NO313703B1 - Diagnostic analysis method and kit - Google Patents

Description

General description of the invention

The invention relates to a kit and a method for classifying bacteria in liquids. In a preferred version of the method, the liquid is milk, and the analysis is carried out for the detection and classification of bacteria in acute mastitis in lactating animals. However, the method is also applicable for the classification of bacteria in any liquid, including liquids that have been obtained by extraction from a sample of a wholly or partially solid material.

The invention includes a kit composed of a) a chemical reagent mixture for treating a sample so that it is dissolved to a level where it is filterable through a filter, but which preserves bacterial residues so that these are retained on a filter, b) a filter unit for collection of residual bacteria after treatment of the liquid, and c) a chemical reagent mixture suitable for the identification and classification of the bacteria present in the liquid sample by analysis of the filter in the filtration unit. The invention also includes a method of treating milk samples with a chemical reagent, transferring the treated sample to a filtration unit, and identifying bacteria or bacterial residues deposited on the filter using a chemical reagent mixture after which the bacteria can be detected by inspection of color changes on the filter with the naked eye, using a spectrophotometer, or by microscopy.

The usefulness of the invention

Bacterial infections constitute a dominant group of diseases in humans and domestic animals, with significant economic and social consequences for the world community. However, since the discovery of penicillin in 1928, people have had access to effective drugs to combat bacterial diseases. Later, a number of antibiotics which together are effective against most forms of bacteria have been developed.

However, the use of antibiotics is not without complications. The extensive use has eventually meant that a number of bacterial strains have developed resistance. Especially in environments where antibiotics are used frequently, for example in hospitals, there have been increasing problems with infections that can no longer be treated with the usual aids. The result was that more and more broad-spectrum drugs have been used, with the result that further resistance develops and that the arsenal of antibiotics that are effective against bacterial diseases gradually shrinks.

This development is becoming a global problem. If the problem develops further, I risk losing control over the resistance problem, and the effectiveness of antibiotics as a means of combating bacterial infections can be greatly reduced. The health consequences of this development can be very extensive. In order to stem this development, the WHO has implemented measures to reduce the consumption of antibiotics, thereby reducing the development of resistance problems, possibly reversing the development.

The problem is exacerbated by the extensive use of antibiotics that are used prophylactically in the livestock and fish farming industry. Trace amounts of antibiotics are also transferred to consumers of fish, meat and milk, thereby spreading the risk of developing resistance from animals to humans. There has also been an increased development of antibiotic allergies among consumers.

Analytical methods that can help determine whether an infection is caused by bacteria, and in the event that this is verified, which class of bacteria, will be decisive tools for whether antibiotics should be used in the treatment in the first place, and possibly to limit the use of antibiotics so that the use of unnecessarily broad-spectrum drugs is avoided.

Mastitis (udder inflammation) is the most loss-making disease in cattle in Norway, with costs incurred due to various forms of production loss: teat tread, retained milk, culling and lost elite milk supplement. The annual loss is estimated at approx. 360 mNOK in 1992 (Veterinærinstituttet and Norwegian Dairies).

About 75% of mastitis cases are caused by bacterial infection in the mammary glands. The symptoms vary from highly febrile animals with a reduced general condition, to animals with no affected general condition, but where changes are detected in the milk. Acute clinical mastitis is the most frequently diagnosed disease among dairy cows in Norway (Sviland, S., 1996).

The Norwegian livestock industry has set itself the goal of reducing the incidence of disease and the consumption of antibiotics in livestock. In May 1996, new guidelines were adopted for

treatment of mastitis in milk-producing animals. These guidelines place greater demands on the choice of antibiotics in the treatment of mastitis than previously. It is added i.a. great emphasis on using penicillin preparations where this is possible. Research results from i.a. Sweden shows that there is no indication for using antibiotics to treat mastitis caused by Escherichia coli.

Optimal use of antibiotics in mastitis therapy requires greater use of differential diagnostic systems that can provide answers while the vet is still in the barn. Only a quick "on-the-spot test" will be able to reduce the consumption of antibiotics because the practicing veterinarian will have to start the treatment before the test results from conventional diagnostics are available.

A good diagnostic method that can guide the veterinarian will help to reduce the spread of infection at the same time that antibiotic consumption can be kept at a low but optimal level. Today, bacteria are detected in milk using traditional cultivation methods. This requires at least a day and does not satisfy the real diagnostic needs. A test that can detect the cause of mastitis already in the barn is important to get the best possible treatment of the infection and will obviously have a market in Norway, as well as in large parts of the rest of Europe.

The present invention represents a rapid test which i.a. is suitable for the detection of mastitis-associated bacteria in milk. The test is based on a simple principle that makes it possible to perform it quickly, and without special aids. The procedure requires less than 8 minutes in total, and can be performed in 3 minutes after moderate exercise. The result appears as a color on a filter that separates bacteria that can be treated with penicillin from bacteria that need a more broad-spectrum treatment. A further step to separate Staphylococci and Streptococci takes less than a minute.

The invention's central advantage is the simplification of the procedure in relation to alternative methods, and that the method, in the event it is used for mastitis, can be used for immediate classification of the bacteria causing the condition with subsequent results for the choice of antibiotics to treat the disease.

An important clinical driving force for carrying out such analyzes in mastitis is that unnecessary suffering in the animals is prevented. Furthermore, the method is suitable for reducing unnecessary antibiotic consumption, which can thereby help limit the spread of antibiotic-resistant bacteria. In addition, an early and precise diagnosis can limit the spread of mastitis-inducing bacteria such as S. aureus to other animals in the herd. Mastitis that is not treated can lead to the slaughter of dairy cows, which represents significant recruitment costs, or that it is treated unnecessarily with a broad-spectrum antibiotic, which means that the delivery of the milk must be discarded for a disproportionately long period of time.

Correspondingly, rapid classification of bacteria in liquids such as urine, blood, water and food extracts will contribute to better environmental and therapeutic measures being taken. For all analyzes carried out on biological fluids such as blood and urine, this can be put in context with the administration of the optimal antibiotic, in the same way as the treatment of mastitis. Infections in the urinary tract in humans and animals are also a source of more use of broad-spectrum antibiotics than necessary. The frequency of unnecessary antibiotic use and the associated negative effects in the form of side effects and the development of resistance can be reduced if a rapid method for classifying the bacteria is available.

The field of invention

Of the products produced for the classification of bacteria in acute mastitis, the so-called The Selma dish (Statens Veterinårmedicinska Anstalt, Sweden) a medium which, after one day's incubation, gives an answer to which of the most commonly occurring bacteria are found in milk. This is a common cultivation methodology where the sample is sown on a dish with different media divided into sectors, and where each medium will limit the growth of bacteria to certain groups.

The so-called The HY test (Hy Laboratories, Israel) is another test for use in mastitis. This is based on a stick with different cultivation media. This detects E. coli and S. aureus, is based on standard cultivation techniques, and requires a 24-hour incubation period. The LIMAST test (Chromogenix AB, Sweden) for use in mastitis is a method based on limulus hemolysate. The presence of bacterial toxins activates enzymes in the lysate, and these have the ability to split substrates that give rise to a color that can either be assessed visually, or read in a spectrophotometer. This test takes 15 minutes to complete, but involves a relatively large number of analysis steps, and the need for special equipment. The method is therefore not suitable for field use and also only results in Gram negative bacteria. Lack of properties to classify the bacteria therefore makes it relatively unsuitable as a tool for guiding antibiotic use.

Other methods for detecting bacteria are dominated by cultivation, followed by analysis under a microscope, possibly after staining the bacteria. Other methods that are becoming increasingly widespread are immunoassays, where antibodies are used that are specific to a single type of bacteria. In addition, genetic analysis by PCR technique is used. However, all of these methods are time-consuming, and in most cases will also require special equipment that reduces their usability as field equipment, or for analyzes in emergency situations. Some specific immunoassays for particular bacteria have been developed as rapid assays. This applies, for example, to Streptococcus A, which can be analyzed in samples from the throat after a few minutes using filtration analyzes or immunochromatographic sticks where an immobilized first antibody captures antigens from a specific bacterium, after which the bacterium is detected with a second antibody that is conjugated to a dye , an enzyme that can give rise to the formation of a colour, or other signaling systems.

Different methods have been used to treat samples before analysis of bacteria. A particular problem has been to find a method for dissolving milk so that the milk can pass through a filter that retains bacteria, after which the bacteria can be stained and the filter inspected under a microscope. A method introduced in 1980 is described by Pettipher et al. in Appl. Environ. Microbiol. 39 pp. 423-429. This uses a mixture of a milk sample with a reagent so that the final concentration of the components is: 0.08 - 0.4% Triton X-100, approx. 2% WA/ Trypsin and chymotrypsin, and 40% milk sample. The sample was then incubated at 50°C for 10 minutes and was followed by filtration on a polycarbonate filter with pore size 0.6 nm where the filter had previously been washed with a warm solution of 0.1 - 0.5% Triton X-100. The filter was then stained with acridine orange, which binds to DNA in the bacteria, after which the bacteria were visualized and counted under a microscope. The use of other dyes such as methylene blue, periodate, bisulphite toluidine blue and phenolalanine blue was reported to stain some other material as well, which made bacterial counting under the microscope difficult. The method therefore requires that the sample is processed so that the bacteria remain sufficiently intact for them to preserve their DNA intracellularly.

Variants of this method are presented where 1) different enzyme mixtures and cleaning of the filter with alcohol are included (Buchrieser and Kaspar (1993) Int.J. Food Microbiol. 20, p.227-236); 2) where the use of citrate-NaOH buffer pH 3.0 is introduced during the filtration to improve filterability (Ubaldina et al., J.Appl.Bacteriol. (1985) 59, pp. 493-499), and 3) where it is used detection with fluorescently labeled antibodies (Tortorello and Gendel (1993) J.Food.Prot. 56, p.672 - 677).

A further optimization was presented by Fernandez-Astorga et al. in J. Microbiol. Meth. (1995), 24, a. 111-115. Here, the incubation time has been reduced to 1.5 minutes, the detergent washing of the filter omitted, and adjustments have been made in the order of the procedure, which together make bacteria easier to visualize for microscopic detection on the filter after staining.

A variation of the method has also been carried out in which the bacteria are detected with a microscope after incubation with a monoclonal antibody conjugated to alkaline phosphatase and formation of an enzyme product which precipitates as a blue dye where the antibodies are attached to the bacteria (Batina et al. J.Appl. Microbiol. (1997) 82, pp. 619-624).

However, none of the methods have achieved the possibility of detecting the bacteria without the use of a microscope after the treatments.

Filtration methods for the analysis of bacteria in samples that do not require treatment with detergents and enzymes have also been presented. Wallis and Melnick present in US 4,336,337 a method for urine in which the bacteria are treated with a chelator and colored before filtering through a filter with a net positive charge, and with a pore size that retains the bacteria. Staining with a cationic dye makes it possible to see the presence of 10<5> - 10<6> bacteria /ml_ with the naked eye. A color method that can distinguish Gram positive and Gram negative bacteria is also presented. In this method, the bacteria are stained at basic pH with a cationic substance, e.g. safranin, followed by the filter being washed with a solution of organic acid at pH approx. 3.0. This washing decolourises Gram +positive bacteria while Gram negative retain their colour.

Similarly, Longoria et al. in US 5,081,017 presented a method in which bacteria in urine or water are treated with a solution which causes them to be fixed in a filter with a net negative surface charge. This is followed by staining, after which the presence of bacteria can be assessed visually as a color on the filter.

Likewise, Romero et al. in J. CIin. Microbiol. (1988), 26, pp. 1378-1382 described a method in which bacterial cultures are filtered and classified by microscopic examination after traditional Gram staining.

Belly et al. likewise describes in US 4,912,035 a method for isolating bacteria in urine or other body fluids by means of filtration or centrifugation, and where any bacteria in the sample are detected spectrophotometrically by combining a redox reagent and a water-soluble metallic dye.

However, none of these works have found a solution where bacteria in milk can be analyzed using a filter method, without the use of a microscope. Based on this, it has surprisingly been found that after adding to milk samples a reagent consisting of detergent, salts, chelator and organic solvents that are completely or partially miscible with water, without the use of enzymes and incubation of the sample over a longer period of time, the milk sample allows pass through a filter. Furthermore, it has been surprisingly found that despite this treatment, bacterial residues will be preserved in such a way that they can be retained on filters with a pore size < 5.0 [im, optimally < 1.0 [im. Furthermore, it has surprisingly been found that in combination with the above-mentioned treatment, these bacterial residues allow themselves to be stained with various reagents in such a way that the presence of bacteria in a number of 10<6> or more per mL sample is reflected as a color on the filter that can be seen with the naked eye, possibly measured with spectrophotometric methods. The amount of bacteria can therefore also be assessed. It has also surprisingly been shown that different staining methods can be used to classify the bacteria into Gram positive and Gram negative types, and that classification through staining of the bacterial residues on the filter corresponds to classification obtained after traditional cultivation and identification of the intact bacteria. An important element of the invention is that the method can be carried out without the use of a microscope.

Unlike Wallis et al. in US 4 336 337 we have also surprisingly found that bacteria or bacterial residues from liquids can generally be retained on certain filters with a neutral charge and colored with a number of dyes without this causing background staining of the filter itself. Any background staining that occurs in certain reagent combinations can optionally be removed with a solution of detergent, for example 1% Triton X-100. In a preferred version of the method, filters made of polysulfone or polysulfone derivatives such as polyethersulfone are used for this purpose. However, certain other types of filter materials, such as polycarbonate, can also be used. The bacteria are stained with a solution of a dye which, in a preferred version of the method, is a solution of amine-substituted phenylthiazines such as e.g. toluidine blue, or amine-substituted phenazines such as e.g. safranin. However, a number of other dyes are also applicable. Among such coloring methods are also colloidal metals covered with oligomeric or polymeric substances containing amine functions. Contrary to Wallis et al. however, we have surprisingly found that treatment of colored bacteria with a solution of pH 2.7-3.5 decolourises Gram negative bacteria, while Gram positive bacteria retain their colour.

DETAILED DESCRIPTION OF THE INVENTION

The invention includes a liquid for treating samples that are expected to contain bacteria, so that these become filterable under conditions that retain bacteria and bacterial residues on a filter with a neutral charge. A liquid which is suitable for dissolving blood and milk contains a detergent, salts, a calcium chelating substance and an organic solvent which is completely or partially miscible with water. Liquids that are suitable for pretreatment of samples that do not contain fats or cells are typically composed of salts and a calcium chelating substance.

There are many detergents that are suitable for use in liquids with cells and/or fats. Among them are the nonionic detergents of which Triton X-100, detergents in the Tween series and the IMonidet series are typical examples. Of ionic detergents, bile acids are the most typical; for example cholate, deoxycholate and chenodeoxycholate, but also detergents of the Saponin type and sodium salts of alkyl sulphates such as sodium dodecyl sulphate, are usable. The detergents can also be present in a mixture, where the combination of nonionic and ionic detergents is particularly effective. The detergents are present in a total concentration of at least 0.05%, typically 0.2-5%. In a preferred version of the invention, a detergent concentration of about 1% is used.

The addition of a chelator that binds divalent cations, or cations with a higher valence, has surprisingly shown a beneficial effect on the dissolution of many samples, including especially milk. The protein fraction in milk in particular is effectively dissolved by using a chelator. Typical examples of effective chelators are ethylenediamino-tetraacetic acid (EDTA), ethylene glycol-bis(p-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), diethylenetriamine-N,N,N,,N",N "-pentaacetic acid (DTPA), 1,2-bis(-o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) or the corresponding tetraacetoxymethyl ester derivative (BAPTA-AM). The chelator is present in a concentration of 0.02 -1 mol/L at a pH value above 8.0.1 a preferred version of the invention uses 0.2 M EDTA at pH > 10.

The addition of salts has shown a beneficial effect on the dissolution and filterability of samples. Salts which can be used for this purpose are typical chlorides, bromides, nitrates, sulphates and phosphates of monovalent ions such as sodium and potassium. In one version of the invention, 0.0.5 - 2.0 mol/L NaCl is used, typically 0.1-0.5 mol/L.

The addition of organic solvents that are completely or partially miscible with water also has a beneficial effect on the dissolution of samples containing fatty substances and/or cells. Alcohols such as methanol, ethanol, propanols, butanols and aromatic alcohols such as phenol and benzyl alcohol can thereby be used. You can also use ethylene glycol, acetonitrile, dimethylsulfoxide, dimethylformamide, tetrahydrofuran, and possibly also together with substances that alone are less soluble in water, such as chloroform, trichloromethane, various alkanes or alkenes, or halogenated derivatives thereof. Several of the possible filter materials available have variable resistance to different solvents. The preferred solutions will therefore vary with the filter type. In a suitable version of the invention, 10% acetonitrile is used when filtering on filters made of polycarbonate, polysulfone or polysulfone derivatives.

A suitable mixture of substances in a liquid that can be used to dissolve milk in the ratio milk: reagent 1:5 is 1% Triton X-100, 0.1 M EDTA pH 12.0, 1.0 M NaCl and 10% acetonitrile. Dissolution is fastest at a higher temperature, such as when the mixture is held under hot tap water for a short period.

The dissolved milk sample is placed on a filter where either a positive pressure is exerted from above, or a negative pressure from below. Both parts can be achieved by using a syringe connected to a closed filter unit from, for example, Millex® or Swinnex®

(Millipore Corp., USA), or equivalent equipment from Pall Gelman Sciences, USA. Negative pressure from the underside can also be achieved by placing the filter in close contact with an absorbent substrate of paper or other liquid-attracting fiber material. In such setups, the concept will be improved by placing above the filter a water-repellent layer with a defined opening, where the water-repellent layer is in contact with the underlying filter material. This will delimit and define the filter surface, and it will give rise to a certain concentration effect of liquid. In such a setup, the filter that is exposed to the sample that flows through can advantageously be smaller than the filter diameters usually used in the above-mentioned filter setups. A favorable diameter is 0.5-15 mm. In the preferred variant of this method, filter openings of 2 - 5 mm are used. It can also be advantageous to place the filter under a water-repellent material that is shaped like a funnel, or like a tube. In this way, larger quantities of liquid can be applied more easily, and liquids containing detergents and organic solvents can also be applied without the liquid flowing outwards, as is often the case with an application on a flat coating of, for example, plastic. Plastic is in all cases a favorable material to use for all the described types of equipment in which filtration can be carried out. The filter with the underlying absorbent layer can also be placed in a container which is connected to the overlying funnel or tubular component. This will ensure that the user does not come into contact with the liquids after they have passed the filter, and the entire device can be thrown away after use.

It can also be advantageous to place a pre-filter with a larger pore size, e.g. 5 - 20 |nm, above the filter where the actual analysis is carried out. Such a pre-filter will remove random large, undissolved particles that are present in the sample, and reduce the risk of clogging or non-specific staining of the analysis filter. Such a pre-filter must be arranged so that it is easy to remove after the first passage of the sample, in order for the rest of the procedure to take place with the filter collecting the bacterial components openly available for visual inspection.

The filter material in the invention itself is both dependent on the solvent used, especially with regard to the content of solvents, and dependent on the color reagent used. In one version of the invention, it has been shown that filters containing polymeric materials of the type polycarbonate, polysulfonate and derivatives of polysulfone such as polyethersulfone are particularly beneficial. Filters with a pore size of 0.2 - 5.0 µm, typically 0.45-1.2 µm, are used.

Bacteria are known to bind a variety of dyes. However, it is a common problem that samples contain a number of substances that also bind these dyes under the same conditions, thereby giving rise to non-specific reactions. Furthermore, there are a number of filter materials that in themselves absorb such dyes, and it is also a widespread problem with the analysis of many types of samples that filters absorb proteins and other dissolved material from milk so that distinction from staining bacteria often becomes impossible. The above-mentioned articles all indicate that successful staining of bacteria on filters can only be performed with intact bacteria. It is therefore surprising that even after the above treatment of milk with a strong concentration of detergent, chelator, strong alkaline pH, and organic solvent, that the bacteria still allow themselves to be specifically stained with a selection of dyes.

Dyes that can be used for this purpose can be amine-substituted pheno-thiazines such as e.g. toluidine blue, thionine, and methylene blue; or amine-substituted phenazine derivatives such as safranine, and neutral-red; or amine-substituted phenyl-dicyclohexadiene derivatives such as gentian violet, methyl green and fuchsin derivatives, or amine-containing porphyrin derivatives or phthalocyanine derivatives such as e.g. alcian blue. However, the dyes are not limited to this range. In principle, the invention encompasses the use of any dye that exposes a positive net charge at pH under the condition where the bacteria expose a negative net charge. In the classic Gram stain, gentian violet or methyl violet is added to the sample. This is then treated with iodine solution, and the preparation is then washed with ethanol. If there are Gram positive bacteria in the sample, they retain their colour, while Gram negative bacteria lose their colour. As a control, the Gram negative bacteria can be counterstained with carbolfuchsin or safranin.

In another variant of this procedure, a method has surprisingly been found in which a number of dyes that bind to the bacteria at neutral or slightly alkaline pH are washed away from Gram-negative bacteria when the sample is washed with a solution with a pH of 2.8-3.5, while the Gram-positive bacteria will retain the color under the same conditions. The decolorization of Gram-negative bacteria is in a particularly preferred method carried out with a mixture of 0.01-0.2 M acetic acid pH 3.0 with 5-70% ethanol. Use of this method can also be improved by the use of prior staining with a mixture of two or more of the above dyes, wherein toluidine blue is present as at least one of the components of the preferred method.

In another variant of the invention, metal colloids are used in conjunction with substances that expose positive net charges at a pH where the bacteria expose negative net charges. Metal colloids are very intense dyes that increase the sensitivity compared to the use of pure dyes by 10-1000x. In a preferred version of the method, colloidal gold is used which is covered with polymeric or oligomeric compounds which are carriers of primary, secondary or tertiary amine groups. Examples of such compounds are polylysine or oligolysine, chitosan (deacetylated chitin), and basic proteins such as e.g. histones. However, the method is not limited to gold colloids, as colloids of other metals and metal salts, and colloids of hydrophobic dyes of the type usually referred to as disperse dyes, can be used for the same purpose.

The interpretation of the results will be such that a color indicating infection with Gram-positive bacteria means that it can be treated exclusively with penicillin. As a further aid for the differential diagnosis of Gram positive bacteria, hydrogen peroxide can be added to a separate part of the milk sample. Catalase activity will cause visible bubble formation in the sample after a few seconds and indicate the presence of Staphylococci, while the absence of bubble formation will indicate Streptococci. This will be of additional value for the treatment as the optimal penicillin regimen is different for these two classes of bacteria.

Detection of Gram-negative bacteria means that if the infection is serious, broad-spectrum antibiotics must be used, such as tetracyclines or sulfa preparations.

A negative result indicates that the infection is not caused by bacteria, but has other causes that have triggered an inflammatory reaction. Treatment with antibiotics is not necessary in such cases.

Some sample materials, such as for example milk, may contain particulate matter, which cannot be dissolved using the reagents presented here. With mastitis, it is relatively common for milk samples to have an atypical appearance, contain solid particles, and have a generally slimy consistency. A direct application of such samples in the described invention will be difficult. But we have surprisingly found that such samples can be used after a successful filtration through a wool-like material or a prefilter with pore sizes > 10 pm, or a combination of these, without significant loss of sensitivity in the subsequent analytical method to detect bacteria by staining. The terminology prefiltration therefore describes a method for treating samples before the samples are analyzed for bacteria, as described elsewhere in this specification.

In a preferred version of this method, both the prefilter and the wool-like material are made of hydrophobic materials. This treatment will retain materials which in some samples tend to clog filters, without significant loss of bacteria in the sample. Such a pre-treatment can either be carried out directly on the samples or after mixing with the described reagents. In both cases, this treatment is applied to the sample before it is placed on a filter.

In one version of this prefiltration method, this prefilter is included in the analytical unit in such a way that it can be removed after the sample, or the sample treated with the described reagent, has passed the prefilter, so that the filter where the bacteria are retained can exposed. In another version of the method, the prefilter is kept in a separate unit which can be used to prefilter samples before or after mixing with the described chemical reagent.

Hydrophobic materials suitable for prefiltration of samples described above include, but are not limited to, polyalkenes such as polyethylene and polypropylene, polyesters, polyvinyl chloride, polyurethanes, polyacrylics, polyacrylamide, polysulfones, polyethersulfones, polycarbonates, and nylon. However, this should be understood to mean that, in general, any hydrophobic material can be used for this purpose. The material can have a shape and appearance like a filter, with a pore size > 5 pm, preferably with a pore size > 10 pm. This material can also be used as a wool-like matrix (wadding) which in a preferred setup is placed inside a container with an inlet and an outlet, which enables a flow of sample material. Optionally, a hydrophobic filter with a pore size > 5 pm, preferably 15-80 pm, can be placed at the outlet of the container, which contains a wool-like matrix.

The procedure of filtration can also be carried out in a device that allows a lateral liquid flow. A suitable composition is shown in Figure 1. The entire filtering unit in this figure is attached to a plastic holder (1). However, such a system can also be mounted inside a plastic unit which, for some applications, can protect the user and the surroundings where the user is located, against contamination from chemicals and/or microorganisms.

All the layers in this unit are in direct physical contact with each other, so that liquids can travel by capillary forces from an application point and on to the other layers.

The test, as if

this is milk can

be mixed with an appropriate volume of 1 mol/L NaCI, 0.1 mol/L EDTA, 0.5% Triton X-100 (pH 12-13), which optionally also contains an organic solvent such as 10% acetonitrile, is placed on the sample filter (2 ). The sample can either be placed on the sample filter (2) by dripping sample solution onto the filter surface (2), or by dipping the part of the stick that makes up the sample filter (2) into the sample solution.

The sample filter should preferably be made of a material that enables the bacteria to flow through, and have a pore size that retains particles that should not migrate to the other layers of the stick. Such a filter material is preferably made of a hydrophobic material, from the same group of materials described for prefilters, and includes, but is not limited to, polyalkenes, polyesters, polyvinyl chloride, polyurethanes, polyacrylics, polyacrylamides, polysulfones, polyethersulfones, polycarbonates and nylons .

The materials used in this test filter should be a filter matrix of the woven or non-woven type, with an average pore size of > 5 pm.

This sample filter (2) can be composed of one or more layers, and in such cases each layer will consist of a defined chemical composition and/or pore size.

The sample that is placed on the sample filter (2) will travel towards the filter membrane (3) using capillary forces, and where any bacteria will be retained. This filter membrane (3) should have some direct overlap with the liquid absorbent (5). If this overlap is large, the filter membrane (3) will essentially function as a vertical filter, while if this overlap is small, the filter membrane (3) will function essentially as a laterally working filter. The filter membrane (3) should preferably be made of polycarbonate, polysulfone or derivatives thereof. The pore size should be in the order of 0.45 - 2.0 pm. The amount of sample solution should be of such a volume that wetting of the liquid absorbent (5) is observed on the opposite side of the contact surface against the filter membrane (3). This filter membrane (3) should also partially overlap with the liquid absorbent (5).

When the liquid is clear, as described, bacteria on the filter membrane can be visualized as previously described, by applying a dye, consisting of, but not limited to, toluidine blue. This coloring substance should preferably be applied directly to the filter membrane (3), and the color must be given the opportunity to travel through the filter membrane (3) and into the liquid absorbent (5).

This step in the process can optionally be followed by a washing step, as previously described, where a detergent solution such as 0.1-5% Triton X-100, or other similar non-ionic or ionic detergents. Furthermore, the filter membrane (3) can be washed with a solution that decolourises Gram negative bacteria, as previously described, i.e. a solution containing an alcohol, such as 10% ethanol, adjusted with acetic acid to a pH of 2.8-3.0.

The final result can be read as the presence or absence of color on the filter membrane (3). Presence of color before decolorizing solution is added indicates presence of bacteria. If this color is retained after the decolorization solution has been added, then these bacteria are of a Gram positive origin, while if the color disappears, then the bacteria are of a Gram negative origin. Amount of color on the filter membrane (3), which is defined as a combination of color intensity and width of the color band, can also be used to estimate the amount of bacteria on the filter membrane (3) and thereby also in the original sample.

The liquid absorbent (5) should have sufficient suction capacity to absorb the total volume of liquid applied to the stick. It can be composed of different materials that can absorb aqueous solutions, or water containing, among other things, a detergent. Suitable materials may be cellulose or glass fiber in a porous form, but this invention is not limited to the choice of such materials

In a special version of the invention, the composition of sticks is built into a plastic holder, with openings that expose the sample filter (2) and filter membrane (3). Application of the sample is carried out in this case through the opening for the sample filter (2), while the other solutions in the analysis are added through the opening for the filter membrane (3), where the final result is also read.

EXAMPLES

Example 1.

Of a milk sample, 1 mL was mixed with 4 mL of a solution containing 0.1 mol/L EDTA adjusted to pH 12 with NaOH solution, 1.0 mol/L NaCl, 0.5% Trition X-100, and 10% acetonitrile. Heating under hot tap water (approx. 50°C) for 2-3 minutes increases the dissolution rate of the milk. The mixture was filtered in a polypropylene filter with an average pore size of about 10 and then drawn up into a syringe which was connected to a Millex® filter unit (Millipore Inc.). The filter unit was equipped with a Supor filter (polysulfone filter from Pall Gelman Inc.) with a diameter of 25 mm. The solution was slowly forced through the filter by a positive pressure from above using the syringe, after which the filter was washed with 1 mL of 1% Triton X-100. Then 1 mL of a solution of safranin-o (2.5 mg/L in distilled water) was added which passed the filter followed by a wash with 1% Triton X-100 solution. The presence of bacteria in an amount of > 5 x 10<6>/ mL in the sample appeared as a reddish color on the filter, while samples containing less than the above-mentioned amount of bacteria appeared as a colorless, white filter. Addition of 1 mL of a solution of 10% ethanol and 0.1 mol/L acetic acid, pH 2.9, decolorized a previously colored filter if the bacteria on the filter were Gram negative. If the bacteria on the filter were Gram positive, the color was not washed off.

To document this, aliquots of milk were added to culture of Staphylococcus aureus (Gram positive) and Escherichia coli (Gram negative) in increasing concentration from 1 x 10<5> to 5 x 10<8> bacteria per ml. Red color was visible on the filters with samples starting at a concentration of 5 x 10<6> bacteria/mL for both bacterial strains. Only the samples with E. coli were decolored with a mixture of 10% ethanol / 0.1 mol/L acetic acid pH 2.9, while the samples with S. aureus retained their color under the same conditions.

Example 2.

The above method was repeated, but the safranin-o solution was replaced with a solution of toluidine blue (4. (3 mg/l in distilled water). Staphylococcus aureus in an amount of > 5 x 106/mL was seen as a blue color on the filter. The color could not be removed from this Gram positive bacterium by washing with 10% ethanol / 0.1 mol/L acetic acid pH 2.9, as described in example 1. Eschericia coli in an amount of > 1 x 10<6>/mL appeared as a blue color on the filter, and the color was removed from this Gram negative bacterium by washing with 10% ethanol / 0.1 mol/L acetic acid pH 2.9, as described in example 1.

Example 3

The methods in examples 1 and 2 were repeated with milk samples from cows diagnosed with acute mastitis. To establish a reference, the samples were sown on media for cultivation, after which determination of bacterial species and number was carried out. Furthermore, the number of somatic cells was determined with the Schalm test, where the number is given on a scale from 0 (little) to 5 (a lot). The same milk samples were treated as described in examples 1 and 2, and the results from this test compared with the results from the reference method.

The result was as follows:

From the experiment, it appears that the method detects bacteria in milk that correspond to cultivation numbers down to the 10<3-> level. This is 2-3 log better than when adding culture to milk, where the sensitivity is now 5 x 10<6> for Gram positive bacteria (S. aureus) and 1 x 10<6> for Gram negative (E. coli). The increased sensitivity is probably due to the fact that the rapid method also detects dead bacteria which are therefore not represented in the culture.

Example 4.

Cultures of Staphylococcus aureus and Escherichia coli were grown in LB medium overnight, and regular milk was mixed with each of the bacterial cultures in a ratio of 1:2. In addition, as a control, a mixture of the same milk to which no bacterial culture had been added was made. From each of the mixtures, 1 mL was taken out and mixed with 4 mL of a solution containing 0.2 mol/L EDTA adjusted to pH 13 with NaOH solution, 0.2 mol/L NaCl, 1% Trition X-100, and 10% acetonitrile . The mixtures were filled into syringes which were then connected to Millex® filtration units containing polycarbonate filters (Isopor®, Millipore Inc.) with pore size 0.4-0.8 and diameter 25 mm. The mixtures were passed through the filters using the syringes with a positive pressure from above. Each of the three cells was then washed with 1 mL of a solution of 0.1% Triton X-100. The filters were then stained as follows: 1 mL of a solution containing 0.5g/L methyl violet 6B and 0.1% Triton X-100 was forced through each of the filters. The filters were then washed by passing 1 mL of 0.1% Triton X-100. The filters that were used to filter samples containing bacterial culture were colored blue. Then 1 mL of a solution of 0.1 g/L iodine - 0.2g/L potassium iodide - 0.1% Triton X-100 was added, which was pushed through the filters using syringes. The filters were then washed with 1 mL of 95% ethanol and washed with 1 mL of 1% Triton X-100. Filters that were used to filter samples with the Gram positive bacteria S. aureus were colored dark blue, while filters that were used to filter samples with E. coli or samples that did not contain bacteria were colored faint pink. 1 mL of a solution containing 0.1 g/L basic fuchsin - 0.04 g/L phenol - 0.05% ethanol - 0.1% Triton X-100 was then added. The solution was forced through the filters using syringes. The filters were finally washed with 1 mL of 0.1% Triton X-100.

Filters that were used for filtering samples with the Gram positive bacteria S. aureus retained the deep blue color, while filters that were used for filtering samples with the Gram negative E. coli were colored red. Filters used for filtering samples to which no bacterial culture had been added were colored white - slightly red, and were clearly different from the filters with bacterial samples.

Example 5.

The methods in examples 1 and 2 were applied to filter units with absorbent paper substrates. The filters were mounted in a plastic holder with a circular opening at the top, through which the filter was exposed. An absorbent paper material was placed under the filter which was clamped in place against the filter when the plastic holder was clamped together. The result was similar where the liquids were forced through the filters using a syringe, as in examples 1 and 2.

Example 6.

Urine samples were treated by mixing with the dissolution medium described in example 1, except that acetonitrile was omitted in this medium, were filtered, treated and stained as described in example 2. The result of the test is presented in the table below. As can be seen from the results, the method indicates both the correct classification and intensity of the infection.

Example 7.

Pure cultures of a number of bacterial species with known identity were grown in liquid medium and added milk (medium:milk = 1:5) to a final number of 10<7-> 5 x 108 bacteria/mL. The samples were analyzed with the rapid test system according to the methods in examples 1 and 2. The result of the test was as follows:

This shows that all species were stained according to the rapid method, and further that the rapid method correctly classified all bacterial species as Gram positive or Gram negative types.

Example 8

Some milk samples from cows diagnosed with mastitis have a slimy and particulate consistency, which makes them less suitable for direct application in the filter method. Such samples will often clog the filter and interfere with subsequent steps of the analysis. Such samples make up an estimated 5-10% of field samples from mastitis cows. A prefiltration of such samples turns them into analyzable robbers in the system described in previous examples.

Prefiltration was carried out by assembling equipment consisting of a plastic syringe with a polystyrene holder positioned so that this covers the exit of the syringe. A polypropylene filter with an average pore size of 80 pm was placed inside this holder, and a layer of polystyrene wadding with a thickness of 2-10 mm was placed over this filter.

Fifteen milk samples with a slimy and chewy consistency were selected to be analyzed directly according to the method described in Example 2, or analyzed after filtration through the pre-filtration equipment. The results from the individual samples are shown in the table below. As the results show, this prefiltering step improves the applicability of the method to almost 100%, without any significant loss in sensitivity.

Example 9

In a practical example of using the test in stick format, a 5 mm wide stick was made by gluing a 10 mm long sample filter to a plastic holder. The sample filter was of the type Sefar Propyltex with pore size 30-100 pm. The sample filter overlapped the filter membrane (Gelman or Pall polysulfone membrane with pore size 0.8 pm) by approx. 2 mm. The filter membrane had a length of 10 mm and was placed over a liquid absorbent (Whatman Grade 17 CHR cellulose) with an 8 mm overlap. The filter membrane was held in place with tape applied partially over the filter membrane, so that about 5 mm of the membrane was still open for addition of liquid and for visual inspection.

Milk was spiked with bacteria (either Staphylococcus aureus or Escherichia coli, grown under standard conditions in broth) to 1 x 10<7> bacteria/mL. This milk was used to examine the functionality of the sticks. The sticks were dipped with the sample filter end down, approx. 5 mm with bacteria added to the milk sample, or milk without bacteria as a control. The liquid had the opportunity to travel up the sticks for so long that the liquid front reached about 2 mm above the filter membrane zone into the liquid absorbent. The sticks were then taken up from the milk samples and placed horizontally. A drop of a 1% Triton X-100 solution was dripped onto the filter membrane and allowed to absorb. A drop of a 0.133 mmol/L toluidine solution was then dripped onto the filter membrane and allowed to absorb. A second drop of a 1% Triton X-100 solution was finally added.

The sticks dipped in milk containing bacteria were colored blue on the filter membrane at a distance of 2 mm from the sample filter, while no color was observed on sticks dipped in milk without bacteria.

Finally, a drop of ethanol adjusted to pH 2.9 with acetic acid was placed on the filter membrane. The sticks containing E. coli were completely decoloured, while the sticks containing S. aureaus retained their blue colour.

Example 10

Sticks were made in the same way as described in example 9, but with sample filters made of other materials. These sticks were made with sample filters made from Porex Polypropylene with a pore size of 70-130 pm, or Porex Polypropylene with a pore size of 40-100 pm. The new sticks were tested as described in Example 9, with consistent results.

In another edition, the sticks as described in Example 9 were modified by surrounding the sample membrane with a layer of Gelman Polypropylene filter with a pore size in the range of 30-70 pm. This polypropylene filter prevented larger particles from reaching the sample membrane, thereby preventing clogging of the sample filter, when mastitis samples were tested on the sticks.

Example 11

Sticks were made as described in examples 9 and 10, using an 80 mm long support material consisting of 0.015 Super White polystyrene with GL-187 acrylic pressure sensitive adhesive material laminated to one side, and applied A74 LB Poly Coated Silicone Release Liner ( Gelmann).

A 60 mm long and 8 mm wide Whatman Grade CD427-07 Development material was glued to one end of the above material and constituted the liquid absorbent. A 15 mm long polysulfone membrane (Gelman) with a pore size of 0.8 pm was placed at the end of the liquid absorbent that was not glued. A 35 mm long 10 µm pore size polypropylene prefilter (Gelman) was placed at the other end of the support material so that it overlapped approximately 5 mm with the polysulfone membrane. The polypropylene filter was also glued to the support material while the polysulfone membrane was held in place with tape that was placed around one side.

Staphylococcus aureus ATCC 25923 and Escherichia coli were grown in culture overnight and milk was added so that the final number of bacteria was 1 x106 - 5x10<8> per ml. 100 µl of each milk sample was mixed with 100 µl 0.1 mol/L EDTA /1.0 mol/L NaCl / 0.5% Triton X-100 Qustert to pH 12.0 with 0.1 mol/L NaOH). This treatment facilitates the fluid flow through the sticks and improves the overall functioning of the system. The function was further improved when the liquid mixture above was heated under hot tap water for approximately two minutes.

A stick compound as described above was dipped into each of the samples until the liquid was absorbed. Next, 100 µl of 1% Triton X-100 was added to the polysulfone membrane on the stick, followed by the addition of 100 µl of 133 µmol/L toluidine blue and 100 µl of 1% Triton X-100.

The presence of bacteria in the samples could be seen as a blue color on the polysulfone membrane in concentrations down to 10<6> bacteria per ml.

Addition of 100 µl of 10% ethanol adjusted to pH 2.9 with acetic acid decolorized the color of sticks containing E. coli, while sticks containing S. aureus retained the color. In this way, the system could distinguish between Gram-positive and Gram-negative bacteria.

Claims (10)

1. A method for detecting bacteria in a milk sample characterized by mixing a milk sample with a reagent with a pH > 8.0 containing salts, one or more chelators that bind di- or polyvalent cations, one or more detergents, and optionally one or several organic solvents that are completely or partially miscible with water - filtering the mixture through a filter with a pore size < 5 µm, - staining bacteria on this filter with one or more coloring substances that have a positive net charge at a pH where the bacteria's outer wall has a negative net charge, and - inspection of the filter for color indicating the presence of bacteria in the sample 2. A method for the detection of bacteria in a sample characterized by - filtering the mixture on a filter with a pore size < 5 |xm, where the filter material is made of polysulfone or a derivative of polysulfone - staining bacteria with one or more dye substances that have a positive net charge at a pH where the bacterial outer wall has a negative net charge - inspection of the filter for color indicating the presence of bacteria in the sample 3. A method according to Claims 1 or 2 for classifying bacteria in a sample, characterized in that this staining is carried out by contacting the described filter with a first coloring substance; followed by washing this filter with a solution of an alcohol and a buffer substance suitable to give the solution a pH in the range 2.7 - 3.5; which thereby classifies any bacteria on this filter as Gram positive if the color of the filter is retained; or classifies any bacteria on this filter as Gram negative if the filter is discolored; and optionally contacting this filter with a secondary dye substance suitable for staining Gram negative bacteria 4. A method in accordance with Claim 1 where the described filter is a polycarbonate, a polysulfonate or a polyethersulfonate filter, with a pore size < 5.0 pm 5. A method according to Claims -1,2 or 3 where the said coloring substance is obtained from the group of chemical compounds consisting of amine-substituted derivatives of a phenothiazine, phenazine, phenyl-dicyclohexadiene, hemocyanin or colloidal metals covered by amine-substituted oligomers or polymers. 6. A method according to Claims 1 or 2 where the described sample or sample diluted in said reagent is prefiltered through a porous matrix made of hydrophobic wool-like material, or a prefilter made of a hydrophobic material where the filter has a pore size > 5 pm , or a combination of a hydrophobic wool-like material and a hydrophobic filter, before application to the described filter. 7. A method according to Claim 3 for the classification of bacteria in a sample, characterized in that the described staining is carried out step by step by - contacting the described filter with a first coloring substance, followed by - washing this filter with a solution containing a alcohol - to classify bacteria on this filter as Gram positive if the color of the filter is retained - to classify bacteria on this filter as Gram negative if the filter is discolored and - possibly contact the aforementioned filter with a secondary coloring substance that is suitable for staining Gram negative bacteria, whereby bacteria on this filter are classified as Gram negative if the filter is colored with this secondary coloring substance. 8. A kit for the classification of bacteria in milk samples, where this kit consists of - a unit containing a filter with pore sizes < 5 pm, and possibly an absorbent material in direct physical contact with this filter - a reagent composed of one or more chelators, one or more salts, at least one detergent, and possibly one or more organic solvents that are completely or partially miscible with water, where this reagent has a pH > 8.0 - a solution containing one or more coloring substances that express a positive net charge at pH < 4, and which is suitable for staining bacteria or bacterial fragments - a washing solution containing an alcohol and a buffer substance, where this washing solution has a pH in the range 2.6 - 3-5 - and optionally a washing solution containing one or more detergents 9. A kit for classifying bacteria in samples, where this kit consists of - a unit containing a filter made of polysulfone or a derivative of polysulfone where the pore size in this filter is < 5 pm, and possibly a liquid absorbent material in direct physical contact with this filter, and - a reagent composed of one or more chelators, one or more salts, at least one detergent, one or more organic solvents that are completely or partially miscible with water, where this reagent has a pH > 8.0, and - a solution containing one or more coloring substances which express positive net charge at a pH < 4, suitable for staining bacteria or bacterial fragments, and - a washing solution containing an alcohol and a buffer substance suitable to give this washing solution a pH in the range 2.6 - 3-5 - and possibly a washing solution containing one or more detergents 10. A kit according to Claims 8 and 9, where this kit additionally contains equipment or methods for removing larger, insoluble materials from the sample, prior to contact with the described filter, characterized by being a unit containing a prefilter with a pore size of > 5 pm, or containing a wool-like material, or containing a combination of a prefilter and a wool-like material, where this prefilter and/or wool-like material is made of a hydrophobic material.