WO1996038584A1

WO1996038584A1 - Assay for microorganisms

Info

Publication number: WO1996038584A1
Application number: PCT/GB1996/001296
Authority: WO
Inventors: Peter Leonard Grant; Simon James Forster; Brian Thomas
Original assignee: Celsis International Plc
Priority date: 1995-05-31
Filing date: 1996-05-31
Publication date: 1996-12-05
Also published as: AU5840496A; GB9510928D0

Abstract

In an assay for coliform or other microorganisms in a sample, which comprises contacting a mixture of the sample and a liquid growth medium with a material that undergoes a detectable change in the presence of the microorganisms to be assayed, the sample is contained in a well whose base is defined by a filter material, and the mixture is formed by placing the well on the growth medium. A preferred embodiment additionally comprises removing the sample and conducting a further assay adapted to distinguish between the microorganisms to be assayed and any other that may have given a false positive.

Description

ASSAY FOR MICROORGANISMS Field of the Invention

This invention relates to a method for enumerating and/or identifying microorganisms. Background of the Invention

Many industries, including pharmaceuticals, need to detect low levels of materials in large volumes of liquid. For example, they rely heavily on classical microbiological techniques to detect microbial contamination. Areas often under surveillance by Quality Assurance personnel include the control of bioburden in incoming raw materials, especially liquids; monitoring of microbial population in the production environment; in-process controls, especially after storage; and final product testing. Often, product is manufactured and stored whilst analysis for microbial content takes place. If contamination is detected, the product may need to be destroyed, and the production line shut down until the source of contamination is found. Often the time taken for microbial analysis is the rate- limiting factor in bringing the plant on stream. This can lead to substantial costs in wasted production or raw materials. Moreover, by traditional methods, results may be produced only after 7-14 days.

The determination of the number of microorganisms present in the various types of water used in a pharmaceutical environment is considered a critical factor in producing many products. Usually, the microbiological specification ranges from 0.01 to 100 cfu per ml of water, depending on the source of the sample. It is also desirable to enumerate and to identify particular microbial species or groups of species, such as E . coli or the colifor group of bacteria. O-A-9319199 discloses an improved assay, the "Celsis Digital" system, for the rapid enumeration of microorganisms or other biological material, in samples containing a very low level of the material. In this assay, a sample in liquid form, diluted as necessary, is distributed and passed through a number of discrete wells adapted to retain the material; and the wells are observed for the presence of the material. Then, for enumeration, the number of wells in which the material is present may be determined as a function of the total number of wells. Alternatively, or in addition, the wells are analysed for the presence of a specific material.

The compartments into which the liquid sample is distributed may be the wells of a multi-well/microtitre plate, with a filter material forming the base of the wells. Such a system allows a very dilute sample to be analysed, the liquid being drawn through the wells under suction, while the analyte is retained on the filter material. WO-A-9319199 discloses the use of colour, for detection, but is primarily adapted to the assaying of organisms by biolu inescence, since a sensitive technique is required if wells are to be analysed containing what may be a single cell. The number of organisms in a well may be increased by adding a growth medium.

US-A-5210022 and EP-A-0470172 describe methods and materials for identifying and differentiating coliforms and E . coli bacteria. The procedure in US-A-5210022 involves inoculation of a test medium substrate capable of forming a solid matrix or solid surface with the sample to be tested and appropriate chromogenic materials, and incubating the test medium as a solid. EP-A-0470172 requires that an enzyme indicator is located at the surface of a solid support, e.g. a plastics material that floats on the surface of the liquid sample and which thus allows the dye-forming substrate to be in contact with air above the liquid.

A problem associated with current tests for coliform bacteria is false positive results due to other organisms. Most methods for determining the presence of coliform bacteria detect by some means the activity of the enzyme

/3-galactosidase, which is characteristically present in coliforms. However, strains of other species, which in general do not express this enzyme, are sometimes found to possess this activity. An example is Aeromonas hydrophila, which is commonly found in environmental water samples. Summary of the Invention

According to the present invention, in an assay for microorganisms. in a sample, which comprises contacting a mixture of the sample and a liquid growth medium with a material that undergoes a detectable change in the presence of the microorganism to be assayed, the sample is contained in a well whose base is defined by a filter material, and the mixture is formed by placing the well on the growth medium.

This method provides various advantages. One is that there is no need for a solid or solidifiable medium (as described in US-A-5210022) . The medium can simply be, for example, a pad holding a nutrient broth.

Another advantage is that material need not be located at a surface (as described in EP-A-0470172) . In embodiments where the detectable change (or signal) comprises an insoluble coloured product, intensification can be achieved by removing the liquid from the wells by suction, thus depositing the colour on the membranous bottom of the wells. There are also advantages common to those described in O-A-9319199, e.g. the use of a single multi-well plate instead of many agar plates, the ability to handle large volumes of liquid samples containing low concentrations of analyte, and the ability to wash and otherwise treat the analyte, without removing it from the well.

Further, this invention provides a ready solution to the problem of false positives, taking advantage of the fact that non-coliform producers of jS-galactosidase can be differentiated from true coliforms by a further test, e.g. for cytochrome oxidase. Coliforms are oxidase-negative, while the vast majority of non-coliform producers of β-galactosidase are oxidase-positive. One embodiment of the current invention allows this confirmatory test to be performed rapidly and conveniently; the coliform count can be corrected to remove false positives. This "memory" system and other aspects of this invention are described in greater detail below.

Description of the Invention

The material that undergoes a detectable change can be any of those that are well known in the art. It is preferred that the detectable change comprises the formation of a coloured, insoluble precipitate. Materials that can be used for such purposes, for example to distinguish E . coli and total coliforms, are fully described in US-A-5210022 and EP-A-0470172. They include Salmon-Gal and X-Gluc. It is preferred that the colour should be retained within the wells. However, some materials that give a useful characteristic colour, e.g. in the well-known and very sensitive indole test, may diffuse out of the wells; this potentially compromises the use of discrete wells in the invention, since the colour may then migrate through the growth medium, and appear in all wells. This problem can be overcome, if necessary, by ensuring that wells are isolated after bringing them and the growth medium into contact, by using a barrier or by clamping on a cooperating member which serves to isolate the wells from each other.

Although the present invention is particularly useful in identifying and enumerating, say, total coliforms and E . coli , it is equally applicable to any groups, species or strains of microorganisms having unique activities that can be detected using a chro ogenic substrate or other material that undergoes a detectable change. Further, if it is desired to distinguish between organisms that have two or more enzyme activities, none of which is unique to a species in itself, but the presence of two or more activities is confined to a particular group, this can be detected by means of the present invention, using two or more appropriate substrates. The present invention is also of wide applicability, in terms of the growth medium that can be used. The use of discrete wells, e.g. in a multi-well plate as described in WO-A-9319199, means that there is no confluence. While any conventional medium may be used, a selective (or inhibitory) medium may be used where it is desired to inhibit the growth of a particular group, species or strain of organism. By using such a selective medium (many examples of which are known) in a first assay, or as the medium for a further assay if the "memory" system is used, a distinction can be drawn between organisms that grow and produce a detectable colour on the one hand, and other organisms that contain the same characteristic enzyme but which are inhibited from growth on the specific medium. Specific viable counts can thus be readily obtained.

A particularly preferred embodiment of the present invention is based on the ability to reduce or eliminate false positive results, using a "memory" system. In this system, an additional step, before or after incubating the mixture (however formed) , comprises removing the sample and conducting a further assay adapted to distinguish between the microorganisms to be assayed and any other that may have given a false positive. The further assay utilises a second chromogen or other material that undergoes a detectable change, in the presence of a second enzyme, associated with another organism but not with the organism which is the subject of the first assay. The second enzyme might be cytochrome oxidase, so that the further assay distinguishes non-coliforms containing jS-galactosidase, such as Aeromonas hydrophila , from coliforms.

The samples in a multi-well plate may be removed and introduced into a further such plate. This operation is well adapted to automation. For example, a multi-pipetting device having an array of pipettes corresponding to the wells can transfer samples simultaneously, and the results in the respective plates can be compared and computed by any instrument that detects the respective results. Accordingly, positive results can be confirmed or additional tests performed using this further feature of the invention, the memory device. This device may comprise an arrangement of probes whereby a sample is removed from each well of the plate and transferred to a further surface or arrangement of containers such that the original spatial relationship of each sample to the other samples and to the original plate is maintained. Further tests can then be performed on this replica of the original plate. In a further embodiment of the invention, the memory device is not simply a passive or non-specific transfer device but is capable of specifically binding to the desired microorganisms. This may be achieved by immobilising, on an appropriate surface or surfaces of the device, specific ligands (which may be antibodies, antibody fragments, nucleic acid probes or any other chemical entity) which are capable of binding to receptors on specific microbes, preferably but not necessarily on the surface of the microbial cells. Bound microbial cells or cell components can then be detected on the memory device by any suitable method. In this way, specific counts can be obtained even in the presence of other microbial species, growth of these other species having occurred in the wells of the plate at the same time as growth of the species for which it is desired to test.

In the first assay at least, and as described in WO-A- 9319199, MPN (most probable number) analysis may be applied if, after distribution of the sample into a multi- well plate, at least one well contains no detectable organisms. The method is thus applied to cases in which observation of the wells indicates that the organisms are present in some, and not in others.

The material to be analysed may be a liquid, in which case it may be distributed with no or minimal pre-treatment (e.g. dilution) . Alternatively, it may be a solid in which microorganisms may be present, or microorganisms collected on a swab or filter, in which case the organisms should be dispersed in a suitable liquid medium.

The invention is applicable to the detection of microorganisms present at any level in a sample. If the initial concentration of the microorganisms in the sample is low, it can be distributed directly into the wells; if high, it can first be diluted appropriately. Dilution will not often be necessary for many environmental samples, e.g. in coliform testing. However, any dilution can be conducted quite quickly, and evidence of retained microorganisms may then be detected directly, without further delay.

After distribution, each well may either contain or not contain material, e.g. 1 microbe. Growth on the medium can then provide a pure, homogeneous culture which itself is valuable. The probability that a well contains a homogeneous culture can readily be calculated. Moreover, the respective wells may comprise different microbes which can be analysed individually, much more accurately and sensitively than is possible by analysis after amplification of all such material in the original sample. The wells can be tested with specific probes, and non¬ specific reactions can be minimised.

Detection preferably involves observation of a colour change. For this purpose, colorimetric reagents may be introduced into the wells with the sample or with the growth medium, or later. By suitable choice of materials, a colour reaction may be observed, e.g. pink or light-blue, according to the microorganisms. The use of discrete wells allows colonies to have growth medium removed, and optionally also a wash liquid, suitably under suction, before introducing any substance that gives a signal in the presence of the microorganisms to be analysed.

Whichever procedure is adopted, a digital (binary) read-out may be available in a very short period, e.g. within 24 hours and often much less. This compares favourably with the longer periods that are required by current techniques.

In a specific, illustrative embodiment, a memory device for use in this invention consists of two components which are:

(1) a "finger plate" having 96 individual fingers that . will "dip" into each of the 96 corresponding wells in a Celsis Digital filter plate, and two side flanges for location in the lid and provision of a grip point for the user; and

(2) a "lid" in which the finger plate is held and which facilitates the movement of the finger plate in the vertical plane. The two components are assembled to provide a single unit.

At the end of each pin, there is a single groove which facilitates the pick-up of liquid containing microorganisms from the well. The groove is designed to pick up approx. 1-2 μl of liquid from the well, the objective being to remove at least one organism from the well at the limit of detection of the system (approx. 1000 cells) . The wells typically hold 20-100 μl of broth.

The remainder of the surface of the pin has a slightly roughened texture, to facilitate contact with and pick-up of microorganisms from colonies which may grow on the membrane filter. The end of the pin is designed to contact the maximum possible area of the membrane.

Associated with use of the memory device is the memory medium (agar) plate. This is a tray designed for transfer of microorganisms by the memory device from the filter plate onto an agar surface, in order to maintain the organisms' viability before a destructive bioluminescence assay is performed. The agar used may be selective or non- selective, depending on the application.

The memory device may be engaged onto the memory agar plate in only one orientation, by the provision of chamferred corner in both components. Thus the correct orientation of the wells is maintained during transfer.

The base of the memory agar plate may be opaque. Preferably, however, it is clear, to allow easy visualisation of colonial growth on the agar.

The following Example illustrates the invention. In particular, it relates to the simultaneous detection and enumeration of E. coli and total- coliform bacteria in a water sample, using a Celsis Digital plate and device of the type available from Celsis International pic and described in WO-A-9319199. Example

A water sample, which may be of any volume, is filtered through a quadrant of a Celsis Digital device (comprising sample manifold and Digital plate) using a vacuum pump. Any microorganisms present are retained on the membranes within the wells of the Digital plate.

The Digital plate is placed on a porous pad which holds a broth (liquid medium) containing a selective agent or selective agents and chromogenic enzyme substrates. The chromogenic substrates preferably comprise Salmon-Gal (6- chloro-3-indolyl-β-D-glucopyranoside) and X-Gluc (5-bromo- 4-chloro-3-indolyl-_/3-D-glucuronic acid) . The membranes within the wells of the Digital plate become saturated with the broth.

The currently preferred medium is

5 g/1 Tryptone

5 g/1 Sodium chloride

1 g/1 D-sorbitol 1 g/1 D-tryptophan

2.7 g/1 Dipotassium hydrogen orthophosphate

2 g/1 Potassium dihydrogen orthophosphate 0.1 g/1 Sodium lauryl sulphate

0.1 g/1 Tween® 20 30 mg/1 5-Bromo-4-chloro-3-indolyl-3-D-glucuronide, cyclohexylam onium salt (X-Gluc) 100 mg/1 6-Chloro-3-indolyl-3-D-galactopyranoside

(Salmon-Gal) 0.238 g/1 Isopropyl-β-D-thiogalactopyranoside (IPTG) q.s. Dimethyl sulphoxide (DMSO) pH 6.80

The pH of the medium is 6.80. The amount of DMSO is sufficient to -dissolve the X-Gluc and Salmon-Gal. The primary nutrient source -is the tryptone; the sorbitol and tryptophan will also be utilised as nutrients. The plate is incubated at 37°C overnight (at least 18 hours) . During this time, growth of any coliform bacteria will lead to the metabolism and breakdown of the Salmon- Gal, one of the products of these reactions being the intensely pink compound 6,6'-dichloroindigo. Similarly, growth of any E . coli will cause the production both of 6,6'-dichloroindigo and of the intensely blue compound 5,5' ,4,4'-dibromodichloroindigo.

At the end of this time, any well which initially contained at least one viable E. coli will be purple (combination of pink and blue) . Any well which initially contained at least one viable non-E. coli coliform will be pink.

Using standard statistical tables, the number of organisms which were present in the original sample can be derived by the most probable number (MPN) method. The number of purple wells gives the E. coli count and the number of purple wells plus the number of pink wells gives the total coliform count.

If desired, the memory device can be used to perform the oxidase test for confirmation of coliforms. As indicated above, a small number of strains of non-coliform bacteria produce jS-galactosidase and thus will produce pink wells. The vast majority of these strains are however oxidase-positive, whereas coliforms are by definition oxidase-negative. The memory device is used to transfer a sample from each of the wells of the original plate onto a pad carrying oxidase reagent. A blue colour on the pad is a positive reaction. The position of the colour on the reagent pad can be related back to the original well; any pink well which also gives a positive oxidase test is a potential false positive. Confirmation of E . coli may be carried out in the wells, by the indole test, by adding any of the variety of reagents commonly used in this test, directly to each well which is positive for E. coli , and noting the appearance of the characteristic colour formed when the reagent contacts indole. Since this test invariably destroys any organisms in the wells, the utility of the memory device in this case lies in the fact that it can be used before the indole test is carried out, to transfer samples to another multi-well plate, agar plate or other receiver. The spatial relationship of the samples to each other is maintained, as described above. The indole is prevented from spreading during growth, by ensuring the isolation of the wells from each other, from the time of initial contact of the wells with the growth medium. The memory device can be used to transfer the pattern of positive wells onto a variety of growth media or storage devices such that a record of all test results can be kept in the form of the microbes themselves with a minimum of extra effort. In this way, the invention provides a significant advantage over many other types of rapid test, in which the sample is either destroyed during the test, or laborious subculturing of each sample is required.

Claims

1. An assay for microorganisms in a sample, which comprises contacting a mixture of the sample and a liquid growth medium with a material that undergoes a detectable change in the presence of the microorganisms to be assayed, wherein the sample is contained in a well whose base is defined by a filter material, and the mixture is formed by placing the well on the growth medium.

2. An assay according to claim 1, which additionally comprises removing the sample and conducting a further assay adapted to distinguish between the microorganisms to be assayed and any other that may have given a false positive.

3. An assay according to either preceding claim, wherein the microorganisms to be assayed are coliform bacteria.

4. An assay according to claims 2 and 3, wherein the other microorganisms contain j3-galactosidase and cytochrome oxidase, e.g. Aeromonas hydrophila .

5. An assay according to any preceding claim, wherein the detectable change comprises the formation of a coloured, insoluble precipitate.

6. An assay according to claim 5, wherein the material that undergoes a detectable change is Salmon-Gal and/or X- Gluc.

7. An assay according to any preceding claim, which additionally comprises removing liquid from the well by suction filtration.

8. An assay according to claim 7, which additionally comprises washing the residue.

9. An assay according to any preceding claim, which is conducted on a plurality of samples contained in respective wells of a multi-well device.