CA1298183C - Method and apparatus for detection and quantitation of bacteria - Google Patents

Abstract

METHOD AND APPARATUS FOR DETECTION
AND QUANTITATION OF BACTERIA

Abstract of the Disclosure A bacterial staining composition, process and apparatus for detection and quantitation of gram negative and gram positive bacteria in fluid samples are disclosed.
The composition comprises a net negatively charged filter onto which bacteria are adsorbed and concentrated, an acid diluent which alters the electrostatic charge of the bacteria, thus enabling adsorption of the bacteria to the filter, a bacterial stain operative at about pH 8 to 12, and a washing reagent which enhances the diffusion of free dye away from the immobilized, stained bacteria. The color intensity at the center of the filter surface manifests the stained bacteria. It is compared to a standard color guide to determine the number of bacteria present in the fluid sample.

Description

~2913~83 ATTORNEY DOCKET NO. 8305 METHOD AND APPARATUS FOR DETECTION
AND QUANTITATION OF BACTERIA

Field of the Invention This invention relates generally to testing procedures for bacteria in fluid samples such as human body fluids and mora 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.

Background of the Invention A need exists for a method to rapidly detect bacteria in urine and various other fluid samples.
Urine specimens are the ma;or 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 ls the predominant form of nosocomial infection. In view of the frequent asymptomatic urinary-tract inections 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-~ract infections ls 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 ' ~2~ 33 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 microbioloyy, light microscopy is required, and the procedurss 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 discovared that both gram negative and gram positive bacteria can be stained for si.mple, rapid detection by means of the aomposltlon 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 interferance common to prior art methods. Furthermore, it was discovered through tests that inexpensive, simple, and rapid quantitative analysis of bacteria is possible employing the prasent staining composition.

Summary of the Invention 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 1~9~83 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) tc 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 presen-t invention to provide a novel 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 ~sa~s3 concentrating 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.
Brief Description of the Drawings So that the manner in which the above recited features, advantages and ob;ects of the present invention are attained and can be understood in detail, more particular description of the invention, briefly summari~ed 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 scopa, 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 speciication wharein:
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.

-~298:18~

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.
Detailed Description of the Invention This invention relates to compositions useful fvr staining both gram negative and gram positive bacteria and to various methods of detecting and quantitating bacteria in fluid samples. Broadly statad, 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.
Sinae 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 stalned bactaria on the filter sur~ace 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 ~1 29 !3~33 invention, rapid and economical detection of urinary tract I 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 filtar sur~aces, 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.
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 ara 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, baateria in urine ttheir 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 ~ilter 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 ~.

~98~L~33 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 diffu~ion of the free dye from this site without removing the stain from the immobil~zed 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 2~ from a variety of materials, e.g., polystyrene. The disk prefarably 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. Locatsd 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 dis~ 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 tha spreading of fluid on the upper ~298i83 surface of the filter pad. The filter pad 5 is positioned ad;acent 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 ilter retainer plate.
The embodiments described above are useful as a single use disposable, portable unit for detection of bacteria and have particular application 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, as shown in Fig. 5, the reaction well - 11 is positioned to accept and retain sample fluid. When ~L~98~33 the device is subsequently placed in a horizontal or near horizontal position as shown in Fig. 6 by 90 degrees clockwise rotation from the position shown in Fig. 5, the fluid flows out of the reaction well and into the reservoir. The fluid then travels through the orifice and upon contast with the filter pad, is absorbed into the pad. This feature is especially usaful in tests requiring incubation or reaction of a bacterial suspension with a chemical substance prior to performance of the bacterial quantitation test. In particular, a test for determination of antibiotic susceptibility may be performed by incubating a bacterial suspension with an appropriate antiblotic 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 m~ltiple 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 o~ the invention have bsen 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 lnvention. The embodiments, composition and methods described herein may be similarly applied to the staining -~:9818;~

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 procadure 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, cr organisms in lag phase stain less intensely ~nd 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.

Example 1 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-0 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 peripharal ring of free dye about 25 mm from the site of the stained bacteria. Such a result is indic~tive of a positive bacteriuria test. Comparison of the intensity of the color of the stained bacteria to a ~298~3 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.

Example 2 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.

Example 3 Employing the procedure ~et forth in Examples 1 and 2, and the preferred embodiment illustrat~d in Fig. 1, experiments were conducted with a variety of organisms using Safranin~0 as a model dye at pH 10 in carbonate-3~ borate-hydroxyl buffer. The results were as follows:

1298~L83 Test Organisms Intensity of Stain*
100,000 Bacteria/ml On Filter Surface Normal urine, no bacteria 0 E. coli +~
S. aureus ++
Proteus vulgaris ~+
Pseudomonas ++
Group A Strep. ++
Group D Strep. ++
Klebsislla pn. +~
*Site of inoculation of sample of urine-diluent onto ths filter surface. Scoring o color intensity: O = white, no color;
: += light pink; +~= pink; ++~=red; ++~+= dark red or magenta.

Example 4 Employing the procedures set forth in Examples 1 and 2, and the preferred ambodiment 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 ollows. The scoring of filter color lntensity is described in Example 3.
E. coli : CFU/ml Added to Color Intensity Normal Urine Of Filter Surface O O
10, 000 50,000 +
100,000 1, 000, 000 f ++
10, 000, ooo +,~,+,~, ~ le 5 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 1298~83 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.
Ater 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 uninhibitad growth control. The color intensity of each site #l through #8 is compared to the controls for interpretation of antimicrobial susceptibility. 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 antlbiotic.

What is Claimed is:

Claims (11)

1. A process for concentrating immobilizing and staining bacteria contained within a fluid sample, comprising:
(a) electrostatically adsorbing bacteria in a fluid sample to a designated portion of a net negatively charged filter;
(b) staining said adsorbed bacteria with a staining dye operative at a basic pH; and (c) diffusing free staining dye from said stained electrostatically adsorbed bacteria to a region of said filter beyond said designated portion, leaving stained bacteria in said designated region for comparative quantitative analysis.

2. The process of Claim 1, including:
diluting a sample fluid with an inorganic acid from a group including hydrochloric acid, nitric acid and sulfuric avoid and having a pH in the range of from 1 to 3.

3. The process of Claim 1, wherein said staining dye is a bacteria staining dye from a group including safranin-O, and basic fuchsin operative in a pH
range of from about 8 to 12 and soluble in buffers at said pH range, said staining dye being in a concentration range of from about 0.1% to about 0.001% and solubilized in a buffer from the group including potassium borate, potassium carbonate and potassium hydroxide at a pH range of from about 8 to about 12.

4. The process of Claim 1, wherein said diffusing is accomplished by washing said stained adsorbed bacteria with a washing reagent composed of an inorganic acid from a group including hydrochloric acid, nitric acid and sulfuric acid in a pH range of from about 2 to about 4.

5. The process of Claim 1, wherein said fluid sample is urine and said process includes:
(a) diluting said urine with an inorganic acid diluent in the pH range of from about 1 to about 3 to solubilize precipitates present in said urine, the composition of the urine-diluent mixture being in the range of from about 4 parts of diluent to about 1 part urine;
(b) depositing said urine diluent mixture onto the surface of said filter;
(c) allowing said urine diluent mixture to absorb into said filter; and (d) said diffusing comprising adding a washing reagent composed of an inorganic acid in the pH
range of from about 2 to about 4 and from a group including hydrochloric acid, nitric acid and sulfuric acid onto the site on said filter of the initial deposit of said urine sample so that the concentrated and stained bacteria are manifest at said site and any free dye deposited at said site with said stained bacteria is diffused remote and distal of said site in the form of a ring of free dye in the range of from about 10mm to about 30mm from the concentrated stained bacteria at said site.

6. A process as recited in Claim 1 wherein said stained and adsorbed bacteria is quantitated by comparing the color intensity of said stained bacteria on said filter to a color guide having colors representing the results of tests from samples having known concentrations of bacteria.

7. Apparatus for concentrating, immobilizing and staining bacteria from fluid samples, comprising:
(a) disk means having a lower surface and forming sample reservoir means intersecting said lower surface and forming an orifice;
(b) filter means;
(c) means retaining said filter means against said lower surface, a portion of said filter means being exposed to said sample reservoir means at said orifice, said filter means extending radially from said opening a sufficient distance permitting radial diffusion of free dye from said filter portion exposed at said orifice to thus leave concentrated and stained bacteria from said fluid sample at said exposed area of said filter means for color comparison indicating quantitation of said stained bacteria.

8. The apparatus of Claim 7, wherein:
(a) said sample reservoir is of upwardly diverging form and defines a sufficient volume for receiving a predetermined volume of said fluid sample, disk means defining lip means about said orifice at the intersection of said orifice and said lower surface, said lip means engaging said filter means;
(b) plate means for securing said filter means against said lower surface; and (c) means retaining said plate means and said filter means in intimate assembly with said disk means.

9. The apparatus of Claim 7, wherein:
(a) said disk means defines reaction well means permitting reaction of said fluid sample prior to deposit of said sample fluid into said reservoir means, upon substantially horizontal positioning of said disk means said sample fluid gravitating from said reaction well means into said sample reservoir means.

10. The apparatus of Claim 9 wherein:
(a) said disk means defines a plurality of spaced sample reservoirs and a plurality of reaction wells each communicating with respective sample reservoirs; and (b) said disc means defines base means orienting said disk means such that fluid samples in said plurality of reaction wells is prevented from entering said sample reservoirs, upon substantially horizontal positioning of said disk means said fluid samples gravitating from said reaction wells into respective sample reservoirs.

11 The apparatus of Claim 7, wherein said retaining means comprises a layer of material covering said filter means and having adhesive peripheral portions there of engaging said lower surface and retaining said filter means in encapsulated assembly with said disk means.