JPH01124767A - Method and apparatus for agglutinating, immobilizing and dyeing bacteria - Google Patents

Abstract

PURPOSE: To effect a rapid germ inspection by attracting germs in a liquid sample to a filter that is electrified to a negative voltage electrostatically and then dyeing it with a coloring matter that functions in an acid region. CONSTITUTION: A filter pad 5 that is formed by such material as cellulose fiber is pinched between a disk 1 where an orifice 3 is formed and a filter retaining plate 6. An urine sample that is diluted to an acid dilution liquid is dripped to the center of the part of the orifice 3 of the filter pad 5 that is electrified to a negative voltage and then safranine O coloring matter solution with pH10 is dripped as a dyeing liquid. Further, HC1 with pH2.5 is dripped and a free coloring matter is diffused. At this time, when a remarkable dyed part appears at a part where the sample is dripped, a germ inspection is positive. The number of germs in a specimen can be obtained from the density of the color at the dyed part.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) This invention relates generally to procedures for testing bacteria in liquid secretions such as human body secretions, and more particularly to the detection of Gram-positive and Gram-negative bacterial species in secretion samples. TECHNICAL FIELD This invention relates to a rapid, efficient and inexpensive testing method and device for determining presence and concentration.

BACKGROUND OF THE INVENTION There is a need for a method for rapidly detecting bacteria in urine and various secretions.

Preparing urine specimens constitutes a major task in diagnostic laboratories. The most common urinary diseases: 5 diseases are urinary system infections (bacteriuria) in children, pregnant women, and patients with diabetes and Meijin disease.
It is. In hospitals, bacteriuria is the main infection caused by hospital life. Considering the frequent occurrence of asymptomatic urinary tract infections, bacterial testing must be simple and economical enough to allow routine testing. Accordingly, there is a need for a simple, rapid, and inexpensive differential test that facilitates diagnosis and expedites treatment of patients with bacteriuria.

A conventional method for detecting urinary tract infections is to inoculate an agar culture plate with urine and incubate the plate at an appropriate temperature (usually 37°C) for 24 to 48 hours.

In this case, bacteria are detected by the formation of colonies on the surface of the agar.

Since difficult-to-cultivate bacteria do not grow in conventional cultures, various agar media are required to detect these bacteria. This procedure is time consuming and expensive.

Durham staining method (Gram 5tain Method)
Although such staining techniques are known in the field of clinical bacteriology, they require light microscopy and the procedure is time-consuming and requires a skilled bacteriologist. Other conventional methods include dyeing the bacteria and then concentrating it by centrifugation or filtration. This method requires a chelating agent and a positively charged small-pore filter, and suffers from clogging and interference with substances such as anionic pigments and blood present in urine.

It has now been unexpectedly discovered that Gram-negative and Gram-positive bacteria can be easily and quickly detected by the composition according to the invention. The condensed bacteria stained by the compositions disclosed within this specification can be easily visualized without the need for light microscopy by specially trained personnel or the use of expensive perishable agar cultures. Detection is possible without This invention allows efficient detection of bacteria and reduces clogging and dye interference that are common in conventional methods. Further tests have shown that the use of the staining composition of the present invention allows constant omega analysis of bacteria at low cost, simple and rapid.

SUMMARY OF THE INVENTION Compositions are provided that stain both Gram-negative and Gram-positive bacteria. The composition includes a diluted inorganic acid solution and a pH-Q
It consists of a dye that dissolves in ~12% and preferentially stains bacteria, a washing reagent consisting of an oxalic acid, and a filter paper or glass filter with a net negative charge. Bacteria are electrostatically fixed and condensed on the filter and stained with a dye solution. Next,! The released dye is removed by a washing reagent.

Since the dye-bearing bacteria become visible, the color intensity of the filter (which is due to the dyed bacteria) and the intensity of the color obtained using known bacterial nitrates, such as Nomogram 7, are determined. Detection and weighing is possible by comparing with a scaled prototype.

This test usually takes less than 1 minute.

Accordingly, the main feature of this invention is to provide a rapid bacterial detection and metering method and apparatus that allows rapid initiation of treatment of patients with bacterial infections.

Another feature of the present invention is a novel method and method for detecting bacteria that can reduce the time required for detection of equipment that may be present in a human secretion sample by simultaneously conducting multiple tests to detect and isolate bacteria. The purpose is to provide equipment.

Another feature of this invention is to detect the presence and concentration of Gram-positive and Gram-negative bacterial species in the secreted fluid → nonpure!
and to provide novel methods and apparatus for certifying IT.

A further feature of the invention is the use of a dye for staining bacteria in conjunction with a filter made of paper, glass, or other suitable material having a net negative charge, which electrostatically fixes and condenses the bacteria prior to staining them. The object of the present invention is to provide a novel method and device for bacterial detection.

The feature of this invention is to visually compare the color intensity of the dyed bacteria on the filter surface with a prototype scale such as a nomograph based on the color intensity obtained using a known concentration of bacteria A. By i
The object of the present invention is to provide a novel method and apparatus for listening to music.

EXAMPLES The present invention relates to compositions useful for staining both Durham-negative and Gram-positive bacteria, as well as various methods for detecting and quantifying bacteria in test fluids. Broadly speaking, the dyeing devices δ and compositions thereof of the present invention have a filter matrix with a net negative charge, a dye capable of staining bacteria in basic pl+, and a free dye removed from the filter fibers but not from the bacteria. It consists of a cleaning reagent. Fixed bacteria that are electrostatically adsorbed to the filter are easily visible and easily distinguishable from the free dye diffused from the stained bacteria by the washing reagent.

Since the color intensity of the filter is proportional to the number of bacteria in the test solution,
Quantitative analysis of bacteria can be performed by comparing the color intensity of the stained bacteria on the filter surface with known standard values.

The compositions and methods of the invention are particularly applicable to the detection and quantification of bacteria in urine. That is, according to the present invention, it is possible to detect urinary tract infection quickly and economically.

In particular, it has been discovered that dyes soluble at pH about 8-12 that stain bacteria work well in the absence of chelating agents. Bacteria are stained by contacting these organic matter electrostatically adsorbed to the negatively charged filter surface with a protein staining dye having a pl+ of about 8 to 12. pl+about 8-12
Any bacterial staining dye that is soluble in can be used. However, the preferred dye is safranin.
0 or basic fuchsin can be used.

Bacteria stained according to the invention, if present,
If the concentration is 0.000/-1 or more, or if it is concentrated, it can be easily detected in the darkness.

The dyed bacteria dispersed in the solution are easily visible upon concentration. Even if the bacteria concentrated to a sufficient concentration are dispersed and stained with f, the presence of IO bacteria becomes obvious immediately.

Precipitation and trapping of bacteria by filtration is an example of an effective method of concentrating bacteria. In the present invention, bacteria in the urine (whose equipotential charge has been altered by the l!ll diluent) are adsorbed to the negatively charged filter surface.

Since this diluted urine solution is spread over a wide area, the bacteria in the solution are electrostatically adsorbed to the filter at the locations where the urine has adhered by applying an effective bacteria concentration method. According to the method of the invention, the bore size of the filter need not be smaller than the bacteria in the test.

The filter surface does not have a net positive charge (it is necessary to use one with a net negative charge. The diluent presented herein reverses the equipotential charge of negatively charged bacteria, making them Therefore, the bacteria in the urine dilute solution are adsorbed to the filter matrix and fixed electrostatically, while
Free dyes, urine pigments, and other urinary compounds diffuse from the confined locations of adsorbed bacteria, thereby allowing detection of dyed organic matter.

The dye used to stain the bacteria at the site of attachment of the diluted urine mixture diffuses out from the fixed, stained bacteria adsorbed on the filter. By adding a washing reagent to the site where the diluted urine solution has adhered, it is possible to promote the diffusion of the free dye from this site without removing the staining of the immobilized 11Ilii1.

In the drawings, FIG. 1 shows an embodiment of the present invention, and this device includes:
It consists of a flat, round or square, rigid or semi-rigid disc 1. The disc may be machined or molded or thermoformed from various materials, such as polystyrene. The disk preferably has dimensions of about 1 to 4 inches in width and has a reservoir 2 formed at or near its center consisting of a concave or rounded recess.

A small diameter and short orifice 3 is formed at the bottom of the reservoir 2 and extends over the entire thickness of the disk. The orifice diameter ranges from about 0.06 to about 0.25 inches.

Although not necessary for operation in accordance with this embodiment, the disk can be provided with an orifice lip 4 projecting from the lower surface around the orifice. This orifice lip 4 allows the liquid to flow out more efficiently to a precise position on the filter band, and also serves to prevent the liquid from spreading on the upper surface of the filter pad. A filter pad 5 is placed close to one side of the disk so that it is in contact with the orifice or orifice rib. The filter pad is made of cellulose, fiberglass or black suitable filter material and exhibits a net negative charge.

The position of the filter pad is maintained by "sandwiching" the pad between the disk and the lower filter holding plate 6. The retaining plate is machined, molded or thermoformed and has four rectangular or rounded sections of slightly larger dimensions than the filter pad. A retaining plate is affixed to the disc to secure the filter pad in place. As a method of attachment, appropriate means such as adhesion, solvent welding, and ultrasonic welding are used. Alternatively, the disk and retaining plate can be joined together by hinges, snaps or threads, and the filter pad can be housed therein.

4-6 illustrate another embodiment of the invention in which an adhesive label or tape 8 is applied to the underside 9 of the disk 10 to secure the filter pad.

According to this, there is no need to provide a lower filter holding plate as in the first embodiment.

Each of the above practical examples is useful as a single-use, disposable, portable unit for detecting bacteria, especially in situations where conventional bacteria detection methods are cumbersome and difficult to implement, for example, for consumer use at home stores. It is applied to the detection of bacteria in liquids such as urine in sales, clinics, screening clinics, etc.

As shown in FIG. 4, the disk 10 is formed with reaction holes 11 extending in the width direction from four conical parts. As shown in FIG.
When the test liquid is on a flat horizontal surface, the test liquid is stored in the reaction hole 11.

Next, by rotating the device from the position shown in Fig. 5 in the direction 90' + Then, it flows into the four conical parts. The liquid then passes through the orifice and contacts the filter pad where it is absorbed.

This feature of the present embodiment is particularly useful in tests that require incubating or reacting a bacterial suspension with a chemical before performing a quantitative test of the bacteria. In particular, when conducting a test to determine the sensitivity B- to antibiotics, if the organic matter is resistant to the antibiotic, bacteria should be exposed to the appropriate antibiotic for a period during which growth of the organic matter would occur. This can be done by culturing a suspension. After 0 hours of incubation, the device was rotated to a horizontal position and the bacteria treated with antibiotics! Settlement tests can be carried out with the dyeing compositions of the invention by draining the 1 suspension into a conical recess and a filter pad.

The disposable multiple testing device shown in FIG. 4 is formed by molding or thermoforming a device using two or more units of this embodiment. This multiplex test device is particularly applicable to tests for determining sensitivity to antibiotics. By pre-immersing an appropriate antibiotic into the reaction hole, simple test preparation and multiple assay of sensitivity to the antibiotic can be performed.

The above embodiments of the present invention are merely illustrative;
It will be appreciated that other embodiments and modifications are possible without departing from the spirit and scope of the invention. ), the compositions and methods of the present invention can be applied to liquids such as 1L of culture medium.
Bacteria can be dyed and detected in blood, saliva, food washing liquid, milk, water, etc., and can also be applied to stain bacteria from these liquids adhering to the filter surface.

The minimum number of bacteria that can be detected by this staining procedure varies somewhat depending on the condition of the bacterial culture medium and the growth phase, and generally, for highly viable bacteria, it is approximately 100,000 pieces/
It is detected at the level of In1. On the other hand, non-viable or slow-growing organic matter is difficult to stain, and therefore a higher level of Apparatus I bacterial concentration is required for detection.

The examples described below are merely illustrative of the invention and are not intended to limit the invention.

Example 1 Using a conventional pipette, 2wI from a patient with bacteriuria
4 urine was collected in a test tube, and an acidic diluted solution of 8F4 (
HC with pH 1.5! J) was added. Four drops of the mixed solution thus obtained were dropped onto the center of the negatively charged filter. Next, three drops of Safranin O dye solution were dropped onto the urine/diluent mixture droplet area on the filter. The Safranin 0 dye solution used here was p
H1o carbonate borate hydroxyl! 1!
This is a solution prepared by adding Safranin O dye to a buffer solution at a ratio of 1:1000. Next, add 3 drops of cleaning solution (pl+
2.5 HCjl) was dropped onto the same area, and the free dye was diffused from the same area. Furthermore, three drops of cleaning liquid were dropped to perform secondary cleaning. Then, the same part has a diameter of 3~
4m prominently stained area (area where stained bacteria exist)
was expressed. At this time, a free colored wood ring with a diameter of 25#W was formed around the dyed area. This indicates that the bacteriuria test is positive. The density of the color of the stained area was compared with the color of the stained area obtained from a sample with a known number of bacteria, and as a result, the number of bacteria in the sample was 1.000,000 per 11 d of urine. Incidentally, as a result of plate culture of this specimen on agar, the same measurement value was obtained, proving that the measurement result by the method of the present invention is correct.

Example 2 Similarly, 2W4 urine is collected from a healthy person (who does not have a urinary tract infection according to conventional tests), and 8
A drop of dilute solution (Hll of pH 1.5) was added. This mixed solution was taken as 4W4 and dropped onto the center of the negatively charged filter. Hereinafter, the same operations as in Example 1 were performed. As a result, no stained area appeared at all in the area where the urine/diluent mixture was dropped, and two diameter areas centered around the same area were found.
Only a 5 m free dye ring was formed. This indicates that the bacteriuria test result is negative. Note that this result has been verified by plate culture of this detection substance on agar.

Example 3 A test was carried out for common bacteria using the procedure described in Examples 1 and 2 and the apparatus shown in FIG. The measurement results are shown in Table 1. Table 1 Types of bacteria.

Normal urine (no bacteria) 0 E. coli
++ Nis Aureas (s, aur)
cus) + +
++Busoidmopus ++Group A Streptococcus +Group 10B Streptococcus
++Klebsiella pneumoniae ＋
+*Measurement results at the dripping area of the urine/diluent mixture on the filter surface 〇-White +=Pale pink →-+=Pink + Juto=Red ++-1-+ = Dark red example 4 Example 1 and Example 2 Bacterial samples of various concentrations (normal urine with various concentrations of E. coli) were tested using the procedure described in 1. and the apparatus shown in FIG. The results are shown in Table 2. In addition, the determination of dyeing strength is based on Example 3.

Table 2 E. coli concentration Stained area Ifi (Number per Id) io, ooo.

50.000 + 100.000 + +1□ooo
, ooo + ten + 10.00.0.
000 + + + +Example 5 Antibiotic sensitivity was measured as follows. First, the fourth
The multiple inspection devices shown in the figure were arranged vertically. and,
One drop of the antimicrobial solution was added to each of reaction holes Nα1-8, and one drop of Polmarin solution and distilled water were added to reaction holes NQ9, No. 10, respectively. Next, the bacterial suspension was diluted to a bacterial concentration of 17! Hit 100.00
Prepare a bacteria-containing solution of
It was dropped onto n1-10. Next, the entire device was incubated at 37° C. for 3 to 4 hours. After this incubation,
As a diluent, 4 drops of p-old, 5 HCJI solution were dropped into each reaction hole. Next, the apparatus was placed horizontally and the mixed solution was moved from the reaction hole into the reservoir. After the solution was absorbed into the filter pad, the dye solution and wash solution were dripped onto each filter according to the procedures of Examples 1 and 2 above. After this staining process was completed, the staining intensity of the stained area on the filter was visually inspected. The formalin-treated sample (reaction hole 9) was pink (++) and served as a zero-hour control. In addition, reaction well 10 exhibited a dark red color (++++) and was used as a non-inhibited growth control. Reaction hole 11
Antibiotic sensitivity was tested by comparing the staining intensity of Q1-8 with the above two controls. If the staining intensity is equal to or stronger than that of the growth control, this indicates that the bacterium has resistance to the antibiotic. On the other hand, if the staining intensity is equal to or weaker than the zero hour control, it indicates that the bacteria are susceptible to the antibiotic.

[Brief explanation of the drawing]

FIG. 1 is a plan view of a filtering device made according to the present invention, FIG. 2 is a sectional view taken along line 2-2 in FIG. 1 is a cross-sectional view showing the filtration device shown in FIGS. 1 and 2 in an assembled state, and FIG. 4 is a plan view showing a filtration device for simultaneously testing a large number of human body secretion samples on the separate actual flow side of the present invention. , FIG. 5 is a sectional view taken along line 5-5 in FIG. 4 showing one position of the filtration device shown in FIG. 4, and FIG. 6 is a view similar to FIG. It is a sectional view showing a posture. 1.10... Disc 2... Reservoir 3... Orifice 4... Orifice lip 5... Filter pad 6... Filter holding plate 8... Tape 9... Bottom surface 11...Reaction Hole 12...Bottom Applicant Applied Polytechnology, Inc. Representative Patent Attorney Hidehiko Okada (2 others) ~Co ~- ≧

Claims (11)

[Claims] (1) A method for aggregating, fixing, and staining bacteria contained in a liquid sample, comprising: a) electrostatically adsorbing the bacteria in the liquid sample to a designated area of a filter that has a net negative charge; b) ) staining the adsorbed bacteria with a dye that functions in an acidic region; c) diffusing free dye from the specified area on the filter, and applying only the stained bacteria to this specified area for comparative quantification; A method for agglutinating, fixing and staining bacteria, comprising: a step of leaving behind; (2) The method for aggregating, fixing, and staining bacteria according to claim 1, which comprises the step of diluting the sample solution with dilute hydrochloric acid, dilute nitric acid, or dilute sulfuric acid having a pH of 1 to 3. (3) The dye is safranin O or basic fuchsin, which is soluble in a buffer solution at pH 8 to 12 and functions in this pH range;
Patent characterized in that it is dissolved in a buffer solution selected from the group consisting of 8 to 12 potassium borate solutions, potassium carbonate solutions and potassium hydroxide solutions, the concentration of which is from 0.1% to 0.001%. A method for aggregating, fixing and staining bacteria according to claim 1. (4) The diffusion is achieved by washing the stained bacteria with a washing liquid, and the washing liquid is dilute hydrochloric acid, dilute nitric acid or dilute sulfuric acid with a pH of 2 to 4. Bacterial agglutination, fixation and staining methods. (5) the sample is urine, and a) the urine is diluted with a diluent of an inorganic acid having a pH of 1 to 3 to dissolve the precipitate in the urine;
b) Dropping the urine/diluent mixture onto the surface of the filter; c) Adsorbing the urine/diluent mixture onto the filter. and the diffusion is carried out by dropping a cleaning solution of an inorganic acid selected from the group consisting of dilute hydrochloric acid, dilute nitric acid and dilute sulfuric acid with a pH of 2 to 4 onto the drip area of the urine/diluent mixture, As a result, the aggregated and stained bacteria are expressed in that area, while the free dye deposited in that area along with the stained bacteria is diffused out of the area, forming a sedimentation ring with a diameter of about 10 mm to 30 mm centered on that area. A method for aggregating, fixing and staining bacteria according to claim 1, characterized in that the method comprises: (6) The number of bacteria in a sample is quantified by comparing the staining intensity of the bacteria with the staining intensity obtained from a sample containing a known number of bacteria. Bacterial agglutination, fixation and staining methods described. (7) A device for flocculation, fixation and staining of bacteria contained in a liquid sample, comprising: a) a disk device having a lower surface, a reservoir device for the sample and an orifice intersecting this lower surface; b) a filter device; c) ) a retaining device for retaining the filter device on the lower surface, a specific area of the filter device communicating with the reservoir device by the orifice, and the filter device radially extending about the orifice. sufficiently wide to allow radial diffusion of free dye from said specific area, thereby leaving aggregated and stained bacteria in said specific area, allowing quantification of bacteria by comparison of staining intensity. A bacterial agglutination, fixation and staining device characterized by: (8) The reservoir device is mortar-shaped and has a sufficient volume to receive the sample, and the disk device has an annular lip at the intersection of the orifice and the lower surface, and the annular lip is 8. The disk device according to claim 7, wherein the retaining device is a plate, and the plate and the filter device are fixed to the disk device by means of a fixing device. Bacterial agglutination, fixation and staining equipment. (9) The disk device has a reaction hole through which a reaction of the sample is performed before the sample is introduced into the reservoir device, and then the disk device is moved to a horizontal position. 8. The bacterial aggregation, fixation and staining device according to claim 7, wherein the sample is poured into the reservoir device from the reaction hole. (10) The disk device has a plurality of separately arranged reservoir devices and a plurality of reaction holes communicating with each of the reservoir devices, and the disk device has a bottom member, and the bottom member allows the The orientation of the disk device is set so that the samples in the plurality of reaction holes do not enter the reservoir device, and then, when the disk device is moved horizontally, the sample is transferred from the reaction holes to each of the reservoir devices. 10. The apparatus for aggregating, fixing and staining bacteria according to claim 9, wherein the apparatus is poured into the apparatus. (11) The holding device is a layered material that covers the filter device, and an adhesive is applied to the peripheral edge of the holding device for adhering to the lower surface of the disk device to hold the filter device to the disk device. The apparatus for aggregating, fixing and staining bacteria according to claim 7, characterized in that: