IL97707A - Devices for carrying out a plurality of tests - Google Patents

Description

DEVICES FOR CARRYING OUT A PLURALITY OF TESTS The present invention relates to a device for carrying out a plurality of assays or tests on a single surface. More particularly the present invention relates to devices enabling the simultaneous performance of a wide range of chemical tests and multiple microbiological assays.

In a first aspect of the present invention there is provided an improved device which can be used as a dry reagent carrier enabling the carrying out of a multiplicity of simultaneous detection tests in an advantageous manner heretofore not achieved with state of art dry reagent chemistry carriers.

As described by Dr. Bert Walter in Analytical Chemistry, Vol. 55, No. 4. April 1983, pp. 498-514, providing a reagent in a dry format for rapid use is by no means new - a familiar example is litmus paper, which dates back to the 19th century. By introducing litmus, a colored extract from several lichens, into a paper matrix, the inventor provided a dry reagent chemistry for testing the akalinity of a solution.

The first major impact dry format chemistries had on clinical testing was the appearance in the 1950s of Ames Clinistix urine reagent strips for testing urinary glucose. By comparing the color developed on the reagent strip with a color chart provided on the product label, the user got a rapid qualitative glucose analysis that otherwise would require a laboratory and skilled personnel. Clinistix reagent strips provided the groundwork for other reagent strips developed by Ames and other manufacturers for testing urine constituents. The appearance of Ames Dextrostix reagent strips in the 1960s for the semiquantitative analysis of blood sugar propagated the development of dry format chemistries for testing blood constituents. With the later introduction of instrumentation, the era of quantitative clinical analysis for glucose with reagent strips began.

In the 1970s, more sophisticated dry chemistries emerged for quantitative analysis of blood constituents. Whereas many of the clinical test formats available share the common trait of requiring reconstitution of reagents prior to use, either manually or automatically, the emerging reagents have a totally new format. In all cases, a complete chemistry is miniaturized into a dispensable dry format. No prior reconstitution of reagent is required, and many manipulations are replaced simply by applying the sample. An analysis is complete in 1-7 min.

The development of dry reagent chemistries is the cumulation of several technologies. These technologies provided crucial knowledge on how to prepare quantitative chemistries in dry media such as thin films, paper matrices and other synthetic porous materials. They also provided knowledge on how to cast thin films of desired porosity with high precision, how to make paper matrices and other porous matrices reproducibly with well-defined characteristics, and how to laminate various materials to each other. The photographic industry has made substantial contributions in advancing the technology of dry reagent chemistries. With the appearance of color photography, the industry had deve Ioped a technology that is based on conducting quantitative Λ chemistries in discrete films arranged in multiple layers. This eventually resulted in instant photography. An instant color print may have as many as 15 layers of film, each with a specific chemical or physical function to perform in developing the photographic image. This very technology was used to develop several dry reagent chemistry formats for clinical testing. The coating and plastic industry provided a variety of techniques for precision casting of thin films, and it has developed lamination techniques frequenty required for bonding various materials together. It also developed a variety of inert materials used in constructing dry reagent chemistries. The paper and fabric industries have developed techniques for making fibrous matrices with reproducible parameters to fit a variety of unique needs. Some of the parameters that can be controlled easily include matrix thickness, fiber density and solvent absorbency, which are important in generating dry reagent chemistries.

Contributions from these industries led to the evolution of dry regent chemistries that were adapted to meet many of the needs in the clinical laboratories.

Dry reagent chemistries have been described for a variety of blood analytes. These include serum metabolites, enzymes, and serum electrolytes as well as therapeutic drugs. Many of these chemistries are available on the market and provide a unique approach to conducting a quantitative analysis of serum analytes. Each dry reagent chemistry provides an integrated ass.ay for a specific analyte that requires only the application of the serum sample.

Thus, e.g., dry reagent chemistries are presently available for nearly all the commonly tested blood metabolites. These include glucose, blood urea nitrogen, uric acid, cholesterol, triglycerides, creatinine, bilirubin, ammonia, and calcium. Analysis of many of these metabolites by conventional means requires several steps that are done, either manually or by automation. The integration of several steps into a single-step analysis is exemplified by the dry reagent chemistries developed for whole-blood glucose analysis.

Two such carriers developed by Boehringer Mannheim Corporation (BMC) and Ames Division, Miles Laboratories, Inc., are described hereinafter to illustrate the present state of the art. Glucose is detected by a glucose oxidase-peroxidase procedure. In both cases, approximately 50 L of whole blood is applied to the surface of the carrier (approximately 0.5 cm x 1 cm), where plasma containing glucose is separated from red blood cells by virtue of the selective permeability of the carrier matrix. After an allotted reaction time 97707/2 -6- (usually 1-3 min.) the red blood cells are removed by washing or wiping, and the color developed is analyzed and translated to blood glucose concentrations.

As with serum metabolites, sophisticated dry reagent chemistries also are available for the analysis of serum enzymes. Dry reagent chemistries have been described for the analysis of creatin kinase, lactate dehydrogenase, aspartate ine transaminase, - o amylase, and alkaline phosphatase. Some of these carriers are available on the market.

In accordance with the present invention there is now provided a solid support for bacterial culture having an absorbent testing surface testing, comprising: a plurality of essentially non- diffusing lines of an essentially water-insoluble antibacterial composition inhibitory to bacteria, subdividing said surface into a multiplicity of individual adjacent test areas, said antibacterial composition serving as an effective barrier for preventing interference between adjacent test areas; wherein said testing surface includes a bacterial growth medium or said surface is supported on a solidifying bacterial growth medium.

Said array of lines can take many forms depending on the nature of the test or assay to be carried out and the desired shape of the test area.

Thus said array can be a plurality of intersecting lines simply subdividing said surface into a multiplicity of test areas, e.g. a simple pair of crossed †ines subdividing the surface into four distinct areas or an entire grid-like array forming nine or twenty-five distinct test areas.

Alternatively, the array of lines can be formed by drawing a single switchback meandering line forming a plurality of closed end channels, the open ends of adjacent channels facing in opposite directions as described hereinafter with reference to the figure and example 2 .

In a preferred embodiment of the present invention at least one of said test areas is provided with at least one soluble reagent deposited thereon.

In especially preferred embodiments at least several of said test areas are individually provided with at least one soluble reagent deposited therein.

The most common applications of this aspect of the invention are for: a) Multiple chemical or biochemical tests applied to a single specimen. b) Simultaneous testing of multiple specimens, and c) Simultaneous initiation of sequential reactions.

The most obvious application of the present device is the replacement of a multiple-area dipstick such as is used in testing of urine. In this embodiment the advantage is in offering a more rapid and sensitive device than a conventional one. In a conventional multiple area device the individual test areas are physically separated and recombined in a single device by attachment onto a stick. In the present device the sole physical element is the absorbent itself (e.g. a filter paper strip) which is totally immersed in the specimen, e.g. urine; hence no extraneous material such as the plastic surface or the glue used in assembling conventional dipsticks, will interfere with the instant uptake of the liquid tested and with the visual clarity of the consequent color reaction.

Furthermore, for some applications the present design is unique. Thus, it allows for the direct testing of small volume specimens which must not be diluted. For example, a single drop of serum or plasma is directly tested, and simultaneously provides the appropriate control, if centered on the line separating a test area from the relevant control area. By diffusing into the two adjacent areas the single specimen provides the result of the test and the direct control for comparison. For multiple simultaneous tests, and controls, the specimen is applied to the point of intersection of the multiple test and control areas. Radial diffusion into the adjacent areas ensures the necessary subdivision for multiple testing and controls, all obtained by the single step of placing a single drop of the specimen at the desired point of intersection.

If desired, the sample can be diluted and subdivided in one step, namely by applying the required volume of the diluent to the specimen placed as described here nbefore.

A similar procedure can be applied to more complex specimens such as a drop of blood. The presence of red blood cells may, however, interfere with the appearance of a color change. This can be avoided by using two identically printed sheets instead of one. One is used for placing the specimen and is termed "sample sheet". The other is impregnated with the test reagents and with the indicator, and is termed "indicator sheet". The samples are applied to the sample sheet which is then placed on a freshly wetted indicator sheet. The printed grids of the two sheets which are identical, are superimposed. While horizontal diffusion is confined by the ink, there is no barrier to the vertical diffusion between the superimposed areas of the sample sheet and the indicator sheet. Thus, while the insoluble components of the complex specimen (e.g. the erythrocytes in the above example) remained trapped in the sample sheet, solutes diffuse and interact, and the reaction is clearly reflected in the color change of the corresponding test area in the indicator sheet.

The advantage of simultaneous testing is in saving time, labor and materials. Beyond that, a strictly simultaneous initiation of a test reaction run with different samples ma be essential for comparative testing, e.g. in screening for the best producer of say, enzyme x, by comparing levels of x in culture supernatants.

The simultaneous initiation is effected by superimposing the sample sheet on a freshly wetted indicator sheet impregnated with the test reagent solution. The reagents, which in the example cited will comprise the substrate of enzyme x, will diffuse simultaneously into all the compartments in the sample sheet, in the indicator sheet or in both.

As is known, a test may require a controlled sequence of steps comprising the orderly addition of reactants at intervals. The present device simplifies the design of such tests as described hereinafter.

An absorbent sheet is printed as before, except for "gates" i.e. tiny openings in the printed grid, left in the horizontal partitions separating adjacent test areas. All vertical partitions are intact, so that no horizontal diffusion will be posssible. Thus the sheet becomes an array of isolated columns, each consisting of a series of potentially interconnecting test areas. The test areas are impregnated with reagents in the order, from bottom to top, in which the reagents are to be added. ' The bottom segment of each column is open (not - n - printed) thus allowing uptake of the test solution into which it is dipped. The solution diffuses into the first test area and continues into the next above it by passing the gate. The width of the gate determines the rate of migration. The time elapsed before the specimen is exposed to the next reagent can be more precisely controlled if the gate is sealed with an appropriate, slowly soluble, substance.

Alternatively, the rate of diffusion may be controlled by the extent of impregnation with the insoluble ink. Partial impregnation will allow eventual diffusion. The delay thus created will allow time for a slow reaction step.

Another especially preferred aspect of the present invention relates to an improved device for carrying out microbiological assays and related procedures utilizing solid growth media.

Solid growth media for cultivating bacteria have been used in diagnostics for over 100 years. The various applications included identification of causative infectious agents, based on the appearance of discrete colonies on differential and selective media, containing, where applicable, reagents and color indicators facilitating biochemical characterization. Similarly, the susceptibil ty of an infecting agent to a variety of antibacterial agents could be assessed.

A reversal of the procedure, namely the use of a bacterial strain susceptible to any such agent enables the detection of the presence of that agent in an unknown sample. Several tests based on this principle are currently used to detect antibacterials in milk, food or in other substances of interest.

Similarly, appropriate bacterial strains and media can be applied to the detection of growth promoting agents or of agents otherwise affecting the growth or the biosynthetic, metabolic or enzymatic activities of the bacterial test strain.

The classical format of such "solid-phase" microbiological or bacteriological assays is the Petri dish containing agar based growth medium. Agar-agar, which is the most commonly used solidifying agent can be replaced with silica or gelatin or other gelling substances. Alternatively, absorbent materials, such as bibulous cellulose in the form of filter paper or cardboard or any other absorbent can be cut as desired and impregnated with liquid growth medium. The solidified, as well as the impregnating, growth medium may take the shape of the Petri dish or any other shape required, to provide the. desired solid base for the assay.

The main advantages of solid base bacteriological assays are simplicity and economy. The economy is further increased by increasing the number of tests per area of the test plate. A single Petri dish can be subdivided to provide for more than one test. However, interference between adjacent test areas is often observed if more than 4-6 samples are applied to one dish. This is a major limitation to the use of the classical format in large-scale screening.

A partial solution has been provided by redesigning the format so as to accommodate more samples per area unit. For example, the Petri dish can be replaced with a tray subdivided to resemble a diminutive ice-cube tray. The resulting compartments are physically separated and each can accommodate one sample. This format is used by Gist-Brocades in their screening test (Delvotest) for antibiotics in milk.

The primary object of the present invention is to allow a similar or greater increase in density of testing areas without requiring any structural barriers. Thus, according to the present invention, no replacement of the standard Petri dish is needed and no new design of any equipment is necessary. In fact, the present invention can be applied to any form or shape of a solid assay and is not subject to limitations inherent to the design or modification of bacteriological culture ware, disposable or reusable.

The principle of the present invention is based on the provision of chemical, rather than structural, barriers for creating multiple test areas. 97707/2 -14- While many types of antibacterial compositions can be used in the present invention, a very simple and readiy available source are the aniline dyes such as Brilliant Green, which can be incorporated into an ink of a pen or dry marker.

In a preferred embodiment according to this aspect of the invention said lines are drawn on a sheet of bibulous cellulose which is then impregnated with liquid bacterial culture. Preferably in this embodiment said testing surface is supported on a solidifying growth medium such as one containing agar-agar.

A further application of the present method is in improving blotting techniques which are widely used in molecular biology, immunology and protein chemistry. A common problem in using e.g. nitrocellulose as a carrier or a blotting surface is the diffusion and eventual cross-contamination of samples. The need for cutting separate channels or creating such channels by mechanical devices can be eliminated by the simple device of imprinting the required pathways into the blotting surface.

The invention will now be described in connection with certain preferred embodiments in the following examples and with reference to the following illustrative figure so that aspects thereof may be more fully understood and appreciated. It is not intended, however, to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as defined by the appended claims. Thus, the following examples which include preferred embodiments will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of formulation procedures as well as of the principles and conceptual aspects of the invention.

Example 1 - An instant multiple test for urine A Whatman No. 1 filter paper pad (60mm x 30mm) is imprinted with a "Stabilo" permanent marker at one of its corners with 3 equidistant lines, each extending from 4mm to 16mm from that corner. The 4 test areas thus created are impregnated, respectively, with (1) a pH indicator, (2) Griess reagent, (3) glucose 97707/2 - 16 - oxidase-peroxidase-c-tcl idine and (4) benzidine reagent.

The ncn-imprinted area of the pad serves as a handle for the user who wets the edge of the imprinted corner with urine. The urine diffuses into the 4 test areas, and the consequent color reactions provide information on the pH, on the presence of nitrites, of glucose and of hemoglobin in the single drop of the specimen tested.

Example 2 - Testing for the presence and properties of β -lactamase in mastitic milk A sheet of Whatman No. 3 filter paper pad is impregnated with a starch-iodine mixture and, after drying is cut into a rectangular strip 2, as shown in the attached figure, having provided at an end 3 thereof two oppositely extended triangular extensions 4, 6.

Using a permanent marker (Pelikan) a meandering switchback line .8 is drawn, which line is water insoluble and which has antibacterial properties due to the aniline dye therein. As shown said switchback line 8 is drawn to define two pairs of closed end channels 10, 12 and 14, 16 extending substantially parallel to each other, the open end of each pair facing in opposite directions and being open to fluid diffusion of solutions deposited on extensions 4, 6 respectively, in the direction of the arrows indicated thereon. 97707/2 - 17 - Spots of benzylpenicillin 18, 20 are deposited at the mouths of channels 10 and 14 and spots of cloxacillin 22, 24 are deposited at the mouths of channels 12 and 16, respectively.

These ^-lactams are potential substrates of β-lactamase. The products of B lactamase reaction will remove iodine from the dark starch-iodine complex and thus cause decolorization of the test area.

The test is then carried out by dipping extension 4 in clean control milk and dipping extension 6 in the specimen milk to be tested. Alternatively, one or more drops of each type of milk could be dripped onto the respective extension whereafter both samples diffuse into the respective channels 10, 12, 14, 16.

If the specimen contains g-lactamase, its activity against benzylpenicillin and cloxacillin is assessed directly by observing decolorization of the respective test areas as compared to adjacent control areas.

Example 3 - Separation of immunoglobulins from a drop of blood A strip of Whatman No. 41 filter paper (60mm x 8mm) is imprinted (marker as in Example 1) with one, interrupted line drawn across 10mm from the edge. The line is broken in the center, creating a 1mm gap.

The area above the gap is impregnated with a suspension of staphylococcal cells of the Cowan I strain. These cells are rich in Protein A, a potent immunoglobulin receptor.

The procedure is started by dipping the edge of the strip in a drop of blood. The edge of the strip is then dipped in physiological saline. The soluble components diffuse up the strip whereas the cellular components are left behind. The soluble fractions reach the imprint and are now forced to diffuse through the gap. The immunoglobulin molecules are captured by the Protein A of the cells immobilized above the gap, whereas all other soluble elements are allowed to diffuse away. Thus the immunoglobulins are separated and concentrated in an area predetermined by the size of the capture spot. The specificity of the antibodies present in the sample can then be easly determined by conventional procedures.

Example 4 - Multiple tests of immunoglobulins in a single drop of blood The test element is a strip similar to that in Example 3. However, the horizontal line is broken in 3 places, so that 3 gaps are formed corresponding to 3 test areas separated by vertical lines, imprinted with insoluble ink, under the horizontal line. The Protein A carrying cells are as in Example 3, but are now deposited above each of the 3 gaps.

The application is as before, but the subdivision causes the specimen to subdivide while taken up by the capillary action of the filter paper. Hence 3 discrete samples are available for testing the binding of threee different antigens, all as a result of dipping the strip in a single drop of blood followed by saline, with no other operation whatosever.

Example 5 A permanent marker, containing Brilliant Green as a component of the ink, is used for drawing a grid (10mm x 10mm squares) on a sheet of filter paper. The sheet is cut into 60mm x 60mm pads. Thus each pad will conveniently fit into a Petri dish. The pads are sterilized, e.g., by dry heat or in an autoclave, and then placed on the surface of a freshly seeded agar plate. The ink prevents growth along the lines of the grid since it contains Brilliant Green, a powerful antibacterial dye. Furthermore, the ink provides a barrier to the diffusion of water soluble solutes across the grid. Thus each little square in the grid will provide a distinct and separate test area on the agar.

While the Petri dish is the commonly available vessel, any other form of solid culture with a conveniently accessible surface can be used. Similarly, while dyes inhibitory to bacteria are convenient to use, the invention is not restricted to colored substances. Obviously a dye can be added to a colorless inhibitor, if desired, to facilitate preparation of grid-pads. The choice of inhibitor will depend on the sensitivity of the bacterial test strain. A combination of non-diffusing inhibitors may be preferred in some tests.

It will be realized that for many purposes a plate with a 25-square grid will be equivalent to 25 plates, providing, e.g. discrete areas for testing the sterility of 25 different specimens. Similarly it will allow rapid estimates of bacterial counts by the conventional methods of varying sample sizes, but with economy that may be important in screening, e.g., for bacteriuria.

Example 6 The procedure of Example 5 is repeated, however, the plate is not seeded, and the grid-pad is impregnated with the desired bacterial culture. The pad is then placed on the unseeded plate or on any other, solid medium, gelled or impregnated. For short term incubations . the medium in the grid-pad will suffice, in which case the plate will only be required to provide protection against contamination and against drying out.

Example 7 The grid-pad suggests a new approach to the sensitivity testing which is a major routine in all hospital laboratories as described hereinafter.

Instead of 6 antibiotic discs, a plain grid-strip consisting of 6 test-areas and 6 grid-strips impregnated with the 6 antibiotics, are provided. All strips contain a colour indicator of metabolic activity. The strips are placed side-by-side and 6 cultures to be tested are streaked across. Thus one plate serves to replace six plates.

A distinct change in color in the antibiotic-free area appears within 3-4 hrs. in most strains. The change thus provides a reference for assessing the sensitivity. profile of the corresponding strain.

The savings in steps, labor, plates and time combine to provide a superior sensitivity testing method.

For large-scale screening the format can be further simplified. Thus, each plate, or a tray of the appropriate dimension, may include a single compound of interest, e. g. , an antibiotic or a sugar, in the medium or in the grid, so that dozens of specimens can be simultaneously tested.

Similar kits can be constructed for testing biochemical traits essential for the identification of a microorganism. This can be illustrated by the example of carbohydrate fermentation profile as a diagnostic aid in identification. It is easy to see that grid-strips can be impregnated with a series of sugars and a pH indicator to provide a kit otherwise identical to that described above. With a heavy inoculum the test can be very rapid.

For very rapid tests, including the example above, the medium in the grid will suffice and the protection afforded by the vessel will not be needed. This will allow wide departures from standard formats, e.g. use of sheets in plastic files.

A solid growth medium, e.g. nutrient agar, can absorb ink and hence can be imprinted with a grid. For practical reasons, the imprinting may preferably involve a template, e.g. a plain paper disc cut to fit a Petri dish and imprinted with the desired grid. The disc is placed at the bottom of a Petri dish into which the agar is subsequently poured. After gelling and having absorbed the print, the molded agar can be easily lifted. Both faces of the agar can thus be used. This format is uniquely suited for maintaining complete physical separation between, e.g. bacteria seeded on one face and reagents applied to the other face. Similarly, two different grids can be applied to the two faces, since the bacteria may be incorporated in the agar. Thus a variety of superimposed effects can be directly observed.

Example 8 - Combined channels for drug sensitivity testing A permanent marker, containing Brilliant Green as a component of the ink, is used for drawing 10 mm wide parallel channels on a sheet of filter paper. Such channels are insulated against cross-contamination by solute or by bacteria (See Example 5). Thus each channel provides a distinct and separate test area when the sheet is cut into convenient size (e.g. 80 mm X 60 mm) pads.

Furthermore, when 2 (or more) "channeled pads" are aligned and brought in wet contact, the combined test areas thus created will allow diffusion between the corresponding super imposed channels, without risk of contamination of adjacent channels.

The unique advantages of such combined test areas are illustrated by the following application. A specimen of urine is tested for the presence of viable bacteria and for their susceptibility to 6 selected antibiotics. The bacteria are collected by centrifugation or filtration, and suspended in 0.3 ml of unbuffered nutrient broth containing glucose and a pH indicator (BIG). The suspension is placed in a trough from which it is soaked up by one of the channeled pads. By this single operation the bacterial suspension is equally subdivided among the 8 channels, with the bacteria concentrated at the bottom of each channel. Furthermore, this "culture pad" will support the growth of such bacteria except where inhibited. The culture pad is brought in contact with an "antibiotic pad", impregnated with 6 antibiotics, one per channel, and providing a "growth control" channel which is not impregnated and an "inhibition control" channel impregnated with a disinfectant. A third pad, soaked in BIG provides a reserve of medium and moisture for more prolonged incubations. In most cases, results can be read within 2 hours (at 37°C), namely, when a distinct change in color is noted in the "growth control" channel" while the "inhibition control" remains unchanged. The effect of other antibiotics is then assessed by visual comparison with the controls.

In a preferred embodiment the 3 pads are prealigned and stapled together between transparent plastic covers to form a "booklet" which is stored in a pad and of "medium pad" is carried out from the respective troughs and the booklet is replaced in the envelope which is then sealed and incubated. In this embodiment handling is minimal, no incubator is needed (a vest pocket has been successfully used) and results are read at a glance.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (12)

97707/2 - 26 - WHAT IS CLAIMED IS:

1. A solid support for bacterial culture having an absorbent testing surface for solid phase bacteriological testing, comprising: a plurality of essentially non-diffusing lines of an essentially water-insoluble antibacterial composition inhibitory to bacteria, subdividing said surface into a multiplicity of individual adjacent test areas, said antibacterial composition serving as an effective barrier for preventing interference between adjacent test areas; wherein said testing surface includes a bacterial growth medium or said surface is supported on a solidifying bacterial growth medium.

2. A solid support for bacterial culture according to claim 1, wherein at least one of said test areas is provided with at least one soluble reagent deposited thereon.

3. A solid support for bacterial culture according to claim 1, wherein at least several of said test areas are individually provided with at least one soluble reagent deposited therein.

4. A solid support for bacterial culture according to claim 1, wherein an opening is provided in at least one of said lines for enabling controlled difusion from a first test area through said opening into a second test area.

5. A solid support for bacterial culture according to claim 1, wherein said first and second test areas are respectively provided with a first and second reagent for effecting sequential reactions. - 27 - 97707/3

6. A solid support for bacterial culture according to claim 1, wherein said antibacterial composition comprises an aniline dye.

7. A solid support for bacterial culture according to claim 1, wherein said antibacterial composition comprises brilliant green.

8. A solid support for bacterial culture according to claim 7, wherein said composition is an ink.

9. A solid support for bacterial culture according to claim 1, wherein said lines are drawn on a sheet of bibulous celulose, which is then impregnated with bacterial culture.

10. A solid support for bacterial culture according to claim 11, wherein said growth medium contains agar-agar.

11. A solid support for bacterial culture according to claim 1, wherein said testing surface is supported on an absorbent support material.

12. A solid support for bacterial culture according to claim 1, wherein said surface is nitrocellulose. For the Applicant WOLFF, BRE6MAN AND GOLLER by: Jb.JuJk