GB2035371A - Petri dish - Google Patents

Abstract

A device for enabling assessment of the activity of biochemical and other compounds in relation to microorganisms or to culture microorganisms for isolation, detection and presumptive identification purposes, comprises a petri dish or other container and numerous discrete gels impregnated with with various compounds which are desirable for particular applications, e.g. for assessments of antibiotic sensitivity or nutritional requirements. In use, the device is usually inoculated by flooding with a fluid containing pure or mixed culture of the microorganisms and incubated at 37 DEG C overnight. Results are observed by a naked eye within 24 hours of the incubation.

Description

SPECIFICATION A device for isolation, differentiation & presumptive identification of microorganisms andlor assessment of the activity of biochemical compounds and other agents in relation to microorganisms I do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which is to be performed to be particularly described in an by the following statement: The invention relates to a device for isolation, differentiation & presumptive identification of microogranisms and/or assessment of the activity of biochemical compounds and other agents in relation to microorganisms. The device is particularly suitable for applications in the field of clinical bacteriology, food microbiology and fermentation technology, epidemiological surveillance and field studies of clinical importance.The device permits a physician to carry out directly in his general practice tests for detection & presumptive identification of microrganisms and concurrent assessment of their susceptibilits to antimicrobic (e.g. antibiotic) treatments by simple means overnight; thus the appropriate treatment can start three days earlier than usually in most common cases.

Effective detection of pathogenic bacteria in clinical specimens requires culturing as soon as possible after coliection. The device described herein is designed to add to the efficiency of culturing and to enable the physician to select instantly a suitable antimicrobic agent if an infection is detected. Taking a urinary tract infection as an example, the current system of processing a patient is as follows: 1 st day : patient visits his physician; 2 nd day : a specimen of urine is delivered to a clinical laboratory; 3 rd day: primary plates are evaluated, infection identified; 4 th day: results of antibiotic susceptibility tests are obtained and posted: 5th day: the physician receives the results and the patients attends to start his treatment.Using the device directly in the general practice, the physician should obtain results within 24 hours of the patient's visit. Thus three days are saved and it is due:to:elimination of transport time; instant analyses for infection and concurrently for "sensitivity", on the spot.

There are already several culture systems which permit immediated culturing of a specimen in the physician's practice, e.g. dip slides, however, these aids aim to deteet an infection and are not designed to provide comprehensive data regarding the antimicrobic treatment.

The conventional method of determining microbial sensitivity to an anti-microbic agent is a diffusion test, i.e. Kirby - Bauer method using paper discs impregnated with antibiotics. These discs are placed on the surface of a solid medium, say, in a petri dish and the plate is incubated. During the period of incubation the antibiotic impregnated in the disc diffuses out into the medium, its concentration being greater at the perimeter of the disc and decreasing with the increase in distance from the edge of the disc.

An alternative is a "dilution test" whereas the concentration of antibiotic is kept constant within within a liquid or solid medium, i.e. the antibiotic is incorporated homogenously in to the medium, either in a test tube or in a petri dish. This alternative is to be prefferred because it enables to establish quantitative categorisation of the susceptibility, however, it is more laborous and less convenient to perform. The liquid medium is used when one sample only is inoculated in the test tubes; the solid medium is used when several samples are inoculated on the surface of one plate. Both media are in use, however, the method is not suitable for a general practice. Also busy clinical laboratories still prefer the disc diffusion method unless they can afford rather expensive instrumentation techniques.

The invention described herein is designed to select a suitable antimicrobic agent by culturing in a general practice or clinical laboratory under the field conditions, conveniently and at a low cost.

According to the present invention a device for isolation & presumptive identification of microorganisms and concurrent assessment of the activity of biochemical compounds and other agents in relation to microorganisms, comprises of multiple discrete gels in one container whereas the gels are individually impregnated with various antibiotics, drugs, microbial substrates and/or reagents and other agents using concentrations individually desired for each particular gel.

The drawing accompanying this specification shows a typical device according to the invention, (Figures 1 and 2 in a base and a lid) The said drawing shows the multiple gels presented in a petri dish having a diameter of approximately 10 cm. The whole system is sterile; the individual gels are arranged in such a pattern as to enable an easy comparision of the results to be read on each particular gel.

The gel in the center (Figure lb) of the dish provides a universal growth supporting substrate which can be used for counts of all viable organisms and for transport of the microrganisms to another laboratory if necessary; five other gels (Figure la) located on the perimeter of the dish provide media necessary to grow, detect and differentiate the microorganisms into groups. The presentation and grouping of these various gels offers a great advantage in comparision with the usual dip slides, in particular it offers larger surface area, say, 3 times larger nad more substrates, i.e. six instead of two.

This enables substantially better differentiation of microorganisms and the device also possesses the ability to grow the delicate pathogens while that is lacked by the dip slides for a variety of reasons. The numerous blobs which are interspaced (Figure ic and Figure 2b) carry various antibiotics and/or reagnets in addition to particular microbial substrates. Each blob is impregnated at a particular desired concentration of the particular antibiotic or drug etc.

In the use, the procedure for handling the device does not differ significantly from usual techniques.

The device is inoculated simply by flooding the with specimen(a portion of it can be conveniently diluted if required) and the excess of the fluid is momentarily drained off; the device is then incubated preferably in an inverted position. Results are recorded after 16-24 hours of the incubation at 35 - 37 "C.

It is noteworthy that all gels in the dish are inoculated simultaneously. If the antibiotic incorporated in a particular blob can suppress the microbs, the surface of the blob will be free of bacterial growth microbial. If the antibiotic cannot suppress the microorganisms which have been ino culated, then the microorganisms will grow and be visible either as discrete colonies or as a confluent growth on the surface. The effective concentration of each antibiotic in the blob can be readily verified and determined experimentally.

The blobs have many advantages, particularly when compared with paper discs methods. The required concentration of each antibiotic is constant, i.e. there is no concentration gradient experienced with the disc diffusion technique. The blobs are detached and therefore mutual cross contamination by the incorporated ingredients or by the microbial metabolites is minimised.

An important advantage of devices according to the invention is the possibility to accommodate a large number of the blobs in one container, in addition to the gels applied to differentiate the microorganisms. This is unlike the situation on a petri dish implanted with individual discs. There the number of these discs is limited usually to 4-6 - 8 in order to avoid their mutual interference because the zones of diffused antibiotics can be relatively large; the blobs can, on the other hand, provide 25 - 50 discrete areas per one petri dish, with no significant interference even if the concentrations of the antibiotics applied are relatively high.

The possibility to apply, ie.e. to incorporate high concentrations of antibiotics per a blob is very important advantage, particularly when synergistic activities of antibiotics are to be determined. By comparing the growth response when two n antibiotics are used separately and in a mixture, it can be assessed if the effect of these antibiotics on the microorganisms in question is, in vitro, synergistic, additive, indifferent or antagonisitc. Another application of an antibiotic at realtively high concentration may be desired in consideration of inoculum variations, i.e. variations of microbial counts in the inoculating fluid, or if high initial levels of antibiotics acting only as antibiotic transport in circulated blood are to be assessed.Another advantage, from a methodology point of view, is that the antibiotic contnet is uniform throughout a blob and any antibiotic can be impregnated at a concentration which is near to the mean antibiotic serum level - in the middle of the application interval; this can contribute to quantitative categorisation of suscepti bility.

Thus the device enables to obtain data which are nearer to the exactly quantifiable agar dilution test than to the routine agar diffusion test, in so far as the methodology is concerned, whereas the device in itself offers simplicity and convenience which is superior to both - diffusion or dilution methods.

The device is time saving - there is no need to place paper discs or similar device onto a petri dish because the blobs are laready there impregnated, sterile and ready to be used. It is labour saving, since the labour required to place the discs and to handle and store disc containers is eliminated. The chances of contaminating the sample to be analysed is reduced because no discs are to be inserted in the petri dish and thus the cover of the petri dish needs not to be opened for that particular purpose. Many variables and potential problems found with the Kirby - Bauer method are eliminated, for example the difficulties associated with the inhibition zones reading and interpretation ofthe results.

When the device is compared with with the agar dilution technique which utilises a multiple inoculation by replica plating, it is obvious that the device is more suitable for individual samples, i.e. for multiple analyses of individual specimens as they are collected and these analyses can start instantly. There is no need for centralised supplies of samples or sophisticated and expensive instruments.

An important factor to be considerde is the possibility of mixed infections. Standard antimicrobit tests require pure culture, i.e. discrete colonies grown up on purity plates. Applying the device one has no way of knowing whether or not the specimen contins for example a mixed infection comprising of a non lactose fermenter and coliforms. This limitation is recognised and the blobs are impregnated with two different microbial substrates, at least. This helps to distinguish effects of various antibiotics upon mixed cultures and if necessary a confirmation test carried out using a pure culture can follow forthwith.

The gels utilised according to the present invention are comprising of a gelling compound or compounds and/or cmpounds which form a gel when mixed with other ingredient. Example of such components are agar, agarose, carrageenan, various gums, gelatin, xanthan gum in a mixture with locust bean gum and many others.

The gels are impregnated with the preparations of the compound or compounds to be tested or utilised, for example by antibiotics, drugs, sugars, amino-acids, enzymes, w vitamins, antibodies, reagents, microbial substrates, blood diagnostic markers and/or other materials, by mixing the particular compound with the gelling component or components and dispensing the mixtures into the container.

The dispensing of the mixtures into a container can be performed using various instruments, for example pipets, rods, tubings, needles etc in such a manner as to obtain multiple discrete gel areas when gelling took place. The device should be sterile and the production is therefore carried out using aseptic methods; for example the components are sterilised where desirable.

The produced device is packed as required for a particular purpose; for example air and moisture proof packaging systems are applied, the container is sealed by adhesives or wrapped or placed in a plastic bag or tighten up, say, by a rubber band etc.

Prior to the packing, the airspace of the device is filled with sterilised air or with other gases, for example with nitrogen, or oxygen or or other vapourised compounds (H20) which have desirable effects upon the stability and performance of the device. This filling can be done by exposure of the device momentarily to a particular atmosphere or vacuum, with a lid off. When anaerobic incubation is desired in conjunction with aerobic one, the device is fitted with a gel attached to the inner side of the container's lid (Figure2a). Following the inoculation of the other gels, this gel is release using flamed or sterilised knife or pinset, transferred on the surface of the central gel which provides the universal substrate and gently pressed to join, i.e. to act as an overlay.

When the device is desired particularly for for antibiotic & chemotherapeutic tests, the design is appropriately modified. For example the blobs can be located also on the inner side of the lid.

The device can be utilised in determining the nutritional requirements of various microorganisms and mutunts. The composition of the gels and their presentation is then modified accordingly.

WHAT WE CLAIM IS: Summary ofthe claims Certain technical features of the invention are part of the prior art, e.g. application of agar and agarose as gelling compounds (Claim 2); impregnation of agar with antibiotics or substrates

Claims (10)

1. (Claim 3); use of a petri dish to culture microorganisms (Claim 4); culturing of microorganisms (Claim 10); packing of poured plates with a plastic film (Claim 7); it is, however, desired to protect such features in combination with the improvements which are characterised by:innovative introduction of numerous impregnated gels in one container, (Claim 1) wherein the gels individually and jointly form a multiple analytical system; formation of multiple discrete gels; (Claim 5) simulateneous inoculation and incubation of the multiple gels; (Claim 10) application of air/moisture proof packaging; (Claim 7) application of gases/vapours; (Claim 8) utilisation of Xanthan gum and Locust beam gum as a gell supporting directly or indirectly microorganisms; (Claim 2) CLAIMS 1.A device for assessment of the activity of biochemical and other compounds in relation to microorganisms and/or for culturing the microorganisms for isolation, detection, presumptive identification and other purposes; whereas the device comprises of a petri dish or other suitable container and numerous gels impregnated with various compounds.
2. 2. A device according to the preceding Claim wherein the gels are comprising of a gelling compound or compounds e.g. agar, agarosa, carrageenan, gums, gelatin and/or compounds forming a gell when mixed, e.g. xanthan gum and locust bean gum.
3. 3. A device according to any of the preceding Claims wherein the gels are impregnated with the preparation or preparations of the materials to be assessed, e.g. antibiotics, drugs, sugars, enzymes, antibodies etc and/or with other desirable compounds e.g. diagnostics, blood, substrates, reagents etc.
4. 4. A device according to any of the preceding Claims wherein the container is a petri dish or a dip slide container or a tray in a bottle or box or any other device or apparatus which would provide a support for the gels and form a closed system.
5. 5. A device according to any of the preceding Claims wherein the gels form multiple discrete areas, in the container.
6. 6. A process of producing the devices in accordance with any of the preceding Claims where the gelling compounds are mixed with the impregnating materials and the mixes individually dispensed into the container to form there multiple gels discrete. or otherwise.
7. 7. A device according to any of the preceding claims wherein the device is packed using air/ moisture proofed systems, e.g. sealed, enclosed in suitable plastic bags, wrapped, tighten up, screwed up etc.
8. 8. A device according to any of the preceding Claims wherein the usual earth atmosphere is altered, for example by application of gases vapours, e.g. nitrogen, oxygen, beta propiolacton, 03, H20, inert gases, SO2 Cr2 and others.
9. 9. A device according to any of the preceding Claims wherein the impregnated gels are distributed in the container to form a multiple system which permits to perform concurrently numerous tests using pure or mixed microbial culture.
10. 10. A method of utilising a device claimed under any of the preceding Claims, comprising of inoculation of the multiple gels with a sample, culturing the device and observing the results by the growth or otherwise in the individual gel areas.