GB2294115A

GB2294115A - Measuring surface contamination by organisms

Info

Publication number: GB2294115A
Application number: GB9507473A
Authority: GB
Inventors: Gerald David Whitlock
Original assignee: WHITLOCK HUGHES Ltd
Current assignee: WHITLOCK HUGHES Ltd
Priority date: 1994-10-11
Filing date: 1995-04-11
Publication date: 1996-04-17
Also published as: GB9507473D0; GB9420486D0

Abstract

Living organisms having ATP-bearing cells are detected at surfaces with a device comprising a porous thin membrane, e.g. filter paper, containing a reagent which releases ATP (adenosine triphosphate) from the organisms and a reagent which reacts with the released ATP to produce bioluminescence for measurement. Contaminating organisms are e.g. bacteria, yeasts or somatic cells. Releasing agents may be trichloracetic acid or a non ionic detergent. Released ATP is reacted with e.g. luciferin/luciferase.

Description

Method of and Apparatus for Measuring Contamination This invention relates to a method of and apparatus for measuring contamination, particularly bacteria, yeast and somatic cell contamination.

Many food, drink, pharmaceutical, water, cosmetic and health care companies and institutions require rapid microbiological testing for bacteria, yeast and somatic cell contamination measurements. At present, they often have to wait up to several days using plate counts for the test results to be known. Such a delay can be costly for processors.

Yeast, mould and bacterial cells (as well as cells contained in meats, soft fruit, and milk) contain concentrations of adenosine triphosphate (ATP) which, when released from cells, will react with luciferase enzymes to produce a luminescent reaction. The photons thus released can be measured using a photomultiplier tube device or other light collecting system.

It is known from GB 2116709 to produce an image of the location of contamination at the surface of an object, by spraying the surface with an agent for releasing ATP from the cells, and with an agent for reaction with released ATP to produce bioluminescence.

Although it may be possible to measure the bioluminescence released, the measurements would only be meaningful if the precise area over which release took place could also be determined.

According to one aspect of the present invention, there is provided a method of measuring the contamination, by living organisms having ATP-bearing cells, at the surface of an object, comprising the steps of: (a) impregnating a thin membrane of porous cellular material (typically filter paper) of predetermined area with an agent for releasing ATP from the cells and with an agent for reaction with released ATP to produce bioluminescence; (b) placing the membrane on the surface of the object; and (c) measuring the bioluminescence produced using a light measuring device.

Preferably, the membranes are supplied pre-impregnated with release and/or reaction agents. In this case, the membranes may be supplied frozen or freeze dried.

They may also be supplied wet particularly if only pre-impregnated with release agent.

Alternatively, the membranes may be impregnated with release and reaction agents prior to use.

The membranes are preferably provided in individual sterile packets.

The individual sterile packets may comprise two parts which can be removed from a membrane by drawing them away from the membrane in opposite directions parallel to the plane of the membrane. In this case, preferably, one major surface of each part overlaps a corresponding major surface of the other part when the parts are in an operative position and, preferably, an opening is provided between the other major surfaces of the two parts when the parts are in an operative position for application of liquid to the membrane. Preferably, the membranes are circular and said one major surface of each part is more than semi-circular so as to prevent the parts slipping off the membrane unintentionally.

Preferably, the bioluminescence produced is measured by a photomultiplier or an array of CCD's.

According to a second aspect of the invention, there is provided apparatus for measuring contamination, comprising a plurality of thin membranes of porous cellular material, typically filter papers, impregnated with or packaged together with an agent for releasing ATP from ATP-bearing cells of living organisms and/or an agent for reaction with released ATP to produce bioluminescence.

Preferably, the membranes are provided in individual sterile packets.

Preferably, the apparatus further comprises light photon detecting apparatus for measuring the bioluminescence produced.

The invention will now be more particularly described by way of example with reference to the drawings, in which: Figure 1 is a sectional view through one embodiment of a thin membrane provided in a sterile packet, Figure 2 is a top plan view of the sterile packet, Figure 3 is a bottom plan view of the sterile packet showing an exposed portion of the membrane, and Figure 4 is a view, partly in section and partly schematic, illustrating light photon measuring apparatus.

The apparatus comprises thin membranes 10 of porous cellular material, typically in the form of filter papers, of predetermined area. These are preimpregnated with an agent for releasing ATP from ATP bearing cells, preferably a microbial extractant (e.g. trichloroacetic acid) or a somatic extractant (e.g. non-ionic detergent) and an agent for reaction with released ATP, e.g. a Luciferin/Luciferase based reagent, to produce bioluminescence. The membranes 10 are supplied in individual sterile packets 11 and stored in a freezer or freeze dried.

Alternatively, the membranes could be impregnated immediately prior to use or impregnated by a user, and stored in a refrigerator at about 4"C.

The membranes 10 are typically circular and the sterile packets 11 comprise two part circular parts 1 la and 1 lb. The parts 1 la and 1 Ib are of flexible, preferably impermeable, material such as polyethylene. The upper surface of the two parts 1 la and 1 ib are each greater than semi-circular in shape and overlap. The membrane 10 is a fairly close fit in the parts 11~ and 1 ib and the membrane 10 therefore prevents the parts 1 la and 1 lib slipping off the membrane unintentionally. An opening 12 is provided between the lower surfaces of the two parts 1 la and 1 ib so that liquid can be applied to the membrane 10 before the membrane is removed from the packet.

In order to apply the membrane to a surface, the lower surface of the packet 11 is presented to the surface. Pressure can be applied to the upper surface of the membrane 10 without direct contact with the membrane. The packet 11 is removed by pulling the two parts 1 la and ilk apart in opposite directions while maintaining slight pressure on the upper surface of the packet. The packet can thus be removed without the need for upward force which may lift the membrane off the surface.

Alternatively or additionally, an impermeable membrane may be applied to the air exposed surface of the porous membrane. This has a number of advantages.

This will insulate the porous membrane from exposure to air and will reduce evaporation. It also offers the prospect of applying external pressure to the porous membrane to cause the porous membrane to conform more closely to a surface to be tested.

Both membranes should, of course, be as transparent as possible to emitted radiation (light photons).

The apparatus also comprises light photon measuring apparatus. A typical example of this apparatus is shown in figure 4 and comprises a housing 14 defining a chamber 20 which is optically open at one side. The one side is defined by a continuous closed edge 24 of a wall 26 of the chamber. A continuous closed elastomeric sealing member 28 extends completely around the said edge so as to provide a light-tight and hermetic seal between the chamber and the surface when the edge of the wall is applied with force against the surface. The chamber is light-tight except at its open side.The apparatus also comprises photon detector means, typically a photomultiplier 36, which is associated with said chamber for detecting individual light photons in the chamber from the surface, means (comprising a pump formed by two telescoping portions 16 and 18 of the housing) for establishing a pressure differential between the inside and the outside of the chamber when the edge of the wall is applied with force against the surface and the light-tight and hermetic seal is formed between the chamber and the surface, and means (comprising an opaque diaphragm 56 and a shutter 60) responsive to said pressure differential for controlling light exposure of the photon detector means. This apparatus is more fully described GB 2218803B.

In order to perform a test, an impregnated membrane is removed from its sterile packet and placed directly on a surface to be tested. This may, for example, be a plastics laminated surface or may be the surface of a cooking utensil. The photon measuring apparatus is then placed over the filter paper (while the filter paper remains in situ) and a measurement is taken.

As an alternative to pre-impregnating the membranes, they may be packaged with a reaction agent and a reaction agent or they may be pre-impregnated with only a release agent and packaged with a reaction agent. The release and reaction agents or the release agent alone as the case may be is then preferably applied to the membrane before removing the membrane from its sterile packet.

It can be advantageous to apply at least the reaction agent to the membrane just prior to use, particularly when testing vertical surfaces. This ensures that the membrane is wet thus allowing the membrane to stick to the surface to be tested.

This apparatus has been shown to work successfully and to provide the following results against colony forming units of a bacterial culture, Eschericha coli.

Using, for example, Whatman filter paper 54 the results indicate the relatively sensitive nature of the test.

2,000,000 cfu's 45,000 c.p.s 250,000 cfu's 32,000 c.p.s 30,000 cfu's 2,560 c.p.s 3,200 cfu's 332 c.p.s 280 cfu's 312 c.p.s 30 cfu's 265 c.p.s where c.p.s = counts per second.

With the limit of sensitivity well within the limit required for many applications the test procedure using the filter paper technique allows a much more convenient delivery of biochemicals to the test surface whilst retaining the elements of the technique of direct measurement of cells on surfaces.

The measurement of the ATP may or may not be delayed for about 1 to 2 minutes to maximise migration of the released ATP through the filter paper and to maximise, therefore, the light signal output from the luciferase cocktail. The major advantage of the system as described, as well as providing ease of use, is that the area under test can be precisely controlled in terms of the even distribution of the extractant and the luciferase enzyme cocktail. The area of test is thus known and the cfu count can be more accurately determined per unit area, and expressed in thus terms. The imprecise nature of adding extractant and enzyme separately by spraying (or with a pipette) where the area is only estimated is thus overcome.

Other filter paper could be used in the test procedure with varying qualities of chemical-holding capacity as well as sensitivity and ease of use. In particular it has been found that filter paper manufactured by Schleicher and Shuel and sold under no.

1573 has also proved to be very satisfactory in that it is highly transparent to bioluminescence.

Claims

1. A method of measuring the contamination, by living organisms having ATP-bearing cells, at the surface of an object, comprising the steps of: (a) impregnating a thin membrane of porous cellular material (typically filter paper) of predetermined area with an agent for releasing ATP from the cells and with an agent for reaction with released ATP to produce bioluminescence; (b) measuring the bioluminescence produced using a light measuring device.

2. A method as claimed in claim 1, wherein the membranes are supplied pre-impregnated with release and/or reaction agents.

3. A method as claimed in claim 2, wherein the membranes are supplied frozen, freeze dried or wet.

4. A method as claimed in claim 1, wherein the membranes are impregnated with release and reaction agents prior to use.

5. A method as claimed in any one of the preceding claims, wherein the membranes are provided in individual sterile packets.

6. A method as claimed in claim 5, wherein the individual sterile packets comprise two parts which can be removed from a membrane by drawing them away from the membrane in opposite directions parallel to the plane of the membrane.

7. A method as claimed in claim 6, wherein one major surface of each part overlaps a corresponding major surface of the other part when the parts are in an operative position.

8. A method as claimed in claim 7, wherein an opening is provided between the other major surfaces of the two parts when the parts are in an operative position for application of liquid to the membrane.

9. A method as claimed in claim 7 or claim 8, wherein the membranes are circular and said one major surface of each part is greater than semi-circular so as to prevent the parts slipping off the membrane unintentionally.

10. A method as claimed in any one of the preceding claims, wherein the bioluminescence produced is measured by a photomultiplier or an array of CCD's.

11. A method of measuring the contamination, by living organisms having ATP-bearing cells, at the surface of an object substantially as hereinbefore described with reference to the accompanying drawings.

12. Apparatus for measuring contamination, comprising a plurality of thin membranes of porous cellular material, typically filter papers, impregnated with or packaged together with an agent for releasing ATP from ATP-bearing cells of living organisms and/or an agent for reaction with released ATP to produce bioluminescence.

13. Apparatus as claimed in claim 12, wherein the membranes are provided in individual sterile packets.

14. Apparatus as claimed in claim 13, wherein one major surface of each part overlaps a corresponding major surface of the other part when the parts are in an operative position.

15. Apparatus as claimed in claim 14, wherein an opening is provided between the other major surfaces of the two parts when the parts are in an operative position for application of liquid to the membrane.

16. Apparatus as claimed in claim 14 or claim 15, wherein the membranes are circular and said one major surface of each part is greater than semi-circular so as to prevent the parts slipping off the membrane unintentionally.

17. Apparatus as claimed in any one of claims 12 to 16, further comprising light photon detecting apparatus for measuring the bioluminescence produced.

18. Apparatus for measuring contamination, substantially as hereinbefore described with reference to the accompanying drawings.