WO2004053482A1 - Method of rapidly staining microorganism and apparatus therefor - Google Patents

Abstract

A method of rapidly staining microorganisms in which use is made of a staining solution heated to temperature other than room temperature. The apparatus for such a microorganism rapid staining comprises multiple liquid storage tanks for reagent wherein a reagent in liquid form is stored; multiple or one liquid storage tank for washing liquid wherein a washing liquid is stored; at least one temperature control device connected downstream of the liquid storage tanks for reagent and capable of controlling the temperature of the reagent at specified one; multiple reagent discharge nozzles connected downstream of the temperature control device and capable of discharging the reagent; one or more washing liquid discharge valves connected downstream of the liquid storage tank for washing liquid and capable of discharging washing liquid; and liquid spray devices connected downstream of the reagent discharge nozzles, washing liquid discharge nozzles, or reagent discharge nozzles and washing liquid discharge nozzles and capable of spraying the reagent and washing liquid over a sample. Further, this apparatus for microorganism rapid staining may be equipped with a recovery storage tank for spent reagent recycling.

Description

 Specification

Microbial rapid staining method and microorganism rapid staining device

 INDUSTRIAL APPLICABILITY The present invention is used in a hospital or clinic or a research institute to quickly perform a staining treatment on a tissue section or a cell (in the present specification, these are collectively referred to as a sample) for microscopic observation. The present invention relates to a method and apparatus for staining microscope specimens. Background art

 At present, microbiological tests conducted in clinical laboratories still have a long-standing culture-based test method, so it takes more than one day to provide clinically useful results to doctors. Takes time. However, infectious diseases that require urgent and appropriate treatment, such as sepsis, meningitis, and pneumonia, are often meaningless in alternate reports. Although new methods have been introduced to overcome this time loss, microscopy, which has been performed as one of the conventional methods, has also been evaluated as a useful and rapid diagnostic method.

 In this microscopic examination, to easily diagnose the causative bacteria of the disease by microscopic observation, a sample smeared on a slide glass is stained. There is acid-fast staining. Of these, Gram stain is widely used as a representative method for microscopy because of its cost-effectiveness and handling flexibility.

The Gram stain method includes the Hucker method, a modified Hucker method, the Bartho 1 omew & Mittwer method, and the like. Use each way Although there are slight differences in the dyes and reagents used, the time required for staining is not much different.For example, according to the modified Hucker method, it takes about 30 to 60 seconds for each stain and about 2 seconds for decolorization. Generally, it takes 0 to 30 seconds, and it takes about 5 minutes to complete the process after washing.

 When the Gram staining method is used for diagnosis of infectious disease or other diseases, the diagnosis is made by identifying the gram-positive bacteria and gram-negative bacteria in the sample. For this discrimination, for example, the following procedure shown in Illustrated Clinical Bacteriology (Second Edition), p. 16 (Tomichi Sakazaki, Bunkodo) is used. First, prepare a sample on which a sample to be subjected to microscopic examination and a sample of positive and negative bacteria to be smeared on a single slide glass.The staining process includes pre-staining, washing with water, medium staining, washing with water, decolorization, After washing with water, post-staining, and washing, the dyeing work is completed, and the sample on the stained slide glass is observed by microscopy as a blue stained gram-positive bacterium and a red stained gram-negative bacterium. ing. In this case, all the steps are performed at room temperature, pre-staining and medium staining are about 6 CV seconds each, and washing, decoloring, and post-staining are each about 30 seconds, and about 5 seconds to complete all staining work. Needs a minute. This time is the same for both the application method and the automatic staining device.

In addition, various manual devices or automatic staining devices have been created in order to improve the efficiency of the above-described tests. For example, “Gram staining apparatus and method” in JP-A-8-43380, “Automatic staining apparatus for microscope specimens” in JP-A-10-91046, and JP 2000 — There is an "automatic coloring apparatus for coloring a sample for microscopic observation" in Japanese Patent No. 3 4 6 7 6 9. However, these devices do not reduce the staining time, but stain a large number of samples at once to improve the efficiency of the inspector. As a result, the specimens collected until the number of units required for the examination are completed are in a waiting state, and it is difficult to give the results of the examination at the point of examination. I got it. As a result, the results of the consultation become clear the day after the consultation, and treatment is started, and it is not possible to respond appropriately to patients with infectious diseases that require urgent treatment.

 The present invention provides a method and apparatus for rapidly staining to appropriately respond to such urgent patients. Disclosure of the invention

 In order to solve the above-mentioned problems, a rapid microorganism staining method according to the present invention uses a staining solution heated to room temperature or a room temperature. '

 Also, the microbial rapid staining device according to the present invention is connected to a reagent storage tank for a plurality of reagents, a washing liquid storage tank for a plurality or a single washing liquid, and a downstream side of the reagent storage tank. A temperature control means for controlling a reagent to a specified temperature; a plurality of discharge valves for a reagent connected to a downstream side of the temperature control means for discharging a reagent; a cleaning liquid connected to a downstream side of the storage tank for a cleaning liquid; One or more cleaning liquid discharge valves for discharging the reagent, the reagent discharge valve, the cleaning liquid discharge valve, or a downstream side of the reagent discharge valve and the cleaning liquid discharge valve, and a reagent, a cleaning liquid, or And at least one liquid ejecting device for ejecting the reagent and the washing liquid to the sample. BRIEF DESCRIPTION OF THE FIGURES

 Fig. 1 shows the conventional method in which the degree of blue enhancement of gram-positive bacteria and the degree of blue enhancement of gram-positive bacteria were stained by nitriding temperature, and the present invention in which the staining solution was heated to temperatures of 30 ° C to 60 ° C. FIG. 7 is a diagram in which the degree of blue enhancement when stained by the method is measured by Microanalyzer and compared.

Figure 2 shows the conventional method in which the degree of red enhancement of Gram-negative bacteria and the degree of red enhancement of Gram-negative bacteria were stained at room temperature, and the staining solution was heated to temperatures from 30 ° C to 60 ° C. FIG. 3 is a diagram in which the degree of red enhancement when stained by the method of the present invention was measured with a microanalyzer and compared.

 FIG. 3 is a diagram showing the staining procedure of the conventional method and the method of the present invention.

 FIG. 4 (a) is a side view of the rapid microorganism staining apparatus of the first embodiment, and FIG. 4 (b) is a top view of the rapid microorganism staining apparatus of (a).

 FIG. 5 (a) is a top view of the microbial rapid staining device of the second embodiment, partially showing through, and FIG. 5 (b) is a cross-sectional view of FIG. 5 (b) — (b). is there. BEST MODE FOR CARRYING OUT THE INVENTION

 Next, embodiments of the present invention will be described, but the present invention is not limited to the following embodiments. First, an embodiment of the rapid microorganism staining method according to the present invention will be described.

 Samples are prepared according to the above procedure adopted in the conventional method. Next, for staining, a method in which the present invention was applied to a modified Hucker method (hereinafter referred to as the present method) was used.

The reagent used for prestaining is crystal violet, the reagent used for medium staining is Lugol's solution, and the two solutions are both heated to 45 ° C, and the reagent for decolorization is an equal volume mixture of acetone and ethanol. It was used at room temperature. These reagents were purchased from Biomelyu Japan. The safranine solution used for post-dyeing was a diluent obtained by diluting a stock solution purchased from Wako Pure Chemical Co., Ltd. five-fold and heated to 45 ° C. Water for washing was used at room temperature. Prepare the pre-staining solution, medium staining solution, decolorizing solution, post-staining solution, and washing water set at each of the above temperatures, and prepare the previously prepared strains spread on the slide glass, etc., except for the washing water. Are covered with each reagent for 1 second, and then immediately washed with water for about 10 seconds. After that, the next process was adopted. These staining procedures are shown in Fig. 3 as the conventional method and the present invention. The procedure is to proceed sequentially from the top of the table to the bottom.

 From FIG. 3, it can be seen that the staining time required for 5 minutes in the conventional method can be reduced to 44 seconds, that is, about 1 minute or less in the method of the present invention.

 By microscopic examination, the sample stained by the method of the present invention was compared with the sample stained by the conventional method, and it was confirmed that equivalent staining results were obtained.

 In order to compare the conventional method and the method of the present invention, the same strain was used for preparing the sample for both the conventional method and the method of the present invention. That is, a plurality of compartments were provided on one slide glass, and two compartments among the compartments were smeared with positive and negative control bacteria, respectively, and the remaining compartments were smeared with strains, and used as samples. In accordance with the standard procedure, the positive control bacteria were Staphylcocccusaeureus ATCC 25923 as standard strains, and the negative control bacteria were EscherichiaicoliATCCC25922 as negative control bacteria. As a sample strain, a Gram-negative bacterium, which is slightly less stainable than a Gram-positive bacterium among clinical isolates, and a bacterium which is said to be particularly difficult-to-stain, were used. Gram-negative bacteria Toshite tooth E n t e r o b a c t e r a e r o g e n e s, Mo r g a n e l l a. Mo r g a n i i, K l e b s i e l l a p n e umo n i a e, P s e u d omo n a s a e r u g i n o s a, C i t r o b a c t e r k o s e i, E n t r o b a c t e r c l o a c a e, K l e b s i e l l a o x y t c a, H a e m o p h i l u s i n f l u e n z a e Roh each bacteria species wo used other. To measure the color enhancement of the stained sample, use the digital image measurement software Microanalysis, manufactured by Nippon Bora Digital Co., Ltd.

The prepared sample was stained according to the conventional staining method shown in Fig. 3, and In the staining according to the present invention, the liquid temperature of pre-staining, medium staining, and post-staining is changed from 30 ° C. to 60 ° C. at 5 ° C. intervals, and washing and decoloring are performed at room temperature. Was set to the value shown in FIG. 3 so that the total staining time was 44 seconds. Various searches for decolorization and post-staining described in the method of the present invention were conducted until the method shown in FIG. 3 was found. For decolorization, the effect of heating and the composition of the solution were examined. For post-dyeing, the effect of the concentration of the solution was also examined, and the solution composition and temperature of the method of the present invention, which exhibited rapid dyeing, were set. In order to examine the effect of heating in the method of the present invention, a number of slide glasses were used, and the average results are shown in FIGS. The sample is stained with the staining solution heated to each of the above temperatures, washed with water at room temperature, decolorized and the sample that has been stained is taken under a microscope, and the image is taken with the digital image measurement software installed on a personal computer.

The degree of color emphasis was measured and displayed by Microananalyzer. FIG. 1 shows the staining of the positive target bacteria as a standard strain on each slide glass, that is, the blue enhancement of the Gram-positive bacteria for each staining solution temperature. The room temperature at the left end in the figure is the measured value of blue enhancement after dyeing for 5 minutes in the method shown in FIG. 3 according to the conventional method. From this figure, it can be seen that the staining with the heated solution at 45 ° C according to the method of the present invention emphasizes the staining of Gram-positive bacteria as compared with the conventional method. FIG. 2 shows the staining of the negative control bacteria as a standard strain on each slide glass, that is, the degree of red enhancement of the gram-negative bacteria at each staining solution temperature. The room temperature at the left end in the figure is the measured value of the degree of red emphasis after dyeing for 5 minutes in the method shown in Fig. 3 using the conventional method. From this figure, it can be seen that the staining with the heating solution at 45 ° C according to the method of the present invention emphasizes the staining of Gram-negative bacteria as compared with the conventional method.

In this figure, it can be seen that the staining of Gram-negative bacteria is more emphasized in the whole range from 30 ° C to 60 ° C than in the conventional method. In addition, the method of the present invention is equivalent to or higher than the conventional method for all the samples of the above-mentioned hard-to-stain bacteria and gram-negative bacteria. Was confirmed to exhibit a staining action. .

 In the method of the present invention, the dyeing solution temperature is set to 45 ° C.This is because the dyeing time is set to 1 second as the minimum time for work, and the dyeing time for pre-staining and mordant colors is set to 3 seconds. If other conditions are set according to the method of the present invention, the temperature of the staining solution can be set in the range of 40 ° C to 50 ° C. According to the present embodiment, when the Gram stain method is used for diagnosis of infectious diseases and the like that require urgent appropriate treatment, the staining solution is set to an appropriate temperature and heated, and the conventional method takes about 5 minutes. By using the method of the present invention, the required staining time was reduced to about 1 minute or less, which enabled not only a more rapid diagnosis, but also a conventional dyeing method, which depends on individual skills. Since it takes only one second for bleaching, the effect of personal skills can be reduced.

 Next, an embodiment of the rapid microorganism staining apparatus according to the present invention will be described. First, a first embodiment of the rapid microorganism staining apparatus according to the present invention will be described. FIG. 4 (a) is a side view of the microbial rapid staining apparatus of the first embodiment, and FIG.

 It is a top view of the microorganism rapid dyeing | staining apparatus of (a).

The microbial rapid staining device 10 is composed of reagent storage tanks 11 a, 11 c, lld, and llf for storing reagents, and washing liquid storage tanks 1 1 b and 11 e for storing washing liquid. Three temperature control devices connected to each downstream side of the reagent storage tanks 11a, 11c, and llf to control the reagent to a specified temperature (the entire temperature control device is not shown), and these temperature control devices Are connected to the downstream of the reagent discharge valves 13a, 13c, and 13f that discharge the reagent, and are connected to the downstream of the reagent storage tank 11d to temporarily discharge liquid. Temporary storage tank 1 2 d that can be stored, reagent discharge valve 13 d connected to the downstream side of temporary storage tank 1 2 d to discharge reagent, and storage tank 11 1 b and lie for cleaning liquid Downstream Temporary storage tanks 1 2b, 1 2e that are connected and can temporarily store liquid, and a cleaning liquid discharge valve that is connected downstream of the temporary storage tank 12d and discharges a cleaning liquid that discharges a reagent 1 3b, 1 3e, reagent discharge valves 1 3a, 1 3c, 1 3f and washing liquid discharge valves 1 3b, 1 3e And a collecting and storing tank 19 for collecting and storing the used reagent and used washing liquid sprayed on the sample 18.

 Reagent storage tanks 11a, 11c, lid, and 11f are accumulator-type reagent storage tanks. Here, the accumulator type storage tank for reagents is one in which a pressure accumulator (not shown) using liquid pressure or air pressure is installed to make the inside of the storage tank for reagents 11a pressurized. Yes, for example, a liquid bag or air bag with elasticity is installed, and a pipe 40 is installed as a pressure source pipe (for example, a water supply pipe), and a coupling 41a is connected to the reagent storage tank 11a. The pressure is supplied via Similarly, pressure is supplied to the reagent storage tanks 11c, 11d, and 11f via the joints 41c, 41d, and 41f. In addition, if it is possible to install the ordinary reagent storage tanks 1 la, 11 c, lld, and 11 f at a position with a high water level until the required pressure is obtained with the liquid ejector 17 Can omit the pressure accumulator. These reagent storage tanks 11a, llc, lid, llf can be filled with different types of reagents, respectively.

The washing liquid storage tanks 11b and 11e are also pressure accumulating type washing liquid storage tanks similar to the reagent storage tanks 11a, 11c, 11d, and llf. Can be pressurized through the piping 40 and the joints 41b and 4le. The pressure accumulator is omitted if it is possible to install the ordinary washing liquid storage tanks 1 1b and 1e at a high water level until the required pressure is obtained by the liquid ejector 17. Can do W

Wear. Washing of the sample with room temperature washing liquid is sufficient, so the washing liquid in the temporary storage tanks 1 2b and 12 e installed below the washing liquid storage tanks 1 1b and 1 1e is sufficient. It is not always necessary to perform temperature control. In addition, tap water, distilled water, alcohol, and the like are used as the cleaning liquid.

 5 The temperature control device (the whole temperature control device is not shown) is composed of the reagent storage tanks 11a, 11c, 11f and the reagent discharge valves 13a, 13c, 13f. A thermometer that measures the temperature of the reagents in the temporary storage sections 12a, 12c, and 12f provided during connection and the temporary storage sections 12a, 12c, and 12f Heating to heat the reagents in the temporary storage sections 12a, 12c, and 12f

It comprises a 10 device (not shown) and a control unit (not shown) for controlling the heating intensity of the heating means based on the measured value of the thermometer. Temporary storage units 1 2a, 1 2c, 1 2 are connected to reagent storage tanks 1 1a, 1 1c, 1 1 f via pipes 14 3, 14 c, 14 f It is used to temporarily store the reagents from the reagent storage tanks 11a, 11c, and 11f.

15 The temperature control of the reagents in the temporary storage sections 12a, 12c, and 12f, which are injected by the heating device or control section of the control device, is performed. This temperature control device shall control the temperature in the temporary liquid storage section 12a, 12c, 12f in the range of 15 to 60 or 45 ° C soil 3 ° C. Is preferred. The reagent may be directly heated by a heating device, or may be temporarily stored.

The 20 parts 12a, 12c, 12f may be heated indirectly by heating. Examples of the heating device include an electric heater.

 The release time of the reagent discharge valves 13a, 13c, and 13f is controlled by a controller (not shown) or the like. For example, when the reagent discharge valve 13a is open, one of the temperature control devices (not shown)

The reagent stored in the 25 o'clock storage tank 12 a is circulated in the order of the pipe 14 a, the reagent discharge valve 13 a, the pipe 15 a, and the header 16. reagent The same applies to the discharge valves 13c and 13f. Similarly to the reagent discharge valves 13a, 13c, and 13f, the release time of the reagent discharge valve 13d and the cleaning liquid discharge valve 13b, 13e is controlled by a controller (not shown). Is controlled. For example, when the reagent discharge valve 13 d is open, the washing liquid stored in the temporary storage section 20 d is supplied with the pipe 14 d, the reagent discharge valve 1 '3 d, the pipe 15 d, and the header 1 They are distributed in the order of 6. The same applies to the cleaning liquid discharge valves 13b and 13e. The reagent discharge valves 13a, 13c, 13d, 13f and the cleaning liquid discharge valves 13b, 13e are fixedly supported by support members (not shown). 4a, 14b, 14c, 14d, 14e, 14f are reagent storage tanks 1 1a, 11c, lld, 11f and storage tank 1 for cleaning liquid When the inside of 1b and 11e is pressurized, it is preferable that the inside can withstand the pressurization.

 The header 16 is a header for collecting the pipes 15a, 15b, 15c, 15d, 15e, and 15f at one place.

 It is preferable that the liquid ejecting apparatus 17 is such that the ejection order, the ejection time, and the ejection amount of the reagent and the washing liquid to be ejected to the sample 18 are controlled by an ejection control device (not shown).

 The sample 18 is, for example, one in which a bacterial strain is applied to a flat surface of a slide glass, and it is preferable to arrange the flat surface so as to be inclined so that the reagent and the washing solution can easily flow downward.

The collection and storage tank 19 collects and stores used reagents and washing liquids, but does not have a collection and storage tank 19 because the reagents and washing liquids used in the present invention are not harmful substances. It can also be used as a device to allow the reagent to flow directly into the sewage. Next, an example of the staining action of the rapid microorganism staining apparatus 10 according to the present embodiment will be described.

 Here, a case where the present invention is applied to a rapid staining method, which is a modified Hucker method, as an example of a staining method will be described.

The reagent storage tank 11a is filled with crystal violet, and a part of the crystal violet is supplied through a piping 14a below the reagent storage tank 11a and a temperature controller (not shown). It is stored in the temporary liquid storage tank 12a. Then, the crystal violet is heated in a temporary storage tank 12a in a temperature control device (not shown) to a range of 15 to 60 ° C, preferably 45 ° C ± 3 ° C. I do. After that, according to the command from the control device (not shown), the opening timing and time of the reagent discharge valve 13a are controlled and the valve port is opened for at least 1 second, and the heated crystal violet is connected to the piping 15a , The header 16 and the liquid ejecting device 17 are circulated in this order, and the strain of the sample 18 is pre-stained by the ejection of the liquid ejecting device 17. The reagent that has been subjected to the pre-staining step is stored in a recovery storage tank 19 provided below the sample 18. After the pre-staining step is completed, the washing step is performed next. Tap water is filled in the wash tank 1 1b adjacent to the reagent tank 1 1a, and part of this tap water is connected to the lower pipe 14 1b of the wash tank. Through a temporary storage tank 12b at room temperature. In accordance with a command from a controller (not shown), the opening timing and time of the cleaning liquid discharge valve 13 b are controlled to open the valve for about 10 seconds, and the tap water in the temporary storage tank 12 b is discharged. The sample is passed through the pipe 15b, the header 16 and the liquid ejector 17 in this order, and the strain of the sample 18 is washed with the liquid ejector 17 by spraying. Used tap water that has completed this washing process is stored in the collection reservoir 19 located below the sample 18. When the water washing process is completed, the process enters the mordant process. For this purpose, the system of the reagent storage tank 11c adjacent to the washing liquid storage tank 11b is used. The reagent storage tank 11 c is filled with Lugol's liquid, and this Lugol's liquid is stored in a temporary storage tank 12 c 內 in a temperature control device (not shown) installed at the lower part. Heat up to 0 ° C, preferably 45 ° C ± 3 ° C. In accordance with a command from a control device (not shown), the opening timing and time of the reagent discharge valve 13 c are controlled, the valve port is opened for at least 1 second, and the heated Lugol solution is piped 15 c and the header 1 6. The liquid is sprayed in the order of the liquid ejecting device 17, and the strain of the sample 18 is dyed by the liquid ejecting device 17. After completion of the mordant process, the reagent is stored in a recovery storage tank 19 provided below the sample 18.

 When the medium dyeing step is completed, the washing step is started. For that purpose, the system of the washing liquid storage tank 11b filled with tap water adjacent to the reagent storage tank 11c is used. Here, a water washing step similar to the water washing step after the pre-dyeing step described above is performed.

When the water washing process is completed, the process proceeds to the decoloring process. For that purpose, the system of the reagent storage tank 11 d adjacent to the washing liquid storage tank 11 e is used. Reagent storage tank 1 1 d is filled with a mixture of equal amounts of acetone and ethanol. Then, this mixed solution is stored at about room temperature in a temporary storage tank 12 d below the reagent storage tank 11 d. This mixture does not necessarily need to be heated. Then, in accordance with a command from a control device (not shown), the opening timing and time of the reagent discharge valve 13 d under the temporary storage tank 12 d are controlled to open the valve port for about 10 seconds, The mixed solution in the temporary storage tank 12 d is circulated in the order of the pipe 15 d, the header 16, and the liquid ejecting device 17, and the bacteria of the sample 18 are decolorized by the ejection of the liquid ejecting device 17. After the decolorization process, the reagent is stored in the collection reservoir 19 located below the sample 18. Is stored.

When the decoloring step is completed, the washing step starts. For this purpose, a system gun of a washing liquid storage tank 11 _e filled with tap water which is in contact with the reagent storage tank 11 d is used. First, part of the tap water in the cleaning liquid storage tank 11e is stored at about room temperature in the temporary storage tank 12e below the cleaning liquid storage tank 11e. Then, in accordance with a command from a control device (not shown), the opening time and time of the reagent discharge valve 13 e are controlled and the valve port is opened for about 10 seconds, and the tap water in the temporary storage tank 12 e is discharged. The pipes 15e, the header 16 and the liquid ejector 17 are circulated in this order, and the strain of the sample 18 is washed with water by the ejection of the liquid ejector 17. The used tap water that has been subjected to the washing step is stored in a collection storage tank 19 provided below the sample 18.

 When the water washing step is completed, a post-dyeing step is started. For this purpose, the system of the reagent storage tank 11 f adjacent to the washing liquid storage tank 11 e is used. The reagent storage tank 11 f is filled with a 5-fold diluted safranine solution, and this 5-fold diluted safranine solution is stored in the lower part of the reagent storage tank 11 f temperature control device ( In the temporary storage tank 12f (not shown), the temperature is raised to 15 to 60 ° C, preferably to 45 ° C ± 3 ° C. After that, in accordance with a command from a control device (not shown), the opening time and the opening time of the reagent discharge valve 13f are controlled and the valve port is opened for at least 1 second to dilute 5 times the safranine solution. The liquid is passed through the pipe 15 f, the header 16, and the liquid ejecting device 17 in this order, and the strain of the sample 18 is post-stained by the ejection of the liquid ejecting device 17. After the post-dyeing step, the reagent is stored in a collection storage tank 19 provided below the sample 18.

When the post-dyeing step is completed, the washing step starts. For this purpose, a system of a washing liquid storage tank 11 e filled with tap water adjacent to the reagent storage tank 11 ί is used. Here, the washing step after the decolorizing step A washing step similar to the above is performed.

 As described above, the dram staining of the strain applied on one slide glass is performed, but the whole Gram staining can be completed in about 1 minute or less. Therefore, it is possible to appropriately respond to urgent patients, and to promptly provide test results.

 In the case of the present embodiment, it goes without saying that only one of the cleaning liquid storage tanks 11b and 11e can be used for cleaning.

 Next, a second embodiment of the rapid microorganism staining apparatus according to the present invention will be described. FIG. 5 (a) is a top view of the microbial rapid staining device of the second embodiment, showing a partially transparent view, and FIG. 5 (b) is a cross-sectional view of (b)-(b) of (a). is there.

Microbial rapid staining device 20 has reagent storage tanks 21 a, 21 c, 21 e, and 21 g for storing reagents, and a cleaning liquid storage tank for storing cleaning liquid (not shown). And three temperature control devices connected to each downstream side of the reagent storage tanks 21a, 21c and 21g to control the reagent to a specified temperature (the entire temperature control device is not shown), and Are connected to each downstream side of the temperature control device and are connected to the reagent discharge valves 23a, 23c, and 23g for discharging the reagent, and the downstream side of the reagent storage tank 21e, and are temporarily connected. A temporary storage tank 2 2 e that can store liquid, a reagent discharge valve 23 e connected to the downstream side of the temporary storage tank 22 e to discharge a reagent, and a cleaning liquid storage tank (not shown) Piping 40, fittings 4 1b, 4 1d, 41 f, 4111 and temporary storage tank connected via pipes 2413, 24d, 24f, 24h for temporary storage of liquid 2 2 b, 22 d, 22 f, 22 h, and one The cleaning liquid discharge valves 23 b, 23 d, 23 f, and 23 h are connected to each downstream side of the storage tanks 22 b, 22 d, 22 f, and 22 h, and discharge the cleaning liquid. Is provided. In addition, microbial rapid dyeing The color device 20 is connected to each downstream side of the reagent discharge valves 23a, 23c, 23e, and 23g, and the reagents are used to sample 28a, 28c, 28e, and 28g. The liquid ejecting devices 27 a, 27 c, 27 e, and 27 g are connected to the downstream side of the cleaning liquid discharge valves 23 b, 23 d, 23 f, and 23 h, respectively. Liquid ejectors 27 b, 27 d, 27 f, 27 h for injecting the cleaning liquid to samples 28 b, 28 d, 28 f, 28 h and liquid ejectors 27 a, 27 b , 27 c, 27 d, 27 e, 27 ί 27 g, 27 h (hereinafter referred to as liquid ejectors 27 a to 27 h) Collection tanks 29 a, 29 b, 29 c, 29 d, 29 e, 29 f, 29 g, 29 h (hereinafter referred to as collection tanks 29 a- Sample 28 a, 28 b, 28 c, 28 d, 28 e, 28 f, 28 g, 28 h (hereinafter, sample 28 a to 28 h Rotation of disk 30 on which can be placed and disk 30 A controllable motor 3 1 degrees, formed by a base 3 2 for fixedly supporting the motor 3 1.

Reagents for reservoir 2 1 a, 2 1 c, 2 1 e, 2 1 g is a reservoir for reagent accumulator type. Here, an elastic liquid bag is installed inside, a pipe 40 is installed as a pressure source pipe (for example, a water supply pipe), and the reagent storage tank 11a is connected via a joint 41a. It supplies pressure. In addition, until the required pressure is obtained with the liquid injection devices 27 a, 27 c, 27 e, and 27 g, the normal reagent storage tanks 21 a, 21 c, If it is possible to install 21e and 21g, the pressure accumulator can be omitted. These reagent storage tanks 21a, 21c, 21e, and 21g can be filled with different types of reagents, respectively. The cleaning liquid storage tank (not shown) is an accumulator type cleaning liquid storage tank. As shown in FIG. 5 (a), the cleaning liquid can be sent to the pipe 40 via the joint 41a. The pressurized cleaning liquid is supplied to the fittings 4 1a, 4 1 It is delivered to each desired part via b, 41 c, 41 d, 41 e, 41 f, 41 g, and 41 i. It should be noted that it is possible to install a regular washing liquid storage tank at a high water level position until the required pressure is obtained with the liquid injection devices 27 b, 27 d, 27 f and 27 h. In such a case, the pressure accumulator can be omitted. In addition, since the washing liquid at room temperature is sufficient for washing the sample with water, it is necessary to control the temperature of the washing liquid in the temporary storage tanks 22 b, 22 d, 22 22, and 22 h that can temporarily store the liquid. Is not necessarily. In addition, tap water, distilled water, alcohol, and the like are used as the cleaning liquid.

The temperature control device (the entire temperature control device is not shown) connects the reagent storage tanks 21a, 21c and 21g to the reagent discharge valves 23a, 23c and 23g. Temporary storage tanks 22 a, 22 c, and 22 g provided along the way, and a thermometer that measures the temperature of reagents in the temporary storage tanks 22 a, 22 c, and 22 g (not shown) ), A heating device (not shown) for heating the reagents in the temporary storage tanks 22a, 22c, and 22g, and a control unit for controlling the heating intensity of the heating means based on the measured value of the thermometer ( (Not shown). The temporary storage tanks 22a, 22c, _22l are connected to the reagent storage tanks 21a, 21c, 21g via the respective pipes 24 &, 24c, 24g. Which temporarily stores the reagents from the reagent storage tanks 21a, 21c and 21g, and is injected by the heating device or control unit of the temperature control device. The temperature of the reagents in the temporary liquid baths 22a, 22c and 22g is controlled. The temperature in the temporary storage tanks 22a, 22c, and 22g is preferably controlled to a range up to 1600 or a range of 45 ° C ± 3 ° C. Les ,. The reagent may be directly heated by a heating device, or may be heated indirectly by heating the temporary storage tanks 22a, 22c, and 22g. Examples of the heating device include an electric heater. Reagent discharge valves 23a, 23c and 23g are not shown Is controlled by the time. For example, when the reagent discharge valve 23 a is open, the reagent stored in the temporary storage tank 22 a of the temperature control device (not shown) is supplied with the pipe 24 a and the reagent discharge valve 23. a, piping 25a are distributed in this order. The same applies to the reagent discharge valves 23c and 23g. Control device (not shown) for the discharge valve 23 e for reagent and the discharge valves 23 b, 23 d, 23 f, and 23 h for the washing liquid as well as the discharge valves 23 a, 23 c, and 23 f for reagent The release time is controlled by such means. For example, when the cleaning liquid discharge valve 23 d is open, the cleaning liquid stored in the temporary liquid storage tank 23 d flows through the pipe 24 d, the cleaning liquid discharge valve 23 d, and the pipe 25 d in this order. Is the thing. The same operation is performed for the reagent discharge valve 23e and the cleaning liquid discharge valves 23b, 23f, and 23h. The reagent discharge valves 23 a, 23 c, 23 e, and 23 _g and the cleaning liquid discharge valves 23 b, 23 d, 23 f, and 23 h are fixed and supported by the support member 33. Have been.

 Piping 24 a, 24 b, 24 c, 24 d, 24 e, 24 f, 24 g, 24 h are reagent storage tanks 21 a, 21 c, 21 e, 21 g When the inside of the liquid storage tank is pressurized, it is preferable that the tank be capable of withstanding the pressurization.

 The liquid ejecting devices 27a to 27h may be controlled by a jet control device (not shown) to control the order, time and amount of jetting of reagents and cleaning liquids ejected onto the samples 28a to 28h. preferable. Although FIG. 5 shows an embodiment in which three injection nozzles are shown, the present invention is not limited to this, and it is sufficient that at least one injection nozzle is provided.

Samples 28a to 28h are, for example, slides coated with a strain on the flat surface, and the flat surface should be inclined so that reagents and washing liquid can easily flow downward. Is preferred. The collection storage tanks 29a to 29h collect and store used reagents and washing liquids. Since the reagents and the washing liquid used in the present invention are not harmful substances, the apparatus may not have the collecting and storing tanks 29a to 29h, and the reagent may be allowed to flow to the sewage as it is. In such a microbial rapid staining device, reagents are separately injected in each of the liquid ejecting devices 27a to 27h, so that each reagent can be separated and collected, so that each reagent can be reused. .

 The disk 30 is capable of placing the samples 28a to 28h radially at regular intervals and the sample 28a to 28h with a slightly inclined flat surface. Around the samples 28a to 28h, holes are provided for allowing the jetted reagents and washing liquid to flow down to the lower collecting reservoirs 29a to 29h.

 The motor 31 has a rotating shaft fixedly connected to the center of the disk 30 and can rotate the disk 30 horizontally about the center thereof. The rotation angle of the disk 30 is controlled by a control device (not shown) connected to the motor 31.

 Next, the staining action of the rapid microorganism staining apparatus of the second embodiment will be described.

Here, a case where the present invention is applied to a rapid staining method based on the modified Hucker method as an example of a staining method will be described. In the modified method of Hucker's rapid staining method, the steps using a chemical solution include pre-staining, medium staining, decolorization, and post-staining.When applied to Fig. 5, the reagent storage tank 21a is used for pre-staining. The system of the reagent storage tank 21c corresponds to medium staining, the system of the reagent storage tank 21e corresponds to decolorization, and the system of the reagent storage tank 21g corresponds to post-staining. On the other hand, the cleaning liquid storage tank 21 b system, the cleaning liquid storage tank 21 d system, the cleaning liquid storage tank 21 f system, and the cleaning liquid storage tank 21 h system all correspond to the washing process. is there.

 Next, each step will be described.

 First, a sample 28a coated with the bacterial strain on the upper surface is placed on the rotating disk 30 below the liquid ejecting device 27a. The reagent storage tank 21a is filled with crystal violet, and a part of this crystal violet is passed through a pipe 24a below the reagent storage tank 21a to a temperature control device (not shown). It is stored in the temporary storage tank 22a. Then, the crystal violet is added to a range of 15 to 60 ° C, preferably 45 ° C ± 3 ° C, in a temporary storage tank 22a in a temperature control device (not shown). Warm up. Thereafter, the opening timing and time of the reagent discharge valve 23a are controlled in accordance with a command from a control device (not shown) to open the valve port for at least 1 second, and the heated crystal biolet is piped 25 a, the liquid ejecting device 27a is circulated in order, and the strain of the sample 28a is pre-stained by the ejection of the liquid ejecting device 27a. The reagent that has been subjected to the pre-staining step is stored alone in the collection reservoir 29a provided below the sample 28a.

Next, the rotating disk 30 is stopped at a position rotated 45 degrees counterclockwise by the motor 31 rotated by a signal from the control device. At this time, the sample 28a to which the strain which has completed the pre-staining step is located at the position where the sample 28b was located at the time of the pre-staining step of the sample 28a (see FIG. 5 (a)). Therefore, the pressurized water (tap water) introduced from the washing water branch joint 41b passes through the washing liquid discharge valve 23b that has started opening operation in response to a signal from the control device, and the piping 25b for about 10 seconds. The sample is jetted toward the sample that has been subjected to the pre-staining step and pasted on the sample 28a located immediately below the liquid jetting device 27b from the liquid jetting device 27b, and performs a water washing process. After the washing step, the washing liquid is stored alone in a collection storage tank 29b provided below the sample 28a. Note that the cleaning liquid may be allowed to flow directly into the sewage. Also, be sure The washing liquid does not need to be heated. The same applies to the following washing steps.

 Next, the rotating disk 30 is stopped at a position rotated 45 degrees counterclockwise by the motor 31 rotated by a signal from the control device. At this time, the sample 28a to which the strain after the washing step has been adhered comes to the position where the sample 28c was in the pre-staining step of the sample 28a (see FIG. 5 (a)). The reagent storage tank 21 c is filled with Lugol's liquid, and a part of this Lugol's liquid is passed through a pipe 24 c below the reagent storage tank 21 c to a temperature control device (not shown). It is stored in the temporary storage tank 2 2 c. Then, this Lugol's solution is added to a range of 15 to 60 ° C, preferably 45 ° C ± 3 ° C, in a temporary storage tank 22c in a temperature control device (not shown). Warm up. Thereafter, the opening timing and time of the reagent discharge valve 23 c are controlled according to a command from a control device (not shown) to open the valve port for at least 1 second, and the heated lugol liquid is connected to the pipe 25. c. The liquid is sprayed in the order of the liquid ejecting device 27c, and the strain of the sample 28a is medium stained by the liquid ejecting device 27c. The reagent that has completed the medium dyeing step is stored alone in the collection reservoir 29c located below Sample 28a.

Next, the rotating disk 30 is again stopped at a position rotated 45 degrees counterclockwise by the motor 31 rotated by a signal from the control device. At this time, the sample 28a to which the strain that has completed the medium staining step is attached is located at the position where the sample 28d was in the pre-staining step of the sample 28a (see FIG. 5 (a)). Therefore, the pressurized water (tap water) introduced from the washing water branch joint 41d passes through the washing liquid discharge valve 23d that started opening operation in response to a signal from the control device, and flows through the pipe 25d for about 10 seconds. Then, the liquid is jetted from the liquid jetting device 27 d toward the sample that has been subjected to the medium dyeing process and that has been stuck on the sample 28 a that is positioned vertically below, and performs the water washing process. After the washing process, It is stored alone in the collection reservoir 29 d installed below the sample 28 a.

 Next, the rotating disk 30 is again stopped at a position rotated 45 degrees counterclockwise by the motor 31 rotated by a signal from the control device. At this time, the sample 28a to which the strain after the washing step was adhered comes to the position where the sample 28e was in the pre-staining step of the sample 28a (see FIG. 5 (a)). The reagent storage tank 21e is filled with a solution obtained by mixing equal amounts of acetone and ethanol as a decolorizing chemical necessary for the decolorization step, and a part of this mixed liquid is temporarily stored in the temporary storage tank 22e. It is stored in. The mixed liquid in the temporary storage tank 22 e passes through the reagent discharge valve 23 e that has started opening operation in response to a signal from the controller, and flows through the pipe 25 e for about 10 seconds. A jetting process is performed from the jetting device 27 e toward the sample stuck on the sample 28 a located vertically below to perform the decolorization process. After the decolorization step, the reagent is stored alone in the collection reservoir 29e provided below the sample 28a. In addition, the above-mentioned mixed solution does not necessarily need to be heated.

Next, the rotating disk 30 is again stopped at a position rotated 45 degrees counterclockwise by the motor 31 rotated by a signal from the control device. At this time, the sample 28a to which the strain that has been subjected to the decolorization step is adhered comes to the position where the sample 28f was attached in the pre-staining step of the sample 28a (see FIG. 5 (a)). Therefore, pressurized water (tap water) introduced from the washing water branch joint 41 f passes through the washing liquid discharge valve 23 f that has started opening operation in response to a signal from the control device, and the piping 25 f From the liquid ejecting device 27 ί via the, the liquid is jetted toward the sample that has been decolorized and pasted on the sample 28 a located vertically below, and the washing process is performed. After the washing process, the washing liquid is stored alone in the collection reservoir 29 f installed below the sample 28 a. Next, the rotating disk 30 is rotated by a signal from the controller. Data 31 stops at a position rotated 45 degrees counterclockwise. At this time, the sample 28a to which the strain after the washing step was adhered comes to the position where the sample 28g was located in the pre-staining step of the sample 28a (see FIG. 5 (a)). The reagent storage tank 2 1 g is filled with a 5-fold dilution of safranine solution as a chemical solution required for the post-staining step, and a part of this 5-fold dilution of safranine solution is used as a reagent storage tank 21 g Stored in a temporary shell tank of 22 g in a temperature control device (not shown) through 24 g of piping at the bottom. Then, the lugol solution is brought into a range of 15 to 60 ° C, preferably 45 ° C ± 3 ° C, in a temporary storage tank 22g in a temperature control device (not shown). Heat. After that, according to the command from the control device (not shown), the opening time and the opening time of the reagent discharge valve 23 g are controlled, and the valve port is opened for at least 1 second, and the heated Lugole solution is connected to the piping 2. 5 g and 27 g of the liquid ejecting device are circulated in this order, and the strain of the sample 28a is post-stained by ejecting the 27 g of the liquid ejecting device. The reagent that has completed the post-staining step is stored alone in 29 g of a recovery storage tank installed below the sample 28a.

Next, the rotating disk 30 is stopped at a position rotated 45 degrees counterclockwise by the motor 31 rotated by a signal from the control device. At this time, the sample 28a to which the strain after the washing step has been adhered comes to the position where the sample 28h was in the pre-staining step of the sample 28a (see FIG. 5 (a)). Therefore, pressurized water (tap water) introduced from the washing water branch joint 41h passed through the washing liquid discharge valve 23h, which started opening operation in response to a signal from the control device, for approximately 10 seconds, and the piping 25h. From the liquid jetting device 27 h via the, a jetting process is performed toward the sample that has been decolorized and that has been stuck on the sample 28 a that is positioned vertically below, and the water washing process is performed. After the washing step, the washing liquid is stored alone in a collection tank 29 h below sample 28 a. In this manner, the disk 30 was attached to the predetermined position of the disk 30 in FIG. For sample 28a, pre-stain, wash, medium stain, wash, decolorize, wash, post-stain, wash and change the Hucker each time the rotating disk is rotated 45 ° counterclockwise as described above. The dyeing process can be completed according to the rapid dyeing method of the method.

 The above shows the staining process of sample 28a, but as shown in Fig. 5 (a), if other samples are also placed on the disk 30, the same procedure as above can be repeated to repeat Can be sequentially stained. For example, the following procedure. First, the sample is placed on the disk 30 at the position of the sample 28a in FIG. 5 (a), and the pre-staining step is performed for 1 second. Next, the sample 28a is rotated 45 degrees counterclockwise by the motor 31 (the sample 28a is placed at the position of the sample 28b in FIG. 5 (a)), and a washing step is performed for 10 seconds. At this time, the next sample cannot be placed at the position of sample 28a in Fig. 5 (a). Because the injection time of the reagent in the pre-staining step is different from the injection time of the cleaning liquid in the washing step, and the same pressure source is used, so simultaneous and simultaneous injection of the reagent and the cleaning liquid cannot be performed. Because.

 Next, the sample 28a is again rotated 45 degrees counterclockwise by the motor 31 (the sample 28a is placed at the position of the sample 28c in Fig. 5 (a)), and the sample in Fig. 5 (a) is rotated. A new sample is placed at position 28a, and the first sample (the sample at position 28c in Fig. 5 (a)) is subjected to the medium staining process, and a new sample (Fig. 5 (a) The sample at the position of sample 28a) is subjected to the pre-staining step.

In this way, when the previously placed sample is in the washing process, no new sample is placed, and in the dyeing and decoloring processes, new samples are placed sequentially. Do the work. As a result, the rapid microorganism staining apparatus of this embodiment can mount up to four samples, and In addition, the steps of pre-staining, washing, medium staining, washing, decoloring, washing, post-staining, and washing are sequentially performed, so that dyeing can be performed quickly. According to the present embodiment, all steps of the gram dyeing operation for one sample can be completed in about 1 minute or less. Therefore, it is possible to appropriately respond to urgent patients, and to promptly provide test results. In addition, since a plurality of samples can be sequentially inspected, the number of sample inspections in a certain time can be greatly increased as compared with the conventional case, and an efficient inspection can be performed. Industrial applicability

 When using the Gram stain method for diagnosis of infectious diseases and other diseases that require urgent and appropriate treatment, the conventional microbial rapid staining method has reduced the dyeing time, which previously required about 5 minutes, to about 1 minute or less. The possibility has arisen to employ a small number of dyeing operations, including one that was not used for cost effectiveness. To further enhance the possibility, a low-cost device with a simple structure was required. This invention also improves the cost-effectiveness, allows one piece to be used for diagnosis quickly and at low cost, increases the chances of appropriate treatment even in an emergency, and promotes prevention of infectious diseases etc. .

Claims

The scope of the claims  1. Rapid microbial staining method using a staining solution heated to a temperature other than room temperature.  2. The method of claim 1, wherein the non-room temperature is in the range of 40 ° C to 50 ° C.  3. The method of claim 1, wherein the non-room temperature is in the range of 45 ° C ± 3 ° C.  4. A plurality of reagent storage tanks for storing reagents,  A plurality or a single washing liquid storage tank for storing the washing liquid,  Temperature control means connected to the downstream side of the reagent storage tank and provided at least one for controlling a reagent to a specified temperature;  A plurality of reagent discharge valves connected to the downstream side of the temperature control means for discharging a reagent;  One or more cleaning liquid discharge valves that are connected to the downstream side of the cleaning liquid storage tank and discharge the cleaning liquid,  The reagent discharge valve, the cleaning liquid discharge valve, or at least one liquid jet connected to the reagent discharge valve and the downstream side of the cleaning liquid discharge valve, for injecting a reagent, a cleaning liquid, or a reagent and a cleaning liquid to a sample. Microbial rapid staining device equipped with a device.  5. The temperature control means comprises: a temporary storage section provided in the middle of the connection between the reagent storage tank and the discharge valve; a thermometer for measuring the temperature of the reagent in the temporary storage section; The microbial rapid staining device according to claim 4, further comprising: a heating unit that heats the reagent in the liquid storage unit; and a control unit that controls heating intensity of the heating unit based on a measurement value of the thermometer. 6. The rapid microorganism staining apparatus according to claim 5, wherein the temperature control means controls the temperature in the temporary liquid part within a range of 15 to 60 ° C. 7. The microbial rapid staining apparatus according to claim 5, wherein the temperature control means controls the temperature in the temporary storage portion to be in a range of 45 ° C ± 3 ° C.  8. The rapid microorganism staining apparatus according to any one of claims 4 to 7, further comprising an ejection control means for controlling an ejection order, an ejection time, and an ejection amount of the reagent and the cleaning liquid ejected from the liquid ejecting apparatus.  9. The disk according to any one of claims 4 to 7, further comprising a disk on which at least one sample can be placed, the disk being rotatable horizontally about a center, and a rotation angle control mechanism for the disk. Microbial rapid staining device.  10. The rapid microorganism staining apparatus according to claim 9, further comprising a plurality of collection and storage tanks for individually collecting and storing each used reagent injected into the sample. 11. A pressurizing means for pressurizing the reagent storage tank, the cleaning liquid storage tank, or the reagent storage tank and the cleaning liquid storage tank to a pressure required by the projection device. The microorganism according to any one of claims 4 to 7, which comprises