Background of the Invention Dipslides or paddles are microbial culture devices which contain agar medium. They are usually dipped into liquid (usually urine) or slurries so that the agar medium comes in direct contact with the liquid or slurry and is seeded with microorganisms. Such dipslides are also useful for other clinical and environmental uses, such as the testing of food products. The term "dipslide", as used herein, should be interpreted to include all kind of surfaces containing microorganism-growth solid or semi-solid media, which are intended for use in growing microorganisms originating from various samples, and this term is by no means intended to be limited to any particular kind of such growth means.

One of the main disadvantages of ordinary dipslides is that when the concentration of microorganisms in the liquid exceeds approximately 100,000 - 1,000,000 colony forming units (CFU) per ml, the colonies become confluent and hard to distinguish and to select for further work. Several attempts have been made in the art of-sampling and inoculation of agar specimens to solve this problem, but all the prior art solutions require the use of additional mechanical parts, resulting in a more expensive and complicated device. A description of the commonly employed Diaslide device can be found, e.g., in Rosenberg, M. et al., J of Clinical Microbiology, Vol. 30, No. 10, Oct. 1992, p. 2686 - 2691, and in U.S. Patent 4,801,547.

Another problem is the opposite problem, viz., that sometime the concentration of microorganisms in the sampled liquid (e.g., water, blood, urine, foodstuff) is so low that the colonies growing on the surface are so few as to be barely or not at all discernible.

Here, again, the art has made various attempt to solve the problem, e.g., by filtration of large volumes, followed by outgrowth of colonies on the filter, but no practical and economical solution has been forthcoming.

It is therefore clear that it would be highly desirable to provide means to overcome the aforesaid drawbacks of prior art devices, and to provide a degree of flexibility in seeding and growing microorganisms on semi-solid growth surfaces. As will be explained in detail below, the present invention provides a dipslide that does not contain any additional or unusual plastic parts, yet enables differential inoculation so that individual colonies can be observed and recovered at a wide range of microbial concentrations.

It is therefore an object of the present invention to provide growth surfaces which can be used with a wide range of microorganism types and concentrations, from different samples, which obviate the problems of the prior art surfaces.

It is another object of the invention to provide a simple and inexpensive method for modifying growth surfaces so as to permit differential inoculation thereon.

It is still another object of the invention to provide diagnostic devices, such as dipslides and paddles, which comprise modified growth surfaces of the kind described above.

Other objects and advantages of the invention will become apparent as the description proceeds.

SUMMARY OF THE INVENTION The method for modifying the plating properties of a growth surface, according to the invention, comprises treating the growth surface so as to affect the liquid retention properties and/or the surface area of said surface. In one preferred embodiment of the invention, the modification of the growth surface is achieved by coating the growth surface with a material, or combination of materials, which affects the liquid retention properties of said surface. According to another preferred embodiment of the invention, cuts are provided in the growth surface so as to improve the liquid retention properties of said surface.

According to a preferred embodiment of the invention the coating material is a material which decreases the wettability of the surface toward hydrophilic liquids. In other words, coating according to this preferred embodiment of the invention causes a greater part of the liquid coming into contact with the surface to run off it, thus leaving on the surface a smaller concentration of microorganisms per given area.

According to a preferred embodiment of the invention the coating material comprises an oil or a wax, or a mixture of both, other hydrophobic materials. A variety of different oils and waxes can be employed for the purposes of the invention, and the skilled person will easily select an appropriate oil or a wax which is suitable for coating the surface which he intends to employ. Illustrative and non-limitative examples of suitable coatings include, e.g., paraffin in a solvent selected from among acetone, ethyl acetate and pentane, the oily phase of melted margarine, cocoa butter in ethanol, and silicones.

Thus, application of such a coating imparts to the surface (e.g., the agar), or to part of it which has been coated, a nonwettable character, so that when urine is applied, it tends to run off rather than to wet the agar. In this instance, the quantity of bacteria seeded onto the agar surface is generally diminished.

According to another preferred embodiment of the invention the coating material is a material which increases the surface area of the growth surface.

In a preferred embodiment of the invention the coating material which increases the surface area of the growth surface is applied as a slurry which is then dried. Because of the nature of the surface to be coated, the slurry is preferably (but non limitatively) made by dispersing the coating material in an aqueous medium or in an alcohol, although other liquid phases, such as acetone or ethyl acetate, can also be used.

The coating material can be any suitable material which, upon drying, results in an increased surface area of the growth surface, and such materials will be easily recognized by the skilled person, for use with a particular growth surface. According to a particular preferred embodiment of the invention, the coating material consists essentially of flour.

In another preferred embodiment of the invention the coating material consists essentially of calcium carbonate. According to still another preferred embodiment of the invention the coating material consists essentially of wax particles, e.g. carnauba wax, suspended in a slurry. Without wishing to be bound by any particular theory, it is the inventor's belief that the crystalline nature of the wax particles leads to a substantial increase in surface area, and that the result is affected by this fact.

In all cases, care should be taken not to coat the surface excessively, so as to allow for availability of the nutrient component (e.g. in the agar) with the microorganism to be grown. Additionally, the surface does not have to be coated uniformly, and in fact, in many instances it is advantageous to provide non-uniform coatings. According to a preferred embodiment of the invention, for instance, the growth surface is selectively coated, so as to provide on the same surface different growth areas, coated by one or more different coating materials, or uncoated. In this way, areas suitable for growing colonies starting from low, medium or high microorganism concentrations are available on the same surface, and useful results (i.e., discernible colonies) will always be obtained, independently from the concentration of the microorganism in the sample being analyzed.

According to another preferred embodiment of the invention the coating of the surface is effected gradually, so as to provide a gradient of coating along the surface.

In one instance, a slurry containing a fine particulate can be applied to the agar surface in a gradient fashion (e.g. by either applying a multiplicity of applications of the same or different treatment, on different areas or heights along the agar surface, or alternatively, by dipping the agar into a solution or slurry and letting it run off so that the concentration of the solution or slurry that remains on the agar after runoff forms a gradient), then a gradient of concentrations of microorganisms will be plated on the agar. Alternatively, one portion of the agar can be treated (e.g., the bottom third), so that two very different levels of seeding are obtained. Alternatively, the agar can be dipped into the coating solution at an angle, so that when the dipslide is subsequently dipped into the microbial sample, the excess water runs down the diagonal interface. This also results in a concentration of colonies along the diagonal.

The invention is also directed to growth surfaces modified by the method of the invention.

Such surfaces, as said, can be of different kinds, and the invention is in no way limited to a specific type of surface. Illustrative surfaces which can be modified according to the invention are an agar medium, a gelatin or an alginate. Generally speaking, any gelling medium modified by the method of the invention, to which a fluid, such as urine, or a slurry, such as homogenized foodstuff, is applied, forms a part of the present invention.

The invention further encompasses devices using the modified surface of the invention, e.g., dipslides or paddles.

Although throughout this description reference is made, for the sake of brevity, mostly to the growth of microorganisms from liquid specimens (as said, mainly from urine), it should be understood that the invention is by no means limited to such diagnostic applications, and it can be exploited for any procedure involving growth on semi-solid medium surfaces, e.g., plating of vegetable, plant or animal cells.

Brief Description of the Drawings In the drawings: Fig. 1 schematically illustrates results obtained on a prior art dipslide; Fig. 2 illustrates a dipslide partially coated at an angle with a coating that diminishes the plating of microorganism, after growth has taken place; Fig. 3 illustrates a dipslide coated with two different coatings; and Fig. 4 illustrates a surface coated with a gradient of growth inhibiting coating.

Detailed Description of Preferred Embodiments The above and other characteristics and advantages of the invention will be more readily apparent through the following detailed description of preferred embodiments thereof.

Example 1 A number of coating experiments were carried out to test different growth inhibiting coatings, according to the following procedures. The surface employed was identical in all cases, and was a commercial dipslide (Oxoid Dipslides - DS103A, C.L.E.D. Medium / MacConkey Agar).

Experiment Protocol 1. Heating the coating materials to the desired temperature.

2. Dipping the Dipslides in the different tested materials.

3. Allowing Dipslides to dry/evaporate at room temperature; checking the agar surface and the cover quality.

4. Preparation of the bacteria: - overnight culture of E. coli in Brain Heart Infusion Broth at 370C.

- collection of urine.

- four 10 sequential fold dilutions of the bacteria in urine to obtain ca. 105 cfu/ml.

5. Dipping the treated Dipslides in the above bacterial dilution (100 ml).

6. Incubation (24h at 370C); counting the colonies.

The experiments carried out and their results are summarized in Table I below.

Table I Treatment Effect on the Effect on Bacterial Agar Surface Outgrowth Margarine' non-homogenous decreased outgrowth emulsion-like cover by factor of ca. 2 fold no outgrowth: total Nail polish2 - fine homogenous bacterial growth concentrated cover inhibition Nail polish2 - 50% fine non- decreased outgrowth (v/v in acetone) homogenous cover by factor of ca. 3 fold Nail polish2 - 25% fine non- decreased outgrowth (v/v in acetone) homogenous cover by factor of ca. 3 fold Alginate3 - 10% granular fine dense increased outgrowth (w/v in ethanol) cover by factor of ca. 1.5 fold Myristic acid isopropyl ester 98% no visible cover none Salt free "Blue Band", Telma, Israel 2 Barbara Wolf 9 Modeling powder (for dental use) - Utan Tillsatser, SVEDIA (Sweden) Example 2 Operating as in Example 1, different coatings and procedures were tested. The results and conditions are summarized in Table II below.

Table II Effect on the Effect on Treatment Agar Surface Bacterial Outgrowth Margarine4 - lower non-homogenous decreased outgrowth phase thick cover by factor of ca. 10 fold Margarine5 - upper thick homogenous decreased outgrowth phase cover by factor of ca. 10 fold Margarine6 70%non-homogenous none Ethanol - 30% thick cover, in clumps Acetone7 non-visible cover none Parafilm8 fine quite non homogenous (in pentane) homogenous cover outgrowth 4 The margarine (Salt free Blue Band, Telma) was melted and following phase separation 100 ul of the lower emulsion-like phase were applied onto each side ofthe dipslide 5 The margarine (Salt free Blue Band, Telma) was melted and following phase separation 100 ul of the upper oil phase were applied onto each side of the dipslide.

6 The margarine (Salt free Blue Band, Telma) was melted and ethanol was added and stirred; the dipslide was dipped in this mixture as described above.

7 Lab-Scan, Ireland 8 Parafilm (American National U.S.A..) squares of 25 cm2 were left for at least o.n. in 10 ml solvent.

Example 3 Operating as in Example 1, different coatings and procedures were tested. The results and conditions are summarized in Table III below.

Table III Effect on the Effector Treatment Agar Surface Bacterial Outgrowth Flour- 10% fme homogenous C' - none; (w/v in water) cover M'- increased Outgrowth by factor ofca. 2.5 Flour - 10% fine homogenous C' - none; (w/v in ethanol) cover M'- increased outgrowth by factor of ca. 2.

CaCO39 fme homogenous C'- increased 10% (w/v in ethanol) cover outgrowth by factor ofca. 2.

Carnauba wax 10%'° turbid homogenous C'- increased (w/v in ethanol) fine cover outgrowth by factor ofca. 2.

9 Merck, Germany 10 Sigma C'=CLED M'=McConkey Example 4 Operating as in Example 1, different coatings were tested. The coatings were made of various proportions of silicone wax 9800 and silicone oil 350 (both ex Goldschmidt, Germany), which were heated to 550C and then used to coat an agar dipslide. The proportions of oil:wax tested were: 1:1, 1:3, 1:10 and 0:100. In all cases slides were obtained with excellent plating behavior and coating border. The result of coating the agar can further be explained by looking at the figures, which schematically illustrate results obtained with actual dipslides coated as described in Examples 1 - 4 above. Fig. 1 illustrates a prior art device, in which the whole of the device is covered with a smear of equally indistinct colonies of microorganisms.

Fig. 2 shows a slide which has been coated in its lower part (B), while upper part (A) has been left uncoated, as in Fig. 1. Coating has been effected while tilting the dipslide at an angle, so as to create a diagonal step (S), along which intensive microorganism growth is seen. This diagonal is useful particularly in samples containing a low concentration of microorganisms, since intensive growth will be achieved along it.

In Fig. 3, a dipslide is seen, which is coated with two different coatings. Coating (A) which diminishes the plating of microorganisms, relative to the uncoated surface (B), and coating (C) which increases it. In Fig. 4 another embodiment of the invention is seen, in which a gradient of concentrations is obtained on the slide, by coating with different coatings having increasing plating reducing power (zone A through D), or by coating different areas a different number of times. Thus, the same slide can efficiently be used with fluids containing a wide range of microbial concentrations, since even if one zone is overplated, a more distant one may yield distinguishable colonies.

Example 5 Operating as in Example 1, different coatings were tested. The coatings were made of mixtures of Abil silicon waxes and oils (Abil 9800, 2440, 100, K4, all obtained from Goldschmidt, Germany) which were heated to 550 C and then used to coat an agar dipslide. The results and coating mixtures are summarized in Table IV below.

Table IV Treatment Effect on the Effect on Bacterial Agar Surface Outgrowth 9800:2440 homogenous, isolated colonies on 1:4 medium- the coating 1:3 thick 100:2440 homogenous isolated colonies on 1:3 medium thick the coating Cocoa butter thin, quite isolated colonies on in ethanol homogenous the coating ca. 1 % K4:2440 thin, quite isolated colonies on 1:3 homogenous the coating 100:2440 smooth, thin isolated colonies on 5:1 quite homogenous the coating All the above description and examples have been given for the purpose of illustration and are not intended to limit the invention in any way. Many modifications can be made in the invention. For instance, different coating materials and combinations can be used, different growth surfaces can be employed, and different shapes of coating surfaces, slides, borders, etc. can be provided, all without exceeding the scope of the invention.