GB2484261A - Water-soluble adhesive tape for sampling a surface for microbial contamination - Google Patents

Abstract

Use of water-soluble adhesive tape for sampling a surface, preferably a porous surface for microbial contamination, in particular, bacterial cells and/or bacterial spores. Preferably, the water-soluble tape is wave solder tape and the porous surface is preferably wood, concrete or fabric. Preferably, after sampling the surface the water-soluble adhesive tape is dissolved in an aqueous solvent to release the collected material for analysis.

Description

t V.' INTELLECTUAL ..* PROPERTY OFFICE Application No. GB 1016271.7 RTM Date:4 January 2011 The following terms are registered trademarks and should be read as such wherever they occur in this document: Perspex Tween ProtoCOL Intellectual Properly Office is an operating name of the Patent Office www.ipo.gov.uk

SURFACE SAMPLING METHOD

The present invention is concerned with use of a water-soluble adhesive tape for sampling a surface for microbial contamination, collecting microbial contaminants, and the detection and/or identification of microbial contamination.

Sampling protocols for the recovery of biological material from surfaces have remained relatively unchanged since the swab-rinsing sampling method was described in 1917. Improvements in the sampling efficiency or recovery of microorganisms from non porous surfaces, for example steel and plastic, have benefited from the development of novel man made materials. Synthetic polymers for example polyester and nylon can replace natural fibres, like cotton, in some commercially available bud or cloth type samplers. Polyurethane macrofoams, similar to those used in domestic sponges, have also been evaluated as swabs and shown to be effective sampling materials for the recovery of spores from steel surfaces. Porous surfaces in particular present significant additional challenges to materials used for surface collectors. They are usually rougher and have a greater surface area than non-porous materials, with deep crevices in which microorganisms can be deposited. They are also more likely to present a more heterogeneous surface topology and composition. Both these factors can reduce the efficiency and precision of swabbing for surface sampling. Porous surface can be categorised into hard porous surfaces, such as wood and concrete, and soft porous surfaces such as fabric and skin. The absorbency of the surfaces may also interfere with sampling. For example, concrete is a highly absorbent surface.

It has been clearly demonstrated that pre-moistening swabs, with an aqueous buffer containing surfactant, prior to sampling maximises the recovery of spores from non

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porous surfaces. However, porous and/or absorbent surfaces wet unevenly, and most of the buffer is absorbed into the surface on the first few strokes of the swab, further reducing the efficiency and precision of the technique.

Sampling, recovery and measurement of biological material from surfaces is an important means for evaluating contamination of surfaces, and also evaluation of clean' surfaces following decontamination. Surface swabbing, and efficient collection and detection of contamination, is of major importance in the healthcare industry and in food manufacturing. Sampling for contamination in hospitals and other public areas is a growing market, with the requirement for more sensitive and accurate and reliable tests ever on the increase.

Traditionally, microbiological swabbing has utilised a cotton wool swab typically requiring the steps of immersing a swab in a suitable buffer solution, swabbing a surface, re-immersing the swab in a buffer solution, processing to release biological material from the swab, and applying an aliquot of the buffer solution to a biological assay for example culturing or a lateral flow test strip.

The present invention thus generally aims to provide an alternative and improved method for sampling surfaces for microbial contamination, especially bacterial cells and spores, and thereby collecting, detecting and/or identifying microbial contamination., The Applicant has now found that a water-soluble adhesive tape is an ideal material for sampling, collecting and recovering microbial contamination from surfaces, and is especially efficient at collecting contamination from porous surfaces. Moreover, its ability to dissolve in aqueous solvents enables efficient release of the collected material for downstream analysis, and thereby detection and/or identification of the contaminant.

Accordingly, in a first aspect, the present invention provides use of a water-soluble adhesive tape for sampling a surface for microbial contamination. The microbial contamination is preferably bacterial cells and bacterial spores which are traditionally very difficult to efficiently sample, collect and identify from surfaces, especially from porous surfaces such as concrete, wood and fabrics.

The Applicant has surprisingly found that a water-soluble adhesive tape is an excellent means of sampling bacterial cells and spores from surfaces, and especially porous surfaces such as concrete and wood. The water-soluble tape is particularly good at sampling vegetative cells which are much less robust than spores and are more likely to lose viability if the sampling method is too physical.

Water soluble tape is used in the electronics industry to mask contacts on printed circuit boards prior to soldering. The tape is designed to be easily removed by immersing in warm water for less than one minute. The tape is made from a water soluble polymer backing and water soluble adhesive.

Adhesive materials have been used for aerobiological sampling for pollen and spores, however the adhesives were water-insoluble. In fact it was stipulated that the adhesives should not be water-soluble (Kapyla M., Grana 1989, 28, 2 15-218).

Hydrophilic adhesive tape has been used in the past for collection of human DNA through removal of surface cells from hairless areas on the body (Li R.C. and Harris H.A., J. Forensic Science 2003, 48, 1-4).

The discovery that water-soluble adhesive tape can be used for efficiently sampling a surface for microbial contamination will lead to the development of low cost systems for interrogating all types of surfaces and terrains, capable of collecting and subsequently analysing a high percentage of all contamination, thus providing improved limits of detection and a high level of confidence in any result. The tape is also an inexpensive alternative to many commercially available swabs.

The water soluble tape has a percentage recovery between 5 and 10 times greater than most commercially available swabs on the majority of surfaces tested.

Microbial contamination from a surface is preferably collected and then the specific contamination detected and/or identified by a suitable biological assay. The identification may for example use a lateral flow test strip or nucleic acid amplification device.

Accordingly, in a second aspect, the present invention provides use of water-soluble adhesive tape in the detection and/or identification of microbial contamination.

The present invention will now be described with reference to the following non-limiting examples and drawings in which Figure 1 is a graph representing the percentage recovery of bacterial spores from a selection of porous and non-porous surfaces. Each point on the graph represents the mean of four replicates for an experimental trial.

Example 1.

Spore preparation. Bacillus atrophaeus (sub species globigii) (BG) spores were prepared by washing three times in sterile distilled water. After the final wash, spores were suspended in 20 ml of water, heat treated to 70°C for 60 mm and then cold shocked on ice, to kill any vegetative cells. The concentration of spores was determined to be 4.7 x 1010 Colony Forming Units (CFU) / ml. This stock suspension was diluted in sterile distilled water to produce 10 ml of a 4.7 x l0 CFU / ml suspension to be used in all the aerosolised deposition experiments.

Swabs and tape. In this study five different sampling materials were used to determine the efficiency of recovery from a variety of porous and non porous surfaces. The materials were cotton tipped swab (Fisher Scientific, UK; cat. no. FB57830), nylon flock tipped swab (Fisher Scientific, UK: cat. no. DIS-275-070G), polyurethane macrofoam-tipped swab (critical swab VWR, UK; cat. no. 149-0269), Dacron tipped Alexeter collection swabTM (Alexeter Technology LLC Chicago, USA; cat. no. K-2025) and a hydrophilic water soluble adhesive tape (3M St. Paul, USA: cat. no. 5414) Surface description. Six different porous and non porous surface materials served as samples on which the BC spores were to be deposited: glass, stainless steel, polyester/cotton fabric, concrete, wood and Formica®. Glass slides (2.5 cm x 8.0 cm) were from Coming Scientific Glassware. Stainless steel coupons (2.5 cm x 8.0 cm) were laser cut from stainless steel 316, fabric pieces (2.5 cm x 8.0 cm) were cut from a laboratory coat (35% cotton, 65% polyester mix), concrete blocks (7.0 cm x 4.5 cm x 3.0 cm) were water jet cut from runway material, wooden blocks (2.5 cmx 8.0 cm) were cut from redwood pine and pieces of Formica® (3.5 cm x 8.0 cm x 1.5 cm) were cut from a standard office desk. A 20 cm2 area, equivalent to the area of the control glass slide, was marked out using a pencil, on the concrete blocks and the desktop pieces. Prior to deposition the glass, steel, fabric and concrete materials were autoclaved whilst the wood and Formica® materials were surface cleaned with 70% (v/v) ethanol.

Deposition chamber. In order to achieve an even deposition of spores on each of the surface types an aerosol deposition chamber was used. This chamber was a 0.5 m3 stainless steel box with a removable Perspex front panel. Aerosols of BG spores were introduced into the chamber via a hand held airbrush paint sprayer (AB-119) through a sealable opening on one side of the box. Samples to be contaminated were placed on removable stainless steel plates within the box.

Preparation of surfaces. The materials to be aerosol deposited were laid in groups of seven on five steel plates placed at pre-determined positions on the floor of the chamber. A total of thirty five individual surfaces were used to test each swab type. In a separate experiment, 15 control glass slides were subjected to aerosol deposition.

For aerosol deposition 10 mL of a 4.7 x i07 CFU / ml suspension was sprayed into the chamber over a 2 mm period. After spraying the chamber was sealed and left for one hour after which the sprayed materials were removed and dried for two hours at 30°C prior to sampling.

Sampling. Each of the 15 control glass slides was placed in a 50 ml Sterilin conical tube containing 10 ml of 10 mM HEPES buffer-0.1% (w/v) Tween 80. All the other sample surfaces were then swabbed with each swab type or sampled with the hydrophilic adhesive tape. All swabs were premoistened with 75 tl of the HEPES buffer prior to sampling. Each test surface was swabbed with a single swab, 5 times along the length, rotated 90 degrees, swabbed 12 times along the width, rotated again 90 degrees, and swabbed an additional 5 times along the length. After swabbing, the swab heads were either removed into individual 50 ml Sterilin tubes containing 10 ml of 10 mM HEPES buffer -0.1% (w/v) Tween 80 or in the case of the Alexeter swab, were returned to the swab sleeve and the rinse buffer from the liquid filled bulb released to cover the swab. For sampling using the adhesive tape, two 1.25 cm x 8.0 cm strips, covering a surface area equivalent to the glass slide were applied to the test surfaces and pressed down firmly using a sterile gloved forefinger. Both strips were then immediately removed using sterile forceps into individual 50 ml Falcon tubes containing 10 ml of 10 mM HEPES buffer-0.1% (w/v) Tween 80. After sampling all the tubes containing either glass slides, cotton bud, macrofoam or flock tips were shaken by hand for 30 s and then subjected to 30 s of vortexing. The Alexeter swab/sleeve was shaken vigorously by hand for 10 s as per the manufacture's instructions. Tubes containing the adhesive tape were incubated for 10 mm at 45°C followed by vortexing for 30 s to completely dissolve the tape. After processing, the recovered BO pores were plated in quadruplicate onto Tryptone Soya Agar (TSA) plates (Oxoid, Basingstoke UK). Plates were incubated overnight at 37°C and colonies were counted the next day using a colony counting instrument (ProtoCol, Synoptics Ltd UK).

Analysis and statistics. Thirty five test surfaces were used in each deposition experiment and seven experimental replicates were performed for each swab type and the adhesive tape. Samples from each test surface were collected and plated in quadruplicate and the mean of each quadruplicate recorded. Percent recovery from a surface was calculated by dividing the mean CPU count from the sampling material by the mean CPU count from the glass slide control samples. Only culturable spores were recorded and no attempt was made to determine the number of non culturable viable spores recovered.

A linear statistical model was used to investigate differences in percent recovery for the surfaces and sampling materials. The R statistical software system was used for the analysis and the model was fitted with a logit transformation of the percent recovery to stabilise the variance within each sampling material and surface.

The linear model included parameters for the surface, sampling material and an interaction between the surface and swab. This interaction was included in the linear model to cover situations where one swab may be more efficient than the other swabs on a particular surface. The importance of each of these model terms was investigated using F-tests and where the results of the test was statistically significant, where the p-value was less than 0.01, a post-hoc Tukey test was subsequently used to identify where the differences occurred. For example, if there was a statistically significant difference in percentage recovery based on the sampling material then the Tukey test would identify the relative performance of the sampling material. The model assumptions were investigated using residual diagnostic plots.

Water-soluble adhesive tape (YR-54l4 water soluble wave solder tape; 3M) was evaluated alongside commercially available swabs for the collection of bacterial spores from a multitude of surfaces. Spores were recovered from swabs by aggressive vortexing and/or shaking, or from the adhesive tape by dissolving in water at 45°C for mm, followed by growth on agar plates.

Having regard to Figure 1 the soluble tape is significantly better at recovering spores from both porous and non porous surfaces than a comprehensive selection of commercially available swabs, especially from porous surfaces such as concrete and This study shows that water soluble hydrophilic adhesive tape is very effective at recovering spores from a range of porous and non porous surfaces. The tape was significantly more effective than any of the swabs tested on all the non porous surfaces and steel. Overall, for all the sampling materials tested the highest recoveries were from the Formica® and steel surfaces. In contrast, the fabric and wood surfaces had low recoveries for all sample materials except the tape.

The tape has some significant advantages over the swabs. For a swab to be effective it must be efficient at both recovering the spores from the surface and subsequently releasing them for analysis. Aggressive recovery techniques, including extensive vortexing or ultrasonic treatment are routinely used to efficiently recover spores from swabs for further analysis. The water soluble adhesive tape however will release all the spore material collected and consequently make available 100% of the material for subsequent analysis. Calcium alginate swabs function in a similar way, and are completely soluble in sodium hexametaphosphate, but their ability to recover culturable bacteria and spores from surfaces is reported to be equivalent to or even poorer than cotton swabs.

The development of environmental sampling techniques for surface swabbing, over many years, has focused entirely on the effective recovery of microorganisms from hard non porous surfaces. Where commercially available swabs have been tested on porous surfaces the recoveries of biological material have been very low.

Consequently, validated protocols for the recovery of microorganisms from surfaces have been restricted to non porous surfaces and this in turn limits the choice of surface to be tested in a contaminated area. This study has identified a novel use for a commercially available soluble adhesive tape for the effective recovery of spores from a variety of porous and non porous surfaces. For recoveries from porous surfaces this material significantly exceeded the performance obtained for the commercially available swabs and for any other previously reported sampling method, including contact plates.

The use of water soluble tapes and adhesives like the one used in this study provides a universal, simple and cost effective way to recover spores and other biological material from a wide range of porous and non porous surfaces. The ability to use a single sampling method on all surfaces simplifies the development and validation of protocols for the recovery and identification of biological material in contaminated environments.

Example 2.

The tape is also better at sampling environmentally sensitive vegetative bacterial cells than the conventional swabs, and especially results in the recovery of fewer non culturable cells Vegetative cells (1.0 x l0), were pipetted, in lO0uL of 10 mM HEPES buffer-0.1% (w/v) Tween 80, onto six concrete blocks similar to those used in Example 1. The concrete blocks were then dried for 2 hours at 30°C. After drying the blocks were divided into 2 sets of three and sampled using either macrofoam swabs or the hydrophilic adhesive tape. Cells were recovered from the swabs and tape as described in Example 1. Cells eluted from the swab and the tape were serially diluted and plated onto TSA plates and incubated, at 37°C, overnight. Colonies were counted as described in Example 1. Percent recovery from a surface was calculated by dividing the mean CFU count from the sampling material by total number of vegetative cells added to each concrete block. Only culturable spores were recorded and no attempt was made to determine the number of non culturable viable cells recovered. The percent recovery of culturable vegetative cells from each of 3 concrete blocks is reported in Table 1. Recoveries using the tape are between 4 and 200X greater than those using the macrofoam swabs.

Table 1. Percent recovery of vegetative cells from concrete. Cells were aliquoted onto each of 3 blocks and dried at 30C prior to sampling.

Percent recovery from concrete blocks Sampling material 1 2 3 tape 0.07 0.03 0.015 macrofoam 0.0003 0.0003 0.004

Claims (10)

1. CLAIMSI. Use of a water-soluble adhesive tape for sampling a surface for microbial contamination.
2. 2. The use according to Claim 1, wherein the microbial contamination is bacterial cells and/or bacterial spores.
3. 3. The use according to Claim 1 or Claim 2 for sampling a porous surface.
4. 4. The use according to Claim 3, wherein the porous surface is wood, concrete or fabric.
5. 5. The use according to Claims 1 to 4, wherein the water-soluble tape is wave solder tape.
6. 6. Use of water-soluble adhesive tape in the detection and/or identification of microbial contamination.
7. 7. The use according to Claim 6, wherein the microbial contamination is bacterial cells and/or bacterial spores.
8. 8. The use according to Claim 6 or Claim 7 for detection and/or identification of microbial contamination on a porous surface.
9. 9. The use according to Claim 8, wherein the porous surface is wood, concrete or fabric.
10. 10. The use according to Claims 6 to 9, wherein the water-soluble tape is wave solder tape.