HUT59956A - Apparatus for microbiological tests - Google Patents

Abstract

Apparatus for performing a plurality of microbiological tests comprising a layer of agar or other nutrient gel filling a plurality of channels which communicate with a common region from which the apparatus may be charged with liquid agar prior to use; characterised in that indicia are provided adjacent said channels to permit a direct visual measurement of the effect of the test substance on the growth of a microorganism on said agar, when said test substance has been allowed to diffuse along said channel from a determined point of application to form a concentration gradient of said substance along said channel.

Description

FIELD OF THE INVENTION The present invention relates to an apparatus for carrying out microbiological assays comprising a plurality of channels in a common region receiving liquid agar agar or other nutrient gels, adapted to remove material from the common domain. The apparatus of the present invention is for the serial testing of the susceptibility of microorganisms to one or more antibiotics.

It is well known to test the susceptibility of a given microorganism to a given antibiotic by delaying the growth of microorganisms on agar-agar or other nutrient-coated plates. The essence of the known method is to pour agar agar or other nutrient medium into a suitable container, such as a petri dish, and allow it to solidify, i.e., to coat the inner surface of the container with a continuous layer. Subsequently, the surface of the agar agar is inoculated with the microorganism to be tested and the surface is covered with an antibiotic impregnated paper disk. In the next step, the agar-agar plate is placed in an incubator and the surface of the agar-agar is kept in a horizontal position until zones of growth inhibition of the microorganisms are formed. During the incubation period, the antibiotic will permeate the medium and thus measure a concentration gradient in the medium that is representative of the amount of antibiotic used to impregnate the paper. Delay zones form a range in which the antibiotic is present in sufficient amount to delay the growth of the microorganism. In these ranges, the jelly-like medium remains clean, while the growth or proliferation of microorganisms is indicated by the formation of opaque zones that are observed in the medium and / or surface of the medium.

Conventional assays consist of comparing the size of the zones of antibiotic susceptibility with those of samples of known sensitivity and resistance, and then classifying the microorganisms into sensitive, transient and resistant ranges.

However, if more than one assay is performed on the same agar-agar plate, interference may occur due to the interaction of different assay media. Unless the agar agar plates used are too large, the antibiotics used in the various assays may diffuse from the individual paper disks into the same zones of agar agar, resulting in overlapping delay ranges. Usually, a maximum of six antibiotics can be tested using a standard Petri dish 9 cm in diameter.

A further disadvantage of the described method is that measurable results are essentially obtained only from measurements and comparisons or calculations. This is an additional sequence of actions ♦

In addition to being time consuming, it can be a source of errors and the risk that the operator may come into contact with hazardous pathogens.

Other methods of assaying for susceptibility to antibiotics include measuring the electrical conductivity or resistance of cultures grown in the presence of the drug or drug designated for the assay, but u <> · κ can serve the same purpose for measuring turbidity (turbidity) in cultures. These measurements are then compared with data on pre-classified crops. In this case, however, calculation and comparison are also required, and continuous reading requires expensive equipment.

These procedures may also require that control assays be performed on multiple media containing various concentrations and possibly combinations of antibiotics to be tested. Therefore, the tests are time consuming and subject to many errors.

SUMMARY OF THE INVENTION It is an object of the present invention to provide an apparatus for easily and reliably evaluating the effect of various pharmaceutical compositions, in particular antibiotics, on cultures of microorganisms.

In order to accomplish this object, we have provided a microbiological control apparatus comprising a plurality of channels for transporting material from a common domain with a common region receiving liquid agar or other nutrient gels, and according to the present invention, the agar-agar —Procedures for direct visual monitoring of the effect of the microorganism on agar or other nutrients are provided, and are arranged along the channel according to a gradient of varying concentration of the test substance as determined from the point of application of the test substance.

It is evident from the above structure that the apparatus of the invention can be used for all control and test assays that measure delay or activation zones characterized by growth of the microorganism. The test substance may therefore be either growth-promoting (e.g., vitamin-like) or growth-retarding (e.g., antibiotic). For example, the proportion of the antibiotic in the sample for use in the assays may be determined by comparing the growth retardation induced by a known amount of a preparation of unknown concentration with a standard solution containing a known amount of a given antibiotic. For the same purpose, dehydrated solids from formulations of a given volume and concentration may be used. The growth-promoting effect can be evaluated in a similar way.

In a preferred embodiment of the device according to the invention, the channels are arranged parallel to each other and communicate with at least one end of the common domain. By moving the common domain through both ends of the channels, it is possible to perform a larger number of tests, for example, formulations containing the same antibiotic in two different concentrations can be tested simultaneously, and sensitivity thresholds or breakpoints can accordingly be determined more easily than channel and common domain contact provided. The common area may also be open externally, through which the apparatus may be filled with liquid agar by placing a removable cover on the apparatus and rotating the apparatus itself so that the common domain is at the highest level.

The proposed apparatus may also be implemented in such a way that the channels extend radially, i.e. radially, from the common region.

The test substance (s) may be introduced into the ducts by conventional means. For this purpose, it is known to cover the surface with a paper-impregnated paper disc. However, it is particularly advantageous if the device according to the invention is adapted to receive at least one substrate that holds the test substance in absorbent form, i.e., a substrate impregnated with the at least one test substance formulation. A preferred embodiment of this is where the carrier in the apparatus of the invention is formed as an absorbent strip crossing the channel.

It is also a very preferred embodiment of the apparatus according to the invention wherein each of the channels is formed as a recess opening, wherein the aperture is adapted to receive an absorbent disk or other substrate impregnated with at least one test substance. The recesses are preferably formed in the central portion of the test substance receiving region, and are of non-essential shape and size to accommodate the test disc impregnated carrier disk or other shaped body. Incorporation may be provided by an upper frame, wall, or protrusion, with the substrate being pushed between the bounding surfaces. This operation can be greatly facilitated by a tapered or cross-sectional introduction element. Otherwise, the substrate may be secured with adhesive in the recess.

In this embodiment of the device according to the invention, it is advantageous for the carrier disc to deliver a well-defined amount of the test substance formulation to the test site. The disk itself can be inserted into the recess before the nutrient necessary for the assay is added.

Another very advantageous embodiment of the apparatus according to the invention is the use of packaging capable of maintaining the sterilized state of the apparatus, wherein

Prior to sealing the package, one or more media or discs impregnated with the test substance or standard sample are inserted into the designated slot (s). The hermetically sealed package is formed in a known manner from a plastic foil or multilayer film, which is then sterilized by known means, such as gamma radiation or X-rays, and is preferably kept in a sterile environment until use.

In a further preferred embodiment of the device according to the invention, the recesses are not provided with substrates for receiving the test substance. Instead, separate components are provided which may provide recesses, preferably with a truncated cone or truncated surface, to form a cover or possibly a recess. With these surface elements, test lines containing recesses are created and have an appropriate aperture so that • · · * ·· · · · ·

- 8 nutrients can be delivered quickly to the desired amount. After removal of the lid or the like, residual depressions may contain formulations containing the test substance, for example, using a micropipette or similar device.

It is also advantageous to use a protuberant, such as a comb-shaped auxiliary device for introducing the test substance or the test substance, wherein the distance of the protrusions, optionally comb teeth, is the same as the recesses in the nutrient. Each tooth or protuberance preferably has an inlet or bore inlet through which a certain amount of fluid can be aspirated using capillary forces. The auxiliary device can then be filled with the test substance formulations at standard concentrations, and a given amount of the formulations may be delivered to selected wells.

A very preferred embodiment of the apparatus according to the invention is that the carrier is formed in the form of a strip, it is laid through the channels and the strip is held in position with the elements formed on the surface, such as protrusions or recesses, troughs.

Preferably, the test material is located at the center of the channels, which has the advantage that it cannot diffuse into the common domain while the delay zones can be appropriately large.

The dimensions of the delay or activation zone can be evaluated visually. In this case, • ·

- 9 markings provide reading basis. Preferably, the markings are in the form of a scale fixed on the surface. In this case, the channels are calibrated, for example, the effect of the test antibiotic on the microorganism can be read directly. This is a sharp difference from the known solutions, since after a single calibration, the device according to the invention does not require any measurements, calculations or comparisons.

Another convenient way of implementing the labels is to form the labels in the apparatus of the invention on an absorbent sheet material, such as paper or the like, which absorbs test material or materials.

It is also extremely advantageous to implement the apparatus according to the invention in which a transparent or translucent material is used to form a channel defining body, such as glass or resin, since in this case the test results can be readily determined by screening.

The invention will now be exemplified by way of example only. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Referring to the accompanying drawings. In the drawing it is

Figure 1 is a perspective view of a preferred embodiment of the apparatus of the invention, a

Fig. 2 is an X-X line cross-sectional view of the embodiment shown in Fig. 1, showing a cover member fitted to the apparatus and in place,

Figure 3: The embodiment shown in Figure 1 is along the Y-Y line.

• · · 4 cross-section of channel-shaped opening with part 4, a

Fig. 4 is a perspective view of a further preferred embodiment of the apparatus according to the invention

Figure 5 is a perspective view of a still further preferred embodiment of the apparatus of the invention, a

Figure 6 is a perspective view of another preferred embodiment of the apparatus of the invention, a

Figure 7 is a perspective view of a material transfer aid for the embodiment of the apparatus of the invention shown in Figure 6;

Fig. 8 is a perspective view of the aperture forming element arranged in accordance with the embodiment of the apparatus of the invention shown in Fig. 5,

Figure 9 is a perspective view of a material transfer aid for the embodiment of the apparatus of the invention shown in Figure 5.

The apparatus according to the invention is provided with channels 2 (FIG. 1), preferably parallel to each other, for carrying out microbiological control tests, which communicate with at least one of the common regions 3 and 4. In the central region of the channels 2, recesses 1 are formed, and in the bottom level there are markings applied in a scaled arrangement. The markings 7 may optionally also be placed on the side walls of the partitions 5 between the channels 2. Partitions 5 are closed between channels 2, while at least one of the common regions 3 and 4 is defined by a wall 6 capable of retaining nutrients not shown in the drawing. The apparatus itself may be made of casting glass or synthetic resin.

The apparatus according to the invention is covered by a cover element 8 not shown in FIG. Its inner surface forms a straight plane, which makes it in good contact with the upper level of the channel defining elements 2 and the partition 5. One side of the apparatus is open, here the common region 4 is not covered, whereby, after positioning the apparatus, the liquid agar or other nutrient can be poured into the channels 2 and the common region 3. The lid 8 is preferably formed with narrow troughs which are located at the level of the partition walls 5 so that air can be removed from the interior of the apparatus. Using this version of the apparatus, the areas designated for the test, such as the ducts 2, can be completely filled, reaching up to the upper level of the partitions 5 and the wall 6 without any air inclusions.

Fig. 3 is a cross-sectional view showing the opening 1 in Fig. 1, bounded by a bottom wall 13 extending from below to the bottom level of the channel 2, which forms a recess in the channel 2. It has 9 discs which are held in place by protrusions 10. The 9 discs are made of paper or other porous material. If desired, it is sufficient to use the disk 9 to close the opening 1, and the lower wall 13 is not required.

Fig. 4 shows an embodiment of the device according to the invention in which the wall 6 also closes the common area 4. In this embodiment, the liquid agar is introduced into the common region 3 and the apparatus is then horizontal.

-12 so that agar-agar forms a uniform depth of layer.

In the embodiment shown in Figure 5, the apparatus according to the invention can be filled with various types of initially liquid material which is the medium of the assay and assayed therein. One possibility is to apply the test substances to the agar surface. For this purpose, a strip of paper not shown in the drawing is first impregnated with the composition containing the test substance and then arranged perpendicular to the longitudinal axis of the channel 2 and overlapping the channels. The strip of paper is then held in place by 11 protrusions. The diffusion of test substances through the paper is prevented by separating the test substance-receiving zones with hydrophobic areas, in which case the areas are impregnated with PVC, silicone or other impermeable composition.

In the embodiment of the proposed apparatus, as shown in Figure 5, it is highly desirable to use guide walls 12 which permit the insertion of an impregnated strip of paper of only proper shape and shape.

It will be apparent from the drawing that the apparatus of the present invention can be constructed in a variety of forms and suitable for testing antibiotic formulations for controlling pathogens of various classes of pathogens, e.g. Equipment to accommodate a particular strip of paper ···· ·· ·

· · · ·, _M ♦ ···· «···» · * · · ·

-13 can be suitably designed by assigning different antibiotic strips to different paper strips and dividing the delay zone into several areas to indicate that the microorganism is resistant, sensitive or transient to the particular composition, and to ensure that the minimum delay concentration (MIC).

For a given drug formulation and for a given zone size, the MIC value from regression analysis can be derived from a series of log ₂ MIC curves plotted against the diameter of the delay zone by baseline measurements using standard tubular dissolution techniques. The assay results are determined by equilibrated agar-agar diffusion and utilized in a large number of different bacterial cultures. The number of cultures can be hundreds. The data obtained by such regression analysis are now known for a broad range of antibiotics, since the number of commonly used antibiotics is very high, all of which have already undergone official regulatory testing for the registration of pharmaceuticals. The apparatus of the present invention is useful for determining MIC values based on the diameter of the zones.

A preferred embodiment of the apparatus for carrying out microbiological assays according to the invention is the embodiment shown in Figure 6. In this case, the liquid agar medium carrying the microorganism is charged into and into the interior space

In contact with agar-agar, the comb-like element shown in Figure 8 is arranged. A comb-like element with a truncated pyramid * ο · ··· · · · ··········

The teeth 14 are aligned with the protrusions 11 in the center line of the interior to form openings in the center line of the protrusions 11.

After the agar-agar has solidified, the comb-like element forming the openings is removed with a gentle motion. There is no risk of surface damage to the agar agar during the operation if the protrusions forming the apertures in the comb element are frustoconical or truncated.

Subsequently, each of the apertures formed by the comb-like element is filled with a specific amount of the antibiotic or other test substance to be tested, preferably in a known manner, with a series of fluid samples of varying concentrations. One or more wells are reserved for samples to be tested containing the antibiotic in unknown concentrations.

After this preparation, the apparatus is incubated. With this, delay zones are developed in relation to openings containing material of known concentration and the typical dimensions of the zones are directly determined. Thus, a standard curve can be obtained that plots the relationship between concentration and zone diameter. Subsequently, the characteristic data of the solution of unknown concentration can be determined by interpolation from the standard curve based on the diameter of the resulting zone.

Alternatively, it is also a good idea to read the zone diameter directly for an unknown concentration solution and compare it to the other zones without plotting the nearest one. If we have a series of standard solutions with a finely divided scale * ·

15 steps, such as gentamicin in steps of 2 mg / L, such a comparison provides a good basis for determining the optimal serum concentration required for treatment in most clinical cases.

A simple method for determining the concentration gradient of a series of pharmaceutical formulations is to use a comb-like element formed by teeth 14 with inlet teeth 14 for exerting capillary action. The dimensions of the inlets 14, which represent small diameter openings, are determined taking into account the requirement

I need a well-defined amount of fluid to reach each tooth through capillary action. The teeth of the comb element can be filled from containers with solutions of standard concentration, and the comb element is inserted directly into the apertures formed in the plate, and the contents of the comb teeth are then added to a known amount of solvent in the apertures. Alternatively, under suitable conditions, the contents of the comb element are dehydrated and the comb element is set aside for later use.

Similar methods may be used to test the effects of vitamins such as folic acid. However, in these cases an increase can be observed in different samples in the areas adjacent to the source of the vitamin, the size of which can be compared to the characteristics of the vitamin source.

The teeth of the comb-like element acting on the capillary may be circular, rectangular, or even square in cross-section, but this is essentially irrelevant;

- Always apply the same amount of fluid to the comb teeth for 16 seconds.

An alternative to the capillary comb-like element may be a capillary comb with an absorbent paper but impervious to paper-absorbent materials. In this case, the comb teeth are the ones that carry a certain amount of dehydrated active ingredient and the desired concentration values can be determined by adding the teeth to a fluid of suitable composition, delivered in equal amounts into the openings.

In addition to the embodiment of the device according to the invention having protrusions 11 and guide walls 12 as shown in FIG. 5, it is also very advantageous to omit the protrusions 11 and guide walls 12 as shown in FIG. 6. 7, using the auxiliary device shown in Figure 7. This accessory includes transverse switch strips 14 'printed with scales 15 suitable for measuring the size of the zones. The switch strips 14 ', which are made of waterproof material, rest on the partitions 5.

In the embodiment shown in Figures 5 and 6, the test substance (s) -containing media is contacted with the agar-agar through protrusions of appropriate height in the channels. These protrusions are not visible in the figures, and are formed by the inner surface of a cover over the agar-agar-covered area.

Marking the edge of the test zone f · · ·

- You can be 17. Make the wind line as recognizable as possible

In other words, an indicator is used which, as a solution, floods the surface of the plate and removes the excess. The layer of trapped cells outside the zone can thus be colored, marked, and the delay zone itself intact. This can also be achieved by using an appropriate oxidation and reduction indicator, since the presence of growing (reproducing) bacterial cells causes a reduction in redox potential. It is also advantageous to apply a fluorescent dye to selected areas.

Based on the size of the zone, nomograms can be generated to determine MIC values, which can be printed on the surface of the cover.

The solutions described above are suitable for providing automation of reading if a suitable optical reader, such as a known design, is used for this purpose. The optical system should be capable of measuring opacity or tracking its change, as it allows electronic processing of the signals received, which can determine the sensitivity class, MIC value, dosage amount required for clinical use, and treatment costs for virtually all pharmaceuticals. All this data can also be computed using computer software and then made available to the printer in a form appropriate to the needs of the application. In addition, computer-aided analysis may provide further evidence for established findings, follow epidemiological processes of drug-resistant strains, and a combination of both local and national data for treatment can improve the efficiency of processes.

A further advantage of particular importance for medical and other efficacy studies is the possibility of automatically identifying the studies currently performed in the proposed apparatus. This is made possible by a support plate 16 arranged adjacent to the nutrient-receiving portion of the device according to the invention, on which an automatic reading mark, for example strips, can be placed. Other information referring to the patient and the sample may also be included on the support sheet.

The apparatus shown in Figure 6 can be used for a variety of tests. Of particular importance is that bacteriophage typing of bacteria can be performed. For this purpose, the transverse strips of paper are brought into contact with the surface of the agar in the channels 2, but only at one side of the length of the channel 2, while the other side is used to control normal growth. In this case, each strip of paper is impregnated with dried phage particles, one phage strain being applied per strip. The number of phage particles is selected in a known manner. The phages are then transferred to the inoculated plate, the paper is removed and the plate is incubated to promote the formation of phage plates. The type of bacteriophage can be determined based on the positive and negative results obtained with a series of papers prepared for typing.

In another application, we have developed a system in which the characteristics of the bacteria are their biochemical reactions • · · · · · ·

- 19 can be explored. The substrates to be modified by the microorganisms being tested are fixed by drying in a manner similar to the papers used for phage typing. The substrates are separated by hydrophobic inhibitory elements and the strips of paper carrying the substrate are left in contact with the surface of the grafted agar-agar. As a result of prolonged contact, substrates diffuse into agar-agar-filled channels and act on growing microorganisms. Where possible, the paper strips are supplemented with an indicator that is capable of indicating changes in the microorganisms. For example, the lactose substrate containing the Andrade indicator turns pink during the lactose fermentation process due to the appearance of acidic products. In the assays used to identify individuals of the Enterobacteriaceae strain, the following sub-strains may be used: glucose, lactose, mannitol, sorbitol, inositol, melibose, sucrose, rhamnose, arabinose, amygdalin, arginine, ornithine, lysine, cifrate sodium. Changes in the metabolism that accompany bacterial growth can also be monitored by adding reagents to the test strips after a certain growth period. For example, in these assays, the Kovács reagent is added to detect benzopyrrole production, alpha-naphthol and potassium hydroxide can be used to produce acetoin, and the presence of cytochrome oxidase can be detected with tetramethyl-p-phenylene diamine, Greiss's reagent indicates nitrate reduction and hydrogen peroxide indicates the presence of catalase. Other indicators • · ··

- 20 f '· are well known in the art and their use is well known in the art.

By analyzing the various embodiments of the device according to the invention, it has become possible to select the dimensions of the channels 2 favorably. Preparatory experiments for paper-strip sensitivity assays were performed using standard methods for the determination of zones using standard strains of individual pharmaceuticals and microorganisms. We then determined the conditions required for the formation of zones of the same size on agar-agar strips for a given microorganism and a disc containing a particular pharmaceutical composition. For example, a standard assay using Isosensitest agar (oxoid), a plate containing 30 M Cephradine and Escherichia coli strain NCTC 10418 yielded a zone 18 mm in diameter. Subsequently, using the same set of discs, the diameters of the zones obtained using the same microorganism grafted on the same agar agar were compared with the results obtained using strips of paper arranged at different depths and widths.

By interpolation, the parameters at which the diffusion from the strips in the agar agar results in the same zone size were determined. These are: 7 mm wide at 5.4 mm depth, and 10 mm wide at 4.1 mm depth. If equipment is used that provides zones of equivalent size for agar-agar diffusion processes, the means for transferring into the resistant, transient and sensitive classes will be directly applicable.

- Standards ensuring the implementation of 21 rankings can be used. A similar series of experiments can be performed to determine the parameters of an apparatus suitable for performing the Kirby-Bauer test technique, as established by the National Committee for Standards for Clinical Laboratory Testing, valid for the United States.

Claims (11)

PATENT CLAIMS An apparatus for carrying out microbiological assays, comprising a plurality of channels (2) for transporting material from a common domain, in a common region receiving a liquid agar agar or other nutrient gel, characterized in that the agar is disposed along the channels (2). signals (7) suitable for direct visual observation of the effect of the microorganism on agar or other nutrients are provided and arranged along the channel (2) according to a gradient of concentration of the test substance spreading from a defined point of application of the test substance. Apparatus according to claim 1, characterized in that the channels (2) are arranged parallel to each other and pass through both ends at the common area. Apparatus according to claim 1 or 2, characterized by channels (2) adapted to receive at least one substrate which holds the test substance in absorbed form. Apparatus according to claim 3, characterized in that the carrier is formed as an absorbent strip crossing the channel (2) and the at least one test substance is arranged on the strip. Apparatus according to any one of claims 1 to 3, characterized in that each of the channels (2) has a surface-to-surface configuration. It is formed as an opening (1) forming a recessed trough, wherein the opening (1) is adapted to receive an absorbent disk or other substrate impregnated with at least one material to be tested. Apparatus according to claim 5, characterized in that it is packaged in a sterilized package and is provided with openings (1) for receiving a test substance or standard sample for testing a series of test materials or standard samples. Apparatus according to any one of claims 1 to 6, characterized in that the channels (2) are provided with removable elements forming troughs defining the walls of the opening (1), in which protuberances can be formed by pouring the agar agar or other nutrient gel. . Apparatus according to any one of claims 1 to 7, characterized in that it is provided with an additional unit for the introduction of test substances, in which protrusions (10) corresponding to pre-formed openings (1) in the agar or other nutrient jelly are provided each of the projections (10) being provided with an inlet (14) capable of absorbing a predetermined amount of fluid by capillary force. Apparatus according to any one of claims 1 to 8, characterized in that the markings (7) are in the form of a scale fixed on the surface. Apparatus according to any one of claims 1 to 8, characterized in that the markings (7) are made of test material. Or absorbent sheet-like material. Apparatus according to any one of claims 1 to 10, characterized in that the markings (7) are arranged in a delay zone adapted to the degree of sensitivity and resistance of a given antibiotic.