Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
  • C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
  • C09B67/0071—Process features in the making of dyestuff preparations; Dehydrating agents; Dispersing agents; Dustfree compositions
  • C09B67/0083—Solutions of dyes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

• the present invention relates to a stain for microorganisms, and more particularly, to a stain capable of staining microorganisms such as bacteria and fungi in a shorter time than conventional methods, and a method for detecting microorganisms using the same.
• the caustic force method and the caustic force method with dimethylsulfoxide do not stain bacteria, so it is difficult to detect Malassezia bacteria, and you must be skilled in measuring under a microscope for detection. There is a problem that must be done.
• the Parker ink monocaustic potash method can stain bacteria, but has a problem that it takes several hours to 10 minutes to stain.
• the type of ink that can be used for dyeing is limited, and the ink itself is not a single component, so that the stainability of bacteria may change due to a change in the ink formulation or the like. There is also a problem.
• an object of the present invention is to solve the problems of the pretreatment in the conventional direct microscopy, and to provide a staining agent for staining microorganisms in a shorter time and a method for staining and detecting microorganisms using the same. It is in. Disclosure of the invention
• the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, by using a dye containing an alkaline substance and a specific dye as a staining agent used for staining microorganisms.
• the present inventors have found that microorganisms can be stained and detected in a short time, and have completed the present invention.
• the present invention provides a stain for a microorganism characterized by containing an alkaline substance and a diazo-based dye or a xanthene-based dye. It is intended to provide a method for detecting a microorganism, characterized in that the microorganism is stained by adding the microorganism, and then the stained microorganism is detected.
• FIG. 1 is a micrograph (X400) showing staining with the staining agent of Example 1.
• FIG. 2 is a micrograph (X400) showing staining with the staining agent of Example 7.
• the microorganism staining agent of the present invention contains an alkaline substance and a diazo dye or a xanthene dye.
• the alkaline dyes used in the dye of the present invention include inorganic hydroxides such as potassium hydroxide, sodium hydroxide, barium hydroxide, potassium carbonate, sodium carbonate, ammonium carbonate, tripotassium phosphate, and phosphoric acid.
• Organic potassium, alkylamines such as methylamine, ethylamine, isopropylamine, etc., methanolamine, ethanolamine, diethanamine, trietano, etc.
• Alkanolamines such as monolamine, isopropanolylamine and diisopropanolamine. These al powers Of the lithium substances, preferred are sodium hydroxide and reduced hydroxide, and particularly preferred are reduced hydroxide.
• the amount of the alkaline substance in the dye of the present invention is 10 to 40% by mass.
• % j preferably 15 to 30%.
• diazo dyes such as Shikago Sky Blue 6B, Evans Bull I, Direct I-15, Trypan Blue, Benzopurpurin 4B, Congo Red etc. are preferable, and particularly, Chicago Sky Blue 6B is preferable.
• the compounding amount of the diazo dye in the dye of the present invention is 0.01 to 2%, preferably 0.1%! ⁇ 1%.
• rhodamine B rhodamine B base, rhodamine 123 hydrate, rhodamine 6G, rhodamine 110, rhodamine 575 and the like are preferable. Particularly, rhodamine B is preferable.
• the amount of the xanthene dye in the dye of the present invention is 0.001-1%, preferably 0.05-0.5%.
• the dyeing agent of the present invention may contain methanol and / or dimethyl sulfoxide in addition to the above components.
• the preferred blending amount of methanol in the dye of the present invention is 0 to 30%, particularly 0 to 20%.
• the preferred amount of dimethyl sulfoxide is 0 to 20%, particularly 0 to 10%.
• the blending of methanol and / or dimethyl sulfoxide described above suppresses the staining of cells, but does not change the staining of microorganisms. Makes detection easier.
• the diazo dyes or xanthene dyes for example, those having low solubility in an alkaline solution such as rhodamine B are preferably added in view of increasing the solubility.
• the dye of the present invention is prepared by dissolving the above alkaline substance in water to prepare an alkaline solution, and then adding a diazo dye or a xanthene dye and, if necessary, methanol and / or dimethyl sulfoxide. This is done by mixing.
• the solubility of the above dyes in alkaline solutions It is preferable to dissolve those having a low value in advance in one or more of methanol and dimethyl sulfoxide, and then mix them with an alkaline solution.
• the staining agent of the present invention thus obtained can stain, for example, dermatophytes such as Trichophyton, fungi such as Candida and Malassezia, and bacteria such as Gram-negative bacteria and Gram-negative bacteria.
• dermatophytes such as Trichophyton
• fungi such as Candida and Malassezia
• bacteria such as Gram-negative bacteria and Gram-negative bacteria.
• the staining agent of the present invention is applied to a sample collected from a site where the presence of these microorganisms is suspected.
• Various samples can be used according to the sampling site. For example, scales, vesicles, vesicle lids, keratin parts of papules, etc. are used to detect Trichophyton in feet and hairs as microorganisms. In the subungual stratum corneum, etc. In addition, in acne ⁇ acne, the diseased hair easily fall out of the focus is used. The amount of the dye of the present invention added to the sample may be about several drops.
• the staining agent of the present invention Since the staining agent of the present invention has high heat stability, it stains microorganisms in a shorter time.After adding the staining agent of the present invention to a sample, the sample is heated at 60 to 80 ° C by a constant temperature hot plate or the like. It can be heated for about 2 to 5 minutes.
• the sample on which the staining agent of the present invention has acted is, for example, placed on a slide glass, covered with a cover glass, and then lightly pressed with a glass rod or the like from above the cover glass to thin the sample. After being spread, it is observed with a microscope or the like at a magnification of 100 to 200 times to detect the presence or absence of staining.
• the parasitoid of fungi such as dermatophytes, Candida and Malassezia orchid in the sample can be observed by microscopic examination at a magnification of about 400 times. This makes it possible to detect its presence.
• the microorganism in the sample is a bacterium such as a gram-positive bacterium or a gram-negative bacterium, the presence thereof can be detected by microscopic examination at a magnification of about 1000 times.
• a particularly preferred form when using a diazo dye examples thereof include those containing 15 to 30% of an alkali substance, 0.1 to 1% of a diazo dye, 0 to 20% of methanol, and 0 to 2% of dimethyl sulfoxide.
• the alkali substance is 15 to 30%
• the xanthene dye is 0.05 to 0.5%
• the methanol is 0 to 30%
• Those containing 5% to 20% of dimethyl sulfoxide can be mentioned.
• Example 1 Example 1
• Chicago Sky Blue 6B (manufactured by Kanto Chemical Co., Ltd.) was added to a 20% potassium hydroxide solution to a concentration of 0.5%, mixed and dissolved to prepare a staining agent.
• Lesion tissue (scale) was collected from the site suspected of superficial mycosis and used as a test sample. This sample was placed on a slide glass, the stain of Example 1 was dropped, and left at room temperature for 20 to 30 minutes to soften the lesion tissue. After the tissue had softened sufficiently, a cover glass was placed over the cover glass, lightly pressed with a glass rod or the like from above the cover glass, spread thinly, and observed with an optical microscope.
• Example 2 Staining and microscopy were performed in the same manner as in Example 2 except that the mixture was left standing at room temperature and heated on a constant-temperature hot plate at 60 to 80 ⁇ for 2 to 5 minutes.
• the following dyes were prepared with various concentrations of methanol (MeOH) and dimethylsulfoxide (DMSO) after fixing the concentration of Chicago Skyble 16B at 0.5% and the KOH concentration at 20%. Then, the degree of staining of specimen cells and the degree of staining of microorganisms were evaluated according to the following criteria. In addition, the characteristics of the coloring agent were comprehensively evaluated. Table 1 shows the results.
• Staining agent A MeOH 0% / DMSO 0%
• Staining agent B MeOH 10% / DMSO 0%
• Staining agent F MeOH 0% / DMSO 1%
• Staining agent G MeOH 10% / DMSO 1%
• Staining agent H MeOH 20% / DMSO 1%
• Staining agent I MeOH 0% / DMSO 2.5%
• Staining agent J MeOH 10% / DMSO 2.5%
• Staining agent K MeOH 20% / DM SO 2.5%
• ⁇ Can be used as a dye.
• X Cannot be used as a dye.
• FIG. 8 shows a photomicrograph at 400x magnification.
• staining red
• FIG. 8 A micrograph at a magnification of 400 ⁇ is shown in FIG. Example 8
• Staining agent O MeOH10% / DMSO10%
• Staining agent R Me0H40% / DMSO10%
• Staining agent S Me0H0% / DMSO2 5%
• the staining agent of the present invention is capable of staining and detecting microorganisms such as fungi and bacteria in a short time. Further, the dyeing agent of the present invention has high storage stability and heat stability, and does not have a problem of deterioration in dyeability due to long-term storage and heating during dyeing.
• the staining agent of the present invention is extremely effective as a staining agent for microorganisms such as fungi and bacteria.

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• Chemical & Material Sciences (AREA)
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• Toxicology (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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• 2003
  • 2003-04-25 JP JP2003121701A patent/JP4370118B2/ja not_active Expired - Lifetime
• 2004
  • 2004-04-08 US US10/554,285 patent/US7294154B2/en not_active Expired - Lifetime
  • 2004-04-08 ES ES04726658T patent/ES2428319T3/es not_active Expired - Lifetime
  • 2004-04-08 WO PCT/JP2004/005085 patent/WO2004097037A1/ja not_active Ceased
  • 2004-04-08 EP EP04726658.0A patent/EP1619256B1/en not_active Expired - Lifetime
  • 2004-04-08 KR KR1020057020146A patent/KR101080897B1/ko not_active Expired - Fee Related
  • 2004-04-19 TW TW093110887A patent/TW200504362A/zh not_active IP Right Cessation

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