WO2008091851A1

WO2008091851A1 - Rapid assay method and device for estimating microbial contamination of different substrata

Info

Publication number: WO2008091851A1
Application number: PCT/US2008/051631
Authority: WO
Inventors: Vladimir Gouli; Rosanna Giordano; Svetlana Gouli
Original assignee: University Of Vermont Of Burlington, Vt
Priority date: 2007-01-22
Filing date: 2008-01-22
Publication date: 2008-07-31

Abstract

The present invention is directed toward a method and device that can rapidly estimate microbial contamination from different substrata, such as solid, soft or gelatinous surfaces, including fruits, vegetables, general agricultural and mass consumption products. The device comprises: a collection device for obtaining a sample of aerobic and anaerobic microbial contamination; a receptacle for facilitating bacterial growth; wherein said collection device is shaped to fit into said receptacle.

Description

RAPID ASSAY METHOD AND DEVICE FOR ESTIMATING MICROBIAL CONTAMINATION OF DIFFERENT SUBSTRATA

CROSS REFERENCE TO RELATED APPLICATION

[0001] The application claims benefit under 35 U.S.C. §119(e) of the U.S. Provisional application No. 60/881,493, filed January 22, 2007, and the content of which is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION:

1. Field of the Invention:

[0002] The present invention relates to a rapid method to estimate microbial contamination from different substrata, such as solid, soft or gelatinous materials, and more particularly, a device for rapidly detecting and identifying both aerobic and anaerobic microorganisms.

2. Description of the Related Art:

[0003] Principal existing methods for the sampling of microorganisms from surfaces include wash and imprint methods. The latter method involves pressing down a sampling tool on a suspect surface and after incubation, determining the numbers and types of microorganisms. U.S. Patent No. 6,207,406 discloses a roller having a sheet of porous plastic foam containing microbial growth media therein that is rolled across the surface and then incubated.

[0004] However, these prior art assay methods do not allow for the retrieval of all contaminants, because some microbial particles have very strong adhesive properties. The existing methods are not capable of removing some of these types of microorganisms from different substrata.

[0005] Another disadvantage of these prior art methods is the inability to estimate microbial contamination from a variety of substrata, especially, soft or gelatinous materials. U.S. Patent Application No. 2005/0272112 teaches using an adhesive strip to obtain a sample representation of the outer layer of the skin. However, using the disclosed adhesive strip to rapidly collect and incubate microbial from a variety of surfaces is not possible. [0006] Thus, there is a need for a method and device to rapidly assay microbial contamination from solid, soft or gelatinous substrata. SUMMARY OF THE INVENTION

[0007] One aspect of the present invention is to provide a method and device that can rapidly estimate microbial contamination from different substrata, such as solid, soft or gelatinous surfaces, including fruits, vegetables, general agricultural and mass consumption products.

[0008] Another aspect of the present invention is to provide a method and device that rapidly detects and identifies both aerobic and facultative anaerobic microorganism.

[0009] Still yet another aspect of the present invention is a method and device that can detect any saprophytic and semi-saprophytic microorganisms from any hard or gelatinous substrate, whether it be natural or synthetic.

[00010] Yet another aspect of the present invention is a method and device that enable efficient removal and multiple analyses in a very short period of time.

[00011] According to these and other aspects, there is provided a method of assaying microbial contamination of substrata. The method includes the steps of providing a collection device for obtaining a sample of the microbial contamination, contacting the surface with the collection device, whereby the collection device simultaneously obtains a sample of at least a portion of aerobic and anaerobic microbial contamination of the substrata, and placing the collection device into a receptacle for facilitating bacterial growth. The receptacle contains a quantity of growth media. The receptacle is incubated and a bacterial colony grown in the receptacle is assessed.

[00012] According to these and other aspects, there is provided a device for assaying microbial contamination of substrata, the device including a collection device for obtaining a sample of aerobic and anaerobic microbial contamination. A receptacle facilitates bacterial growth and contains a quantity of growth media. The collection device is shaped to fit into the receptacle such that the collection device has continuous contact with the media.

[00013] These and other features, aspects, and advantages of the present invention will become more apparent from the following detailed description of the preferred embodiment relative to the accompanied drawings, in which:

BRIEF DESCRIPTION OF THE DRAWINGS

[00014] Fig. 1 is a top view of the collection device of the present invention.

[00015] Fig. 2 is a cross-section of the device taken along line I-I of Fig. 1.

[00016] Fig. 3 is a top view of the collection device and growth receptacle of the present invention. [00017] Fig. 4 is a cross-section of the growth receptacle and device taken along line II-II of Fig. 3.

[00018] Fig. 5 is a graphical representation of the method of the present invention vs. prior art assay methods.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[00019] The present invention relates to a method and device for rapidly collecting, detecting and identifying both aerobic and facultative anaerobic microorganisms, and determining the intensity of microbial contamination that could constitute a source of disease for animals or humans. The device of the present invention can detect any saprophytic and semi-saprophytic microorganism from any hard or gelatinous substrate, whether it be natural or synthetic. Examples of such surfaces are fruits, vegetables, meat (cooked or fresh), cheese, wood, plastic, metal, etc. it should be appreciated that collection of microorganisms from other surfaces is contemplated by the present invention.

[00020] Referring to Figs. 1-3, the device 10 for assaying microbial contamination of substrata consists of a collection device 12 for obtaining a sample of microbial contamination. Collection device 12 can be an adhesive tape having an adhesive surface 14 on a side that is designated to remove microorganisms from any substrata. The collection tape 12 can be Scotch 375^® manufactured by 3M of St. Paul, MN. It should be appreciated that although the collection device of the present invention is referred to as an adhesive tape, other types of adhesive collection devices are contemplated by the present invention. [00021] With some surfaces, such as varieties of plastics, i.e., plastic plates/dishes, bags, patent leather, or polished metal, other variants of tape should be utilized. For example, with such surfaces it is best to utilize a removable variant of scotch tape, such as Magic Tape^® manufactured by 3 M of St. Paul, MN.

[00022] As will be described further herein, collection tape 12 is sized and configured to fit within a receptacle 20. As shown in Fig. 4, receptacle 20 can contain a quantity of growth media 24. The collection tape 12 is designed to have the same shape as the receptacle and is sized to fit within the receptacle such that the collection tape has continuous contact with media 24.

[00023] Referring back to Figs. 1 and 2, collection tape 12 includes a plurality of perforations or holes 16 located on one half thereof for growing aerobic bacteria therein. The other half 18 of the collection tape is un-perforated for simultaneously growing the anaerobic bacteria. Although the perforations are shown as being located on one-half of the collection tape, it should be appreciated that the perforations can cover a larger or smaller area on the tape. However, for favorable bacteria growth, the perforations should cover at least one half of the tape.

[00024] Perforations 16 can have a diameter in the range of 0.5 - 1.5 mm and can be located at a distance of approximately 5 mm from each other to allow for the maximum number of perforations on the one half of the tape. Once again, it should be appreciated that a variety of perforation diameters and separation distances are contemplated by the present invention depending on the size and shape of the collection device.

[00025] In a preferred embodiment of the present invention, receptacle 20 can be a standard Petri dish. Collection tape 12 can be a disk of material sized and shaped to fit within the Petri dish. For example, Petri dishes having a diameter of 36 mm, 55 mm, 86 mm or more can be used.

[00026] Different nutrient media can be used for screening different groups of bacteria.

General microbial analysis can be estimated using universal media rich in complex nutrient elements, for example, Sabouraud dextrose agar and yeast, Sabouraud dextrose agar supplemented with egg yolk and milk, blood agar, bacto-agar with beef extract, and bacto- agar with liver extract. Other selective medias can be used to encourage the growth of specific groups or species of bacteria.

[00027] The method of the present invention allows for rapid assaying of microbial contamination of the substrata. The collection tape 12 is tightly pressed on the surface to be tested. The tape should be pressed firmly to insure that contact is made over the surface, especially when probing irregular surfaces.

[00028] The tape, having simultaneously obtained a sample of at least a portion of aerobic and anaerobic microbial contamination of the substrata, is placed within the receptacle 20.

As discussed supra, the receptacle facilitates bacterial growth by containing a quantity of growth media. The amount of media contained within receptacle 20 depends upon the size of the receptacle. However, at least 1 ml of media or sterile water should be deposited on the surface of the collection tape to insure continuous contact and eliminate air bubbles.

[00029] The receptacle is then incubated. Petri dish receptacles and tape should be incubated between 26-28 ⁰C. During incubation the bacterial colony is grown. Due to the construction of the collection device, aerobic and anaerobic contaminants will simultaneously grow under the perforated and un-perforated areas respectfully.

[00030] After incubation, the number of bacterial colonies grown on the tape to indicate the degree of contamination tested is assessed. As discussed supra, the method of the present invention can estimate the microbial contamination of solid, soft or gelatinous substrata. More particularly, microbial contamination of fruits, vegetables, agricultural and/or mass consumption products.

[00031 ] The number of bacterial colonies determined can be assessed to indicate the degree of contamination. The degree of contamination can be scaled. For example, less than 90-100% of perforation having microbial colonies could correspond to a contamination level 10; 90-80% - level 9; 80-70 - level 8; 70-60% - level 7; 60-50% - level 6; 50-40% - level 5; 40-30% - level 4; 30-20% - level 3; 20-10% - level 2; and 10-1% contamination - level 1. [00032] Further identification of the microorganisms can be accomplished using several different methods. For example, traditional, i.e., morphological, serological, immunofluorescent, and morphological analyses, and modern, i.e., DNA analysis. It should be appreciated that the most effective and compatible method of identification being DNA analysis, since this permits the identification of selective microbial colonies. [00033] Fig. 5 is a graphical representation of the average number of bacteria that can be removed from substrata using the device of the present invention as compared to the prior art imprint and wash assay methods and devices. As can be seen from the graph, the rapid assay method and tape of the present invention had a ten-fold higher detection capacity than the prior art methods.

[00034] Although the present invention has been described in relation to particular embodiments thereof, many other variations and modifications and other uses will become apparent to those skilled in the art. It is preferred therefore, that the present invention be limited not by the specific disclosure herein, but only by the appended claims.

Claims

What Is Claimed Is:

1. A method of assaying microbial contamination of substrata, the method comprising the steps of: providing a collection device for obtaining a sample of the microbial contamination; contacting the surface with said collection device, whereby said collection device simultaneously obtains a sample of at least a portion of aerobic and anaerobic microbial contamination of the substrata; placing said collection device into a receptacle for facilitating bacterial growth, said receptacle containing a quantity of growth media; incubating said receptacle; and assessing a bacterial colony grown in said receptacle.

2. The method of claim 1, wherein said collection device comprises a tape having an adhesive portion and the step of providing the collection device further comprises providing a tape that has the same size and shape as said receptacle.

3. The method of claim 1, wherein said receptacle is a Petri dish and the step of providing the collection device further comprises providing a tape that has the same size and shape as the Petri dish.

4. The method of claim 1, wherein the step of providing the collection device further comprises perforating said collection device with a plurality of perforations.

5. The method of claim 4, wherein the perforations are formed on one-half of the collection device and are spaced approximately 5mm apart.

6. The method of claim 5, wherein the perforations are sized from 0.5-1.5mm.

7. The method of claim 1, wherein said contacting step further comprises tightly pressing said collection device against the substrata.

8. The method of claim 2, wherein said contacting step further comprises tightly pressing said tape against the substrata.

9. The method of claim 1, wherein said incubating step further comprises growing said bacterial colony in a media having rich complex nutrient elements.

10. The method of claim 9, wherein said media is selected from a group consisting of Sabouraud dextrose agar and yeast, Sabouraud dextrose agar supplemented with egg yolk and milk, blood agar, bacto-agar with beef extract, and bacto-agar with liver extract.

11. The method of claim 1 , wherein said incubating step further comprises growing said bacterial colony in a selective media such that a growth of a specific group or species of bacteria occurs.

12. The method of claim 1, wherein said incubating step further comprises incubating said receptacle at a temperature between 26°-28°C.

13. The method of claim 1, wherein said assessing step further comprises determining a number of bacterial colonies grown on said collecting device.

14. The method of claim 13, wherein said assessing step further comprises corresponding said number of bacterial colonies determined to a degree of contamination.

15. The method of claim 1, wherein said assessing step further comprises determining an identification of any microorganisms.

16. The method of claim 15, wherein said identification determination is performed by a DNA analysis.

17. The method of claim 15, wherein said identification determination is performed by an analysis selected from a group including morphological, serological, immunofluorescent, and morphological analyses.

18. The method of claim 1, wherein said assessing step comprises estimating the microbial contamination of solid, soft or gelatinous substrata.

19. The method of claim 18, wherein said assessing step comprises estimating the microbial contamination of fruits, vegetables, agricultural and/or mass consumption products.

20. The method of claim 4, wherein said incubating step further comprises growing aerobic bacteria in said perforations and anaerobic bacteria in the un-perforated area of said collection device.

21. A device for assaying microbial contamination of a substrata, said device comprising: a collection device for or obtaining a sample of aerobic and anaerobic microbial contamination; a receptacle for facilitating bacterial growth, said receptacle containing a quantity of growth media; wherein said collection device is shaped to fit into said receptacle such that said collection device has continuous contact with said media.

22. The device of claim 21, wherein said collection device comprises a tape having an adhesive surface.

23. The device of claim 22, wherein said receptacle is a Petri dish.

24. The device of claim 23, wherein said tape is circular and has a diameter sized to fit within the Petri dish.

25. The device of claim 21 , wherein said collection device has a plurality of perforations for growing the aerobic bacteria therein and an un-perforated area for growing the anaerobic bacteria.

26. The device of claim 25, wherein at least one-half of said collection device is perforated.

27. The device of claim 22, wherein said tape has a plurality of perforations on one half for growing aerobic bacteria therein and an un-perforated area for growing anaerobic bacteria.

28. The device of claim 27, wherein the perforations are spaced approximately 5mm apart.

29. The device of claim 27, wherein the perforations are sized from 0.5-1.5mm.

30. The device of claim 21, wherein the media is a universal media having rich complex nutrient elements.

31. The device of claim 30, wherein said universal media is selected from a group consisting of Sabouraud dextrose agar and yeast, Sabouraud dextrose agar supplemented with egg yolk and milk, blood agar, bacto-agar with beef extract, and bacto-agar with liver extract.

32. The device of claim 21, wherein the media is a selective media, such that a growth of a specific group or species of bacteria occurs