Method and Apparatus for Detection and Quantitation of Bacteria

This invention relates generally to testing procedures for bacteria in fluid samples such as human body fluids and more particularly to a method and apparatus for efficient, less cost, rapid testing procedures for identifying the presence and concentration of gram negative and gram positive bacteria strains in fluid samples.

A need exists for a method to rapidly detect bacteria in urine and various other fluid samples.

Urine specimens are the major work load of the diagnostic laboratory. The most common urological disease is urinary-tract infections in children, pregnant women, diabetics and geriatric patients, (bacteriuria). In hospitals, bacteriuria is the predominant form of nosocomial infection. In view of the frequent asymptomatic urinary-tract infections bacteriuria tests must be sufficiently simple and economical to permit routine testing. A need, therefore, exists for simple, rapid, inexpensive screening tests to facilitate diagnosis and ensure prompt treatment of bacteriuric patients.

The conventional method for detection of urinary-tract infections is by inoculation of urine onto agar culture plates and incubation of these plates at optimal temperatures (usually 37 degrees C.) for 24-48 hours; bacteria are detected by formation of colonies on the agar surface. Fastidious bacteria may not grow on conventional culture media, and a variety of agar media may be required to detect these bacteria. This procedure is time consuming and expensive.

Although staining techniques, such as the Gram Stain Method, are known in clinical microbiology light microscopy is required, and the procedures are time consuming and require a skilled microbiologist. Other prior art processes involve staining of bacteria followed by concentration by centrifugation or filtration. These processes require chelating agents and positively-charged small pore filters, and suffer clogging and interference from anionic pigments, blood and other substances which may be present in urine.

It has now been unexpectedly discovered that both gram negative and gram positive bacteria can be stained for simple, rapid detection by means of the composition of the present invention. Concentrated bacteria stained with the composition described herein are readily visible and can thus be rapidly detected without resorting to light microscopic examination by specially trained personnel, or the use of expensive, perishable agar cultures. The present invention allows the efficient detection of bacteria while reducing clogging and pigment interference common to prior art methods. Furthermore, it was discovered through tests that inexpensive, simple, and rapid quantitative analysis of bacteria is possible employing the present staining composition.

A composition for staining both gram negative and gram positive bacteria is provided. The composition comprises an inorganic acid diluent, a dye soluble at a pH of 8 to 12 which preferentially stains bacteria, a washing reagent composed of an inorganic acid, and a filter paper or glass filter of a net negative charge. Bacteria are electrostatically immobilized and concentrated on the filter and are stained with the dye solution. The free dye is then removed with the washing reagent. The bacteria, which retain the dye, become visible and thus may be detected and quantitated by comparing the color intensity of the filter surface (due to the stained bacteria) to a nomograph or other calibrated standard based on color intensities obtained using known amounts of bacteria. The total test time is usually less than one minute.

It is therefore a primary feature of the present invention to provide a nove- method and apparatus for rapid detection and quantitation of bacteria to thus enable rapid initiation of treatment of patients for bacterial infections.

It is also a feature of this invention to provide novel method and apparatus for bacteria detection wherein a number of tests may be conducted simultaneously for detection and quantitation of bacteria to thus minimize the time period for detection of bacteria that might be present in samples of body fluid.

It is another feature of this invention to provide novel method and apparatus for identifying the presence and concentrations of both gram negative and gram positive bacteria strains in fluid samples.

Among the several features of this invention is contemplated the provision of a novel method and apparatus for bacteria detection through employment of a dye for staining of the bacteria together with a filter composed of paper, glass, or any other suitable material having a net negative charge for electrostatically immobilizing and conoentrating the bacteria on the filter prior to staining.

It is another feature of this invention to provide a novel method and apparatus for quantifying the concentration of bacteria in a fluid sample by means of visible comparison of the color intensity of stained bacteria on the filter surface with a nomograph or other calibrated standard based on color intensities obtained using known concentrations of bacteria.

So that the manner in which the above recited features, advantages and objects of the present invention are attained and can be understood in detail, more particular description of the invention, briefly summarized above, may be had by reference to the embodiments thereof which are illustrated in the appended drawings.

It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

Preferred embodiments of this invention have been chosen for purposes of illustration and description and are shown in the accompanying drawings forming a part of this specification wherein:

FIG. 1 is a plan view of filter apparatus constructed in accordance with the present invention.

FIG. 2 is a sectional view taken along line 2--2 of FIG. 1 and showing the various parts of the filter in exploded relation.

FIG. 3 is a sectional view showing the assembled filter apparatus of FIGS. 1 and 2.

FIG. 4 is a plan view of filter apparatus representing an alternative embodiment of this invention and providing for simultaneous multiple testing of a body fluid sample.

FIG. 5 is a sectional view taken along line 5--5 of FIG. 4 which illustrates one position of the filter apparatus of FIG. 4.

FIG. 6 is a sectional view similar to that of FIG. 5 showing another position of the filter apparatus.

This invention relates to compositions useful for staining both gram negative and gram positive bacteria and to various methods of detecting and quantitating bacteria in fluid samples. Broadly stated, the staining apparatus and composition of the invention comprises a filter matrix having a net negative charge, an acid diluent, a dye capable of staining bacteria at basic pH, and a washing reagent which removes the free dye from filter fibers but not from the bacteria. The immobilized bacteria, electrostatically adsorbed to the filter, are readily visible and easily distinguished from free dye which is diffused away from the stained bacteria by the washing reagent.

Since the color intensity of the filter is proportional to the number of bacteria in the fluid sample, quantitative analysis of bacteria may be accomplished by comparing the color intensity of the stained bacteria on the filter surface to a known standard.

The composition and methods of the invention have particular application to the detection and quantitation of bacteria in urine. By means of this invention, rapid and economical detection of urinary tract infection is possible.

More particularly, it has been discovered that dyes soluble at pH 8 to 12 which stain bacteria will do so in the absence of chelating agents. Bacteria are stained by contacting these organisms, which are electrostatically adsorbed to negatively charged filter surfaces, with a protein staining dye at about pH 8 to 12. Any dye capable of staining bacteria which is soluble at about pH 8 to 12 may be employed. However, preferable dyes are Safranin-O or Basic Fuchsin.

It is known that bacterial cells vary in width or diameter with a range of about 0.3 um (microns) to 0.8 um. Filters commonly used in the industry and in prior art for efficient retention of bacteria typically have a pore size of 0.2 unm to 0.45 um. For use in conjunction with the teachings of this invention, a filter having a pore size of 1.0 um to 25 um may be used, depending on the fulid being processed and the presence of other particulates which may effect the flow, diffusion or fluid absorption rate. Such a filter in the pore size range of 1.0 um to 25 um would not be expected to efficiently retain bacterial cells by porosity alone or in the absence of adsorption phenomena.

Bacteria stained in accordance with the present invention may be readily detected if present at a concentration of 50,000 or higher per milliliter, or at lower amounts if concentrated. Stained bacteria which are dispersed in solution will, upon concentration, be readily visible. When concentrated bacteria at sufficient concentration are stained without dispersion, the presence of bacteria is immediately manifest.

Sedimentation and entrapment of bacteria by filtration are examples of effective means for concentrating bacteria. In this current invention, bacteria in urine (their isoelectric charge having been modified by an acid diluent) are adsorbed to a negatively charged filter surface. As the urine-diluent solution diffuses over a wide area, the bacteria therein electrostatically adsorb to the filter at the site where the urine is deposited, providing an effective method for concentrating bacteria. In accordance with the method of this invention, the pores of the filter are not required to be smaller than the bacteria under test.

Filter surfaces which have a net negative charge rather than a net positive charge must be employed. The diluent indicated in this application will reverse the isoelectric charge of negatively charged bacteria, altering the same to a positive charge. Thus, the bacteria in the urine-diluent solution will be immobilized electrostatically by adsorption to the filter matrix whereas free dye, urine pigments and other urinary compounds will diffuse away from the restricted area site of the adsorbed bacteria allowing detection of the stained organisms.

Dye used to stain bacteria at the site of the deposit of the urine-diluent mixture is diffused away from the immobilized stained bacteria adsorbed to the filter. Addition of the washing reagent at the site at which the urine-diluent solution was deposited enhances the diffusion of the free dye from this site without removing the stain from the immobilized bacteria.

Referring now to the drawings, a preferred embodiment of the invention is illustrated in FIG. 1 and consists of a flat, round or square, rigid or semi-rigid disk 1. This disk may be machined, molded or thermoformed from a variety of materials, e.g., polystyrene. The disk preferably measures about one to four inches in diameter or width and features a conical or round depression or reservoir 2 at or near the center of the disk. Located at the bottom of the reservoir the disk defines a short bore forming a small diameter hole or orifice 3 which extends through the entire thickness of the disk. The orifice diameter may be in the range of from about 0.06 inch to about about 0.25 inch.

Though it is not an absolute requirement for performance of the procedure, the disk may feature an orifice lip 4 protruding from the lower surface around the orifice. This orifice lip allows for more efficient flow of fluids into a precise location on the filter paper pad and serves to inhibit the spreading of fluid on the upper surface of the filter pad. The filter pad 5 is positioned adjacent to the lower surface of the disk so that the filter pad is in contact with the orifice or orifice lip. The filter pad may be composed of cellulose, fiberglass or other suitable filter material and exhibits a net negative electrostatic charge.

The position of the filter pad is maintained by "sandwiching" the pad between the disk and a lower filter retainer plate 6. The lower plate may be machined, molded or thermoformed, and features a square or round depression 7 slightly larger in size than the filter pad. The lower plate may be attached to the disk to secure the filter pad in place. This attachment may be by any suitable means such as by an adhesive, solvent welding, or sonic welding. Alternatively, the disk and lower plate may be designed to hinge, snap or screw together to house the filter pad.

In another embodiment of the invention, as evidenced by FIGS. 4-6, an adhesive label or tape 8 is applied to the lower surface 9 of the disk or plate 10 to secure the filter pad, thus eliminating the need for a lower filter retainer plate.

The embodiments described above are useful as a single use disposable, portable unit for detection of bacteria and have particular applioation for detection of bacteria in fluids, e.g., urine, in situations where conventional methods for bacteria detection are cumbersome or unfeasible, e.g., over-the-counter for consumer home use, physicians' offices, screening clinics, etc.

The disk may be formed to define a reaction well 11 which extends transversely from the tapered reservoir as illustrated in FIG. 4. When the device is placed in a vertical position, with its base 12 resting on any flat horizontal surface the reaction well 11 is positioned to acoept and retain sample fluid. When the device is subsequently placed in a horizontal or near horizontal position by 90 degrees clockwise rotation from the position shown in FIG. 4 as evidenced by FIG. 6, the fluid flows out of the reaction well and into the reservoir. The fluid then travels through the orifice and upon contact with the filter pad, is absorbed into the pad. This feature is especially useful in tests requiring incubation or reaction of a bacterial suspension with a chemical substance prior to performance of the bacterial quantitatio test. In particular, a test for determination of antibiotic susceptibility may be performed by incubating a bacterial suspension with an appropriate antibiotic for a period of time during which, if the organism is resistant to the antibiotic, growth of the organism will occur. After the incubation period, the device is placed in a horizontal position to allow the antibiotic-treated bacterial suspension to flow to the reservoir and filter pad, thus allowing for quantitation with the staining composition of the current invention.

A disposable multiple test device is illustrated in FIG. 4 which may be made by molding or thermoforming a device featuring two or more units of the embodiments described above in connection with FIG. 4. This multiple test device has particular application in a test for determination of antibiotic susceptibility. The appropriate antibiotics may be pre-loaded into the reaction well thus allowing for simple test set-up and multiple determination of antibiotic susceptibility.

Although specific embodiments of the invention have been shown and described, it is understood that other embodiments adaptations and modifications may be utilized without departing from the true spirit and scope of the invention. The embodiments, composition and methods described herein may be similarly applied to the staining and detection of bacteria in other fluids, such as culture media, blood, spinal fluids, food washings, milk and water, as well as to staining bacteria from such fluids which have been deposited on filter surfaces.

The minimum quantity of bacteria which can be detected by this staining procedure varies somewhat with the condition and growth phase of the bacterial culture. In general, actively growing, viable bacteria are strongly detected at levels of about 100,000/ml. Non-viable organisms, or organisms in lag phase stain less intensely and thus higher levels of bacteria are required for detection.

The following examples are illustrative of the invention and are not to be taken in a limiting sense.

Two drops of urine (using a conventional dropping pipette) from a known positive bacteriuric patient was added to 8 drops of acid diluent, pH 1.5 HCl in a test tube. Four drops of the mixture was added to the center of a negatively charged filter. Three drops of Safranin-O dye at 1:1000 in pH 10 carbonate-borate-hydroxyl buffer was added to the filter surface at the site of inoculation of the urine-diluent mixture. Three drops of washing reagent, pH 2.5 HCl was added to the same site to allow diffusion of free dye away from the site of inoculation. Three additional drops of washing reagent was used as a second rinse. The results manifested a 3-4 mm diameter intensely stained filter surface at the site of inoculation with a peripheral ring of free dye about 25 mm from the site of the stained bacteria. Such a result is indicative of a positive bacteriuria test. Comparison of the intensity of the color of the stained bacteria to a known amount of bacteria added to urine indicated about 1,000,000 bacteria per ml urine. Plating of the sample on agar culture verified these results.

Two drops of urine (using a conventional dropping pipette) from a healthy volunteer (free of urinary-tract infection by conventional assay) was added to eight drops of acid diluent, pH 1.5 HCl in a test tube. Four drops of the mixture was added to the center of a negatively charged filter. The remainder of this experiment was conducted exactly as described in Example 1 above. Upon observation, the filter surface at the site at which the urine-diluent solution was deposited was completely white and the free dye ringed the filter at about 25 mm from the site of the inoculation. Such a result is indicative of a negative bacteriuria test. This result was verified by plating of the sample on agar culture media.

Employing the procedure set forth in Examples 1 and 2, and the preferred embodiment illustrated in FIG. 1, experiments were conducted with a variety of organisms using Safranin-O as a model dye at pH 10 in carbonate-borate-hydroxyl buffer. The results were as follows:

Employing the procedures set forth in Examples 1 and 2, and the preferred embodiment illustrated in FIG. 1, experiments were conducted adding different final concentrations of bacteria/ml to normal urine, and proceeding to test 4 drops of the urine-diluent mixture with results as follows. The scoring of filter color intensity is described in Example 3.

A test for determination of antibiotic susceptibility may be performed as follows: The multiple test device illustrated in FIG. 4 is placed in a vertical position. Antibiotic solutions (one drop each) are dispensed into reaction wells #1 through #8. One drop of formalin solution is dispensed into well #9 and one drop of distilled water is placed into well #10. A broth suspension of bacteria is diluted to yield approximately 100,000 bacteria/ml, and one drop of this dilution is added to each reaction well #1 through #10. The device is then incubated for three to four hours at 37 degrees C. After the incubation period, 4 drops of pH 1.5 HCl diluent are added to each reaction well. The device is placed in a horizontal position to allow travel of the fluid from the reaction well into the reservoir. After the fluid is absorbed into the filter pad, the dye solution and washing reagent are placed on each filter site as described in Examples 1 and 2 above. After completion of the staining process, the filter pad is visually examined for color development. Formalin treated site #9 manifests a pink (++) color and represents the zero-hour control. Site #10 manifests a dark red (++++) color and represents the uninhibited growth control. The color intensity of each site #1 through #8 is compared to the controls for interpretation of antimicrobial susoeptibility. Color intensity equal to or greater than the growth control indicates that the organism is resistant to the antibiotic under test. Color intensity equal to or less than the zero-hour control indicates that the organism is susceptible to the antibiotic.