprises a single site initiated polyolefin elastomer.

18. The method of claim 1, wherein the biological indicator is further exposed to related contaminants.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0030] The drawing depicts one non-limiting example of the placement of open-cell foam material into a body of water, as a step in the collection and removal from the water of biological specimens and/or contamination.

DETAILED DESCRIPTION OF EXAMPLES

[0031] Methods of removing and detecting the presence of biological contaminants from a body of water or the air are disclosed. As a first step, an open-cell foam material (or other foam materials, as described elsewhere herein) can be placed into water or into the air, or water or air can be passed though the material. The placement can be at one or more locations in the body of water or air, and at one or more depths or heights in the body of water or in the air. After desired exposure times, one or more separate portions of the open-cell foam material are removed from the water or air. The presence in the removed separate portions of one or more biological contaminants that were removed from the water or air by the open-cell foam material are then determined, typically by standard testing procedures well known in the art for the particular type of biological contaminant(s).

[0032] There are several different preferred water testing methods with the open-cell foam. Non-limiting examples include the following. In a first example, a grab sample can be taken by placing a piece of the open-cell foam in a sample jar and then partially or fully filling the jar with water. The foam can be removed for testing after any desired exposure time. If necessary to help preserve specimens that are collected by the foam, the container with water and foam can be placed on ice until the foam is ready to be tested; however, ice is not necessarily required. In a second example, the open-cell foam can be placed directly into a stream or body of water to be tested. Exposure times can vary; non-limiting examples are 5, 10, or 20 minutes. The foam is then removed from the water and tested. In a third example, cumulative testing can be accomplished by placing the foam into water to be tested, and then periodically removing portions of the foam at different exposure times.

[0033] The methods are effective both to determine the presence of biological contaminants in the water or air, and also to remove such contaminants from the water or air. The methods thus can be used for contaminant detection and/or filtration or remediation.

[0034] The drawing depicts three groups of strips or blades of open-cell foam material 12, 14 and 16. Each group has multiple strips that are held together at about their centers. The groups are fastened to a line 32 that is held on the bottom 24 of water body 20 by weight or anchor 30. In this example group 16 floats on the water surface 22, while groups 12 and 14 are held at different depths below the surface. This disclosure allows for the placement of open-cell foam material at any one or more heights of a body of water and/or the air, and at one or more locations in the body of water or air. Various non-limiting methods of exposing the open-cell material to water or air are described herein; any such method can be used as desired or as necessary depending on the body of water or the air mass, and/or the testing regime that is desired under the circumstances.

[0035] After desired exposure times, one or more portions of the foam material are removed from the water or air. This can be done by clipping or cutting a piece of foam, or removing an entire group or other portion or separate piece of foam, for example. The exposure times can be from seconds to minutes to hours to days to weeks to months, depending on the particular testing regime. Since the open-cell foam absorbs and adsorbs biological contaminants, the removed portions of the foam can be tested for particular biological contaminant(s) that are expected or are being investigated. The foam can act as an accumulator for these biological contaminants. Also, the different locations and different exposure times allow for a tailored review of biological contaminants, their locations, and their movement within the water or air.

[0036] The subject materials have been used in testing of potable water. Test methods and results follow.

[0037] Results of uses of the biological indicator in water are disclosed in the appendices 1-5 of the priority Provisional application, which are incorporated by reference herein in their entireties. A brief discussion of those appendices follows.

[0038] Appendix 1 that was part of the Provisional Application that is incorporated herein by reference (four pages) is a report from an independent testing laboratory that details the study design, procedures, and results, for comparison of grab samples (prior art) to testing using the open-cell foam of the present disclosure in potable water. The results prove that the open-cell foam acts as a biological indicator, as it is effective to remove and detect Legionella at low levels, where conventional grab samples can show non-detects when in fact Legionella is present.

[0039] Appendix 1 included the following:

[0040] A purpose of this study was to identify an effective method for the extraction of Legionella from an open-cell foam environmental indicator sampling device. Replicate sponge devices (i.e., pieces of the open-cell foam) were indirectly inoculated with a mixed suspension of fresh Legionella cultures at three target concentrations: low (1-10 CFU/mL), medium (10-100 CFU/mL) and high (100-1,000 CFU/mL). The recovery and detection procedure of the pathogen was evaluated using a non-ionic surfactant (Polysorbate 80) in conjunction with a maceration extraction process and nutritive media (BCYE agars) culturing following a modification of the Centers for Disease Control and Prevention (CDC) Procedures for the Recovery of Legionella from the Environment, January 2005. A summary of the study design is presented in Table A below.

TABLE-US-00001 TABLE A Legionella Recovery Study Design Summary Maceration Mixed Legionella Target Target Extraction suspension Matrix Level Concentration Procedure Surfactant Legionella pneumophila Sterile Tap Low 1-10 CFU/mL Blending Polysorbate ATCC.sup.1 33152 Water 80.sup.3 Legionella dumoffii Medium 10-100 CFU/mL QL14012.sup.2-1A Legionella micdadei High 100-1,000 CFU/mL QL145022-1A .sup.1ATCC: American Type Culture Collection .sup.2QL: Q Laboratories, Inc. Culture Collection .sup.3The polysorbate was Tween? 80, which is a registered trademark of Croda Americas, Inc.

[0041] The study included three replicate open-cell sponge samples indirectly inoculated for each target contamination level with Legionella species. For each contamination level, one liter of sterile tap water was inoculated using a mixed suspension of the Legionella cultures that had been diluted to the targeted levels. To simulate real-world environmental sampling, each open-cell device was submerged and allowed to absorb the contaminated water for 3-5 minutes. During submersion, the sponges were mixed in a bobbing motion using sterile pipettes. The sponges were then placed into the original sample glass vial and approximately 200 mL of the contaminated water added and the lid tightly capped. Samples remained at ambient temperature (20-24? C.) for approximately 24 hours prior to analysis.

Legionella Extraction and Detection

Extraction

[0042] All metals rings and zip ties were aseptically removed from each sponge sample prior to transferring all sample contents to a sterile laboratory blender jar. A one milliliter volume of a sterile, non-ionic surfactant, Tween? 80, was added to each blender jar to facilitate the release of any Legionella organisms that may be present within the pores of the sampling device.

[0043] Open-cell sponge samples were blended for two minutes and the jars allowed to rest for approximately ten minutes, which provided sufficient time for the sponge particulate matter to float to the surface. The liquid portion of each blender jar was aseptically transferred to sterile conical tubes and centrifuged at 5500?g for thirty minutes at ambient temperature (20-24? C.). All but five milliliters of the supernatant was aseptically removed and discarded into approved biohazard containers.

Detection

[0044] The remaining five milliliters of sample was homogenized by vortex and an aliquot spread plated onto BCYE, PCV, GPCV and PCV (-) microbiological agar plates and incubated aerobically at 35?1? C. to encourage the proliferation of Legionella organisms. The presence or absence of typical Legionella colonies based on morphology and/or fluorescence was determined after 72 to 96 hours of incubation. If any agar plates did not appear to contain typical colonies, incubation was extended for an additional seven days.

[0045] Typical colonies from each contamination level replicate were re-struck to selective and non-selective media. Typical colonies were then confirmed via serological latex agglutination and molecular identification using the Bruker MS Biotyper.

[0046] The results obtained from this method development study indicate that overall, the extraction procedure had positive outcomes for removing Legionella microorganisms the open-cell foam environmental sampling device. The novel open-cell foam sponges evaluated in this study were inoculated at levels as low as about 4 (e.g. 3.5) CFU/mL, or as high as approximately 250 CFU/mL. Inoculation of the device paralleled actual sampling procedures employed in the field. Whether the pathogen is present at a level of a few cells or many thousands of cells per milliliter, the ability to capture, extract, and detect the organism reliably and consistently is paramount to maintaining the good health of the building occupants. The detection of Legionella is dependent upon the sampling device or procedure used in addition to the laboratory method employed. One cannot be successful without the other.

[0047] The cultural detection and confirmation of Legionella at all levels for all replicates demonstrates the method has applicability as a viable option for Legionella analysis in routine water samples. See Tables B and C for detailed inoculum and recovery results.

TABLE-US-00002 TABLE B Inoculum Results Mixed Legionella Mixed Inoculum Extraction suspension Matrix Target Level Concentration Procedure Surfactant Legionella pneumophila Sterile Tap Low 3.5 CFU/mL Blending Polysorbate 80 ATCC 33152 Water Legionella dumoffii Medium 20.6 CFU/mL QL14012-1A Legionella micdadei High 247.5 CFU/mL QL145022-1A

TABLE-US-00003 TABLE C Detailed Recovery Results Examination for Typical Legionella Confirmation Contamination PCV GPCV PCV PCV Slide Agglutination Test Bruker Level/Replicate BCYE A B A B () BCYE.sup.a () SBA 1 2-15 L. spp. Result Biotyper ID Low A + + ? + + ? + + ? ? + + ? ? Positive Legionella pneumophila Low B + + + ? + ? + + ? ? + ? + Positive Legionella pneumophila, Legionella dumoffii Low C + + + + + ? + + ? ? + + ? ? Positive Legionella pneumophila Medium A + + + + + ? + + ? ? + ? + Positive Legionella pneumophila, Legionella dumoffii Medium B + + + + + ? + + ? ? + ? + Positive Legionella pneumophila, Legionella micdadei Medium C + + + + + ? + + ? ? + + ? ? Positive Legionella pneumophila High A + + + + + ? + + ? ? + + ? ? Positive Legionella pneumophila High B + + + + + ? + + ? ? + + ? ? Positive Legionella pneumophila High C + + + + + ? + + ? ? + ? + Positive Legionella pneumophila, Legionella micdadei Sterility Control ? ? ? ? ? ? ? ? ? NA NA NA Typical NA Negative Control ? ? ? ? ? ? ? ? ? ? ? ? Typical NA Positive Control + + + + + ? + ? ? + ? + Typical Legionella pneumophila .sup.aTwo typical Legionella colonies picked for serological confirmation and molecular identification

[0048] The procedure to extract Legionella from the open-cell foam environmental sampling device was adapted based on previous works for detecting Legionella from environmental samples. The positive outcomes of this study following the procedures presented above, as well as experience working with similar sampling devices, has prompted possibilities of streamlining the method to better suit the workflow in a routine laboratory environment. Blending the device requires sterile laboratory blender jars with sharp blades and potentially poses a safety risk if not performed in a careful manner and in a Biological Safety Cabinet (BSC). One alternative to blending is to place the sampling device into a common sterile laboratory blender bag with Tween? 80 and extract the bacteria by homogenizing with a laboratory paddle blender. This procedure would not only decrease the time required for processing the sample but also allow for the use of readily available disposable sterile materials used by a majority of testing laboratories. Selecting blender bags would have the additional benefit of increasing the ease of use factor, thereby improving laboratory technician efficiency.

[0049] The inoculation method utilized in this laboratory study followed the prescribed, real world best practices for correctly sampling with the sponge device: the glass jars containing the sponges were filled with the sample water to be tested.

[0050] Appendices 2-5 that were part of the Provisional Application that is incorporated herein by reference (two pages each) included reports from independent lab testing from Flint Mich.where bacteria has been a continued challenge with the water distribution system and potential reported human health effects. For the testing with the open-cell foam biological indicator the lab used the following tests and methods:

TABLE-US-00004 TEST METHOD Aerobic Plate Count (APC) FDA Bacteriological Analytical Manual Coliform AOAC 991.14 Legionella Centers for Disease Control (01/2005) Microbial Identification Bruker MALDI Biotyper (Q Labs SOP #10- MIDL-METH-001A)
For the testing for the water grab samples the lab used the following test and method:

TABLE-US-00005 TEST METHOD Aerobic Plate Count Standard Methods for the Examination of Water and (APC) Wastewater, 22.sup.nd Edition

[0051] Appendix 2 shows lower to <10 or <1 (non-detect) APC counts on grab samples while the open-cell foam biological indicator (Waterbug) shows APCs in the millions and identifies bacteria of concern. The following is from appendix 2.

[0052] The following results were obtained from the samples submitted for assay:

Methodology

[0053]

TABLE-US-00006 TEST METHOD Aerobic Plate Count Standard Methods for the Examination of (APC) Water and Wastewater, 22.sup.nd Edition

Results

[0054]

TABLE-US-00007 Sample No. IDENTIFICATION OF SAMPLE APC/mL 1 1608640-01A 4,400 (Upstairs Bath Grab for Bacteria/Fungi) 2 1608640-05A <10 (Water Meter Grab for Bacteria/Fungi)

Methodology

[0055]

TABLE-US-00008 TEST METHOD Aerobic Plate Count (APC) FDA Bacteriological Analytical Manual Identification Gram Stain & VITEK

Results

[0056]

TABLE-US-00009 Sample IDENTIFICATION APC/ No. OF SAMPLE sponge Identification 1 1608640-03B 3,800,000 Acinetobacter junii, (Upstairs Bath Brevibacillus WaterBug Grab) 2 1608640-07B 2,900,000 Pseudomonas aeruginosa/ (Water Meter Pseudomonas putida, WaterBug Grab)

[0057] Appendix 3 shows no APC count on the grab sample while the open-cell foam biological indicator (Waterbug) shows an APC count of >570,000 and identifies bacteria of concern Pseudomonas aeruginosa.

[0058] The following results were obtained from the samples submitted for assay:

Methodology

[0059]

TABLE-US-00010 TEST METHOD Aerobic Plate Count Standard Method for the Examination of (APC) Water and Wastewater, 22.sup.nd Edition

Results

[0060]

TABLE-US-00011 Sample No. IDENTIFICATION OF SAMPLE APC/mL 1 1609132-04A (Amber Grab for Bacteria/Fungi) <1

Methodology

[0061]

TABLE-US-00012 TEST METHOD Aerobic Plate Count (APC) FDA Bacteriological Analytical Manual Identification Gram Stain & VITEK

Results

[0062]

TABLE-US-00013 Sample IDENTIFICATION APC/ No. OF SAMPLE sponge Identification 1 1609132-03B (WaterBug >570,000 Pseudomonas aeruginosa Grab 5 mins)

[0063] Appendix 4 shows lower to <1 (non-detect) APC counts on grab samples while the open-cell foam biological indicator (Waterbug) shows APCs>570,000 and identifies bacteria.

[0064] The following results were obtained from the samples submitted for assay:

Methodology

[0065]

TABLE-US-00014 TEST METHOD Aerobic Plate Count (APC) FDA Bacteriological Analytical Manual Identification Gram Stain & VITEK

Results

[0066]

TABLE-US-00015 Sample IDENTIFICATION No. OF SAMPLE APC/mL Identification 1 1609134-01A (Water <1 N/A Meter Amber Grab for Bacteria/Fungi) 2 1609134-05A (Shower 3,100 Bacillus simplex Amber Grab for Bacteria/Fungi)

Methodology

[0067]

TABLE-US-00016 TEST METHOD Aerobic Plate Count (APC) FDA Bacteriological Analytical Manual Identification Gram Stain & VITEK

Results

[0068]

TABLE-US-00017 Sample IDENTIFICATION APC/ No. OF SAMPLE sponge Identification 1 1609134-03B (Water >570,000 Pseudomonas fluorescens Meter Grab WaterBug) 2 1609134-07B (Shower >570,000 Acinetobacter species Grab WaterBug)

[0069] Appendix 5 shows low APC counts (11,000 and <1) while the open-cell foam biological indicator (Waterbug) shows APC counts of 150,000 and >570,000.

[0070] The following results were obtained from the samples submitted for assay:

Methodology

[0071]

TABLE-US-00018 TEST METHOD Aerobic Plate Count (APC) FDA Bacteriological Analytical Manual Identification Gram Stain & VITEK

Results

[0072]

TABLE-US-00019 Sample IDENTIFICATION APC/ No. OF SAMPLE sponge Identification 1 1609133-03B 150,000 Delftia acidovorans (Murphy Water Meter WaterBug Grab) 2 1609133-07B >570,000 Brevundimonas diminuta/ (Murphy Shower vesicularis Grab WaterBug)

Methodology

[0073]

TABLE-US-00020 TEST METHOD Aerobic Plate Count (APC) FDA Bacteriological Analytical Manual Identification Gram Stain & VITEK

Results

[0074]

TABLE-US-00021 Sample IDENTIFICATION OF No. SAMPLE APC/mL Identification 1 1609133-01A (Murphy Water 11,000 Rhodotorula sp. Meter Amber Grab for Bacteria/Fungi) 2 1609133-05A (Murphy <1 N/A Shower Amber Grab for Bacteria/Fungi)

[0075] A second report from an independent testing laboratory details the study design, procedures, and results, for the evaluation of the ability for six different types of the subject open-cell foam sampling devices (i.e., WaterBugs) in recovering and releasing select bacteria from a water source. In this study, a bulk lot of sterile tap water was inoculated with Pseudomonas aeruginosa. Traditional grab samples consisting of three (3) replicate 100 mL volumes were collected to establish starting baseline bacterial counts for evaluation purposes. WaterBug sampling devices, comprised of six different design formulations, and in replicates of three, were submerged for a total of 20 minutes. During submersion the inoculated water was periodically mixed to maintain homogeneity and even distribution of the bacteria. After 20 minutes had elapsed, each WaterBug was transferred individually to a sterile stomacher bag. Customary laboratory procedures for extracting bacteria from matrices involve the use of a laboratory paddle blender, or stomacher. One point of focus for this study was to determine the stomaching time for optimal recovery; therefore an aliquot from each bag was removed after being stomached for 30 seconds, 1 minute, and 2 minutes. At each time point, the aliquot was diluted as appropriate and the concentration of target organism determined using standard microbiological plate count techniques. Final bacterial counts of the inoculated water were determined after the WaterBugs were removed by obtaining three 100 mL traditional grab samples and enumerating as previously described. A summary of the WaterBug formulations tested and study summary is presented in Table A below.

TABLE-US-00022 TABLE A Pseudomonas Retention and Release Study Design Summary Plating WaterBug Target Extraction Medium/ Formulation Matrix Organism Procedure Incubation A: Sterile Pseudomonas Stomaching MacConkey agar Open-cell Tap aeruginosa (30 s, 1 min, 35? C. for 24 ? 2 EMA Water ATCC 15442 2 min) hours B: Closed-cell EMA C1: Open-cell LDPE/8452 C2: Open-cell EVA/8452 Large-cell C3: Open-cell EVA/8452 Small-cell D: Open-cell urethane
Pseudomonas aeruginosa Extraction and Enumeration

Extraction

[0076] Prior to submersing the WaterBugs, 3?100 mL grab samples were taken from the inoculated sterile tap water. The WaterBugs were removed after 20 minutes of submersion in the inoculated sterile tap water and were stomached for 30 seconds, 1 minute, and 2 minutes. An aliquot of sterile tap water was removed at each time point. An additional 3?100 mL grab samples were taken from the inoculated sterile tap water once the WaterBugs had been removed.

Enumeration

[0077] The grab samples and the aliquots of the inoculated sterile tap water removed at the three pre-determined time points for each of the WaterBug formulations was plated onto MacConkey agar in duplicate. The dilutions were spread plated and incubated at 35?1? C. for 24?2 hours. Typical colonies were enumerated and recorded as CFU/plate, then averaged and multiplied by the dilution factor to determine the amount of microorganisms present in the inoculated sterile tap water sample at the beginning and end of testing as well as the concentration recovered from each of the different sponge design formulations.

[0078] The average CFU/mL, expressed as normalized values (Log.sub.10), recovered from each WaterBug design formulation was compared to the average initial grab samples prior to submersion to obtain percent recovery at each time point in the bacterial extraction process (30 sec., 1 min., 2 min.). Of the six WaterBug formulations tested, Type A: Open-cell EMA demonstrated the highest retention and subsequent release of the inoculating organism at 88.8% after a 1 minute stomaching time period. Type B: Closed-cell EMA demonstrated the lowest retention and release after 2 minutes of stomaching at 70.5%. Type C2: Open-cell EVA/8452 Large-cell was the only formulation to show an increase in percent recovery at the final stomaching time point. This may suggest that it performed best at retaining liquid and bacteria compared to the other formulations; however, the concentration of trapped bacteria that were released was less than other designs on average. Comparing the difference of means between the initial grab sample counts and mean Log.sub.10, counts for each sampling time point demonstrates significant differences (>0.5 Log.sub.10) with several of the design formulations. Tables B and C present the results of the percent recovery and the difference of means.

TABLE-US-00023 TABLE B Grab Sample Recovery Results Grab Samples Average CFU/mL Log.sub.10 CFU/mL Initial 3.9 ? 10.sup.2 2.5911 Final 3.3 ? 10.sup.1 1.5185

TABLE-US-00024 TABLE C Sponge Formulation Statistical Data 30 sec. Stomach 1 min. Stomach 2 min. Stomach % Mean % Mean % Mean Sponge Log.sub.10 Recov- Differ- Log.sub.10 Recov- Differ- Log.sub.10 Recov- Differ- Formulation CFU/mL CFU/mL ery.sup.1 ence.sup.2 CFU/mL CFU/mL ery.sup.1 ence.sup.2 CFU/mL CFU/mL ery.sup.1 ence.sup.2 A: Open-cell 2.0 ? 10.sup.2 2.3010 88.8 0.2901 2.0 ? 10.sup.2 2.3010 88.8 0.2901 1.7 ? 10.sup.2 2.2304 86.1 0.3607 EMA B: Closed-cell 9.7 ? 10.sup.1 1.9868 76.7 0.6043 1.0 ? 10.sup.2 2.0000 77.2 0.5911 6.7 ? 10.sup.1 1.8261 70.5 0.7650 EMA C1: Open-cell 1.3 ? 10.sup.2 2.1139 81.6 0.4772 1.2 ? 10.sup.2 2.0792 80.2 0.5119 1.3 ? 10.sup.2 2.1139 81.6 0.4772 LDPE/8452 C2: Open-cell 1.4 ? 10.sup.2 2.1461 82.8 0.4450 1.3 ? 10.sup.2 2.1139 81.6 0.4772 1.6 ? 10.sup.2 2.2041 85.1 0.3870 EVA/8452 Large-cell C3: Open-cell 1.0 ? 10.sup.2 2.0000 77.2 0.5911 1.1 ? 10.sup.2 2.0414 78.8 0.5497 1.0 ? 10.sup.2 2.0000 77.2 0.5911 EVA/8452 Small-cell D: Open-cell 1.6 ? 10.sup.2 2.2041 85.1 0.3870 1.2 ? 10.sup.2 2.0792 80.2 0.5119 1.1 ? 10.sup.2 2.0414 78.8 0.5497 Urethane .sup.1% recovery calculated using the Log.sub.10 CFU/mL mean average at each sampling time point and the initial grab sample Log.sub.10 CFU/mL mean average .sup.2A mean difference absolute value of greater than 0.5 indicates a statistical significant difference between counts

[0079] The invention is not limited by the above description but rather is supported by it. Other options will occur to those skilled in the art and are within the scope of the following claims.