CN102344887A - Indicator tube for indicating microbe growth and application thereof - Google Patents
Indicator tube for indicating microbe growth and application thereof Download PDFInfo
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- CN102344887A CN102344887A CN2010102427107A CN201010242710A CN102344887A CN 102344887 A CN102344887 A CN 102344887A CN 2010102427107 A CN2010102427107 A CN 2010102427107A CN 201010242710 A CN201010242710 A CN 201010242710A CN 102344887 A CN102344887 A CN 102344887A
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- inditron
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- indicator
- saccharoid
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- 0 CC(CC=C1)C=C1c(**1-c2ccccc2)n[n]1-c1ccccc1 Chemical compound CC(CC=C1)C=C1c(**1-c2ccccc2)n[n]1-c1ccccc1 0.000 description 1
- BEIHVSJTPTXQGB-UNJMICPPSA-N c(cc1)ccc1/C(/N=N\c1ccccc1)=N/Nc1ccccc1 Chemical compound c(cc1)ccc1/C(/N=N\c1ccccc1)=N/Nc1ccccc1 BEIHVSJTPTXQGB-UNJMICPPSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/08—Flask, bottle or test tube
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/16—Particles; Beads; Granular material; Encapsulation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
Abstract
The invention relates to an indicator tube for indicating microbe growth and application thereof. As a general container, the indicator tube of the invention employs a drying method to conduct coloration for indicating storage and transportation of particles; so that indicating particles are maintained at a drying state be for usage, and influence of humidity on a redox indicator (e.g. tetrazole salt) is avoided, and the redox indicator is not susceptible to degradation or color changing. The invention also provides a kit for detecting microbe growth. The indicator tube of the invention is easily used, has good versatility, high sensitivity and low toxicity.
Description
Technical field
The invention belongs to the microorganism detection field; More specifically, the present invention relates to the inditron and the application thereof of indicator microoraganism growth.
Background technology
Clinically pathogenic bacterium to obtain the matrix of energy mainly be carbohydrate, oxidation or glycolysis through sugar release energy, and with form (ADP, the ATP) storage power of high-energy phosphate bond.The type of bacterium living beings oxidation is divided into breathes and fermentation.In the bioid process, the hydrogen that the nutrition of bacterium (like sugar) is taken off through the desaturase effect needs the transmission transhipment through hydrogen carriers in the middle of a series of (like nicotinamide adenine dinucleotide, coenzyme II, flavoprotein etc.), gives hydrogen acceptor with hydrogen at last.With the inorganics is the biological oxidation process of hydrogen acceptor, is called breathing, wherein is the title aerobic respiration of hydrogen acceptor with the molecular oxygen; And be the title anaerobic respiration of hydrogen acceptor with mineral compound (like nitrate salt, vitriol).Be the fermentation that is called of hydrogen acceptor with various organism in the bio-oxidation, most of pathogenic bacterias only carry out aerobic respiration or fermentation.Can reduce during most bacterial growths fermentation colourless chlorinated triphenyl tetrazole (2,3,5-triphenyltetrazolium chloride; TTC) be red San Ben Jia Za (TTF) (as shown in the formula); Form red colony, San Ben Jia Za is more stable, can be by airborne oxygen autoxidation.
Tetrazole MTT (Tetrazolium) method is often used in the growth of cell or the detection of cytoactive; Its principle is that redox reaction takes place succinodehydrogenase and the ectogenic MTT on the viable cell plastosome; Be reduced into water-fast bluish voilet crystallisate be deposited in the cell (as shown in the formula), and dead cell does not have this function.Zi Se Za crystallisate in the DMSO ability dissolved cell, enzyme-linked immunosorbent assay instrument is measured absorbance value in the 490nm wavelength then, just can reflect the quantity of viable cell indirectly.
In a word, all can the generate energy metabolism when viable cell, bacterial growth, institute's energy requirement in the metabolism, the overwhelming majority obtains through biological oxidation, the serial redox reaction that bio-oxidation is promptly taken place in the biomass cells under the effect of enzyme.Redox reaction taking place the hydrogen proton transfer will take place, therefore, can claim that again redox indicator (Redox indicator) detects through the desaturase hydrogen acceptor.
Redox indicator is one type of desaturase hydrogen acceptor, and essence is one type of organic reagent that self has redox property, can change in specific electrodes current potential generation obvious color, and its oxidized form and reduced form have distinct colors.
Redox indicator commonly used comprises: 2,3, the 5-triphenyltetrazolium chloride (2,3,5-triphenyltetrazolium chloride; TTC), 3-(4 ', 5 '-dimethyl--2-thiazolyl)-2,4-phenylbenzene tetrazole bromide, tetrazolium bromide; The thiazole bromide blue tetrazolium, tetrazolium bromide, Thiazolyl blue, 3-(4,5-dimethyl--2-thiazole)-2; 5-phenylbenzene bromination tetrazole, 3-(4,5-dimethyl--2-thiazolyl)-2,5-phenylbenzene tetrazole bromide, bromination-3-(4; 5-dimethyl--2-thiazolyl)-2,5-phenylbenzene tetrazole, 3-(4,5-dimethyl--2-thiazolyl)-2,5-phenylbenzene-2H-bromo tetrazolium (MTT); 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-tetrazolium chlorine (2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-tetrazolium chloride, INT), tetranitro tetrazole blue (2,2 '; 5,5 '-tetra-(p-nitrophenyl)-3,3 '-(3-dimethoxy-4-diphenylene)-ditetrazoliumchloride, TNBT), nitrogen ditetrazolium chloride (2; 2 '-di-(p-nitrophenyl)-5,5 '-diphenyl-3,3 '-dimethoxy-4,4 '-diphenylene)-ditetrazolium chloride; NBT) 2,2 '-p-diphenylene-3,3 ', 5; 5 '-tetraphenylditetrazoliumchloride (neotetrazolium chloride) (NTC), OTC, PTC, MTC, WST etc.
Wherein, tetrazole (Tetrazolium) material is a modal reagent in the redox indicator.This type redox indicator very sensitive detects reducing substances; Itself be reduced into the Shen Se Za class material that to distinguish easily under the visible light, in the cultivation of biochemistry detection, cell cultures and conventional bacterium, evaluation, susceptibility, obtained using widely.
But there is following shortcoming in the tetrazole material:
1, tetrazolium is unstable, and is comparatively responsive to temperature, humidity and light, and very easily degraded especially is prone to generate coloured product in the aqueous solution, and environment also must not be above one month even its storage liquid is kept at low temperature (20 ℃).The degraded of tetrazolium or variable color greatly reduce the sensitivity of detection; Therefore; General recommendations tetrazolium solution fresh and using up as early as possible, this has seriously limited the purposes of tetrazolium, especially in the application that needs aspect the cultivation of certain quality guaranteed period, evaluation, susceptibility reagent or the product.
2, in fact, the tetrazole material is either large or small all toxic to bacterial growth, and research shows; Tetrazolium TTC content was at 0.005% o'clock, and the red indicating effect of Gram-negative bacteria is best, and also less to bacteria effect; But to most of gram-positive microorganisms (like mycobacterium etc.); Present stronger restraining effect, if reduce working concentration again, it is not good to the positive bacteria indicating effect.For example, carry out the food microorganisms colony count as if agent that TTC is given instruction and measure, the defective food misjudgement that mikrobe is exceeded standard is protection food.
3, can not join indication its growth, especially mycobacterium tuberculosis in the substratum of some living slowly bacterium (like mycobacterium, fungi etc.) in advance.
Should use very extensively in cultivation, evaluation, the susceptibility of cell cultures and quick growth bacterium by tetrazole; But, mycobacterium especially mycobacterium tuberculosis is restricted on using; Reason is: the nutritional condition that the growth of (1) mycobacterium tuberculosis requires is very harsh; Responsive more to the tetrazole material, the amount of adding is big suppresses its growth, the little change in color of can't see; (2) mycobacterium tuberculosis growth extremely slow (the breeding monobasic cycle needs 18-20 hour), energy metabolism is very low on the one hand, and growth can not make indicator reduction colour developing at all when initially having only a small amount of bacterium to measure.On the other hand, the cultivation of 37 ℃ of several days, tens days even twenties days, the tetrazolium in the substratum is easy to degraded, has reduced the indication ability of tetrazolium.
Therefore, usual method is at present, tetrazolium is not joined in the substratum earlier; But earlier mycobacterium tuberculosis is carried out for some time the cultivation of (general 7-10 days); Isometric to certain bacterium amount (this amount bore hole can have been seen basically), add tetrazolium this moment again, colour developing after 2-4 hour; Confirming the life or death of bacterium, or the bacterium amount is carried out quantitatively (measuring the optical density(OD) absorption value).
To the problem that above tetrazolium bacterial indicator growth occurs, resolution policy need be found in this area, with the step that simplifies the operation, improves detection efficiency and detection accuracy.
Summary of the invention
The object of the present invention is to provide the reagent and the method that detect mikrobe.
In first aspect of the present invention, a kind of inditron of indicator microoraganism growth is provided, it is characterized in that described inditron comprises:
One container; And the particle that places this internal tank, described particle comprises: 50-600 purpose saccharoid and the redox indicator that is attached to this saccharoid surface.
In a preference, the size of described saccharoid is the 100-400 order; It more preferably is the 150-300 order; Be 200 orders best.
In another preference, the shape of described saccharoid is selected from (but being not limited to): sphere, elliposoidal, cube shaped, cuboid, cylindricality, taper or irregularly shaped.
In another preference, described redox indicator is the organic reagent that self has redox property, can change in specific electrodes current potential generation obvious color, and its oxidized form and reduced form have distinct colors.
In another preference, described particle surface also is attached with the material and/or the stablizer (preferably for stablizing the reagent of redox agent) that can carry out proton transfer.
In another preference, described redox indicator is selected from (but being not limited to): tetrazolium, and methylene blue (methylene blue) is born reddish black (Resauzurin) or its combination; And/or
The described material that can carry out proton transfer is selected from (but being not limited to): and the azophenlyene Methylsulfate (Phenazine methosulfate, PMS), the azophenlyene sulfovinate (Phenazine ethosulfate, PES) or its combination; And/or
Described stablizer is mineral acid or organic acid (not comprising hydrofluoric acid), preferably is selected from (but being not limited to): boric acid, Hydrocerol A or succsinic acid or its combination.
In another preference, described tetrazolium is selected from: 2,3, and 5-triphenyltetrazolium chloride (2,3; 5-triphenyltetrazolium chloride, TTC), 3-(4 ', 5 '-dimethyl--2-thiazolyl)-2,4-phenylbenzene tetrazole bromide; Tetrazolium bromide, the thiazole bromide blue tetrazolium, tetrazolium bromide, Thiazolyl blue, 3-(4; 5-dimethyl--2-thiazole)-2,5-phenylbenzene bromination tetrazole, 3-(4,5-dimethyl--2-thiazolyl)-2,5-phenylbenzene tetrazole bromide; Bromination-3-(4,5-dimethyl--2-thiazolyl)-2,5-phenylbenzene tetrazole, 3-(4,5-dimethyl--2-thiazolyl)-2; 5-phenylbenzene-2H-bromo tetrazolium (MTT), and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-tetrazolium chlorine (2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-tetrazolium chloride, INT), tetranitro tetrazole blue (2,2 '; 5,5 '-tetra-(p-nitrophenyl)-3,3 '-(3-dimethoxy-4-diphenylene)-ditetrazoliumchloride, TNBT), the nitrogen ditetrazolium chloride (2,2 '-di-(p-nitrophenyl)-5; 5 '-diphenyl-3,3 ' dimethoxy-4,4 '-diphenylene)-dit etrazolium chloride, NBT), 2; 2 '-p-diphenylene-3,3 ', 5,5 '-tetraphenylditetrazolium chloride (neotetrazolium chloride) is (NTC); OTC, PTC, MTC, WST etc.
In another preference, described saccharoid is a silica gel particle.
In another preference, described container is a test tube.
In another preference, described container is aseptic, exsiccant container.
In another preference, described granulopexy is in container bottom or inwall, or places container with the on-fixed mode.
In another preference, described inditron also comprises: the lid of a salable said container.
In another preference, be non-flat at the bottom of the pipe of described inditron.
In another preference, at the bottom of the pipe of described inditron round bottom or arcuate bottom; Perhaps, narrow at the bottom of the pipe of described inditron.
In another preference, narrowing at the bottom of the described pipe is that diameter at the bottom of the vial is significantly less than the diameter of the mouth of pipe and pipe shaft; As little more than 30%; More preferably little more than 50%; More preferably little more than 70%.
In another preference, described particle is the 0.02-0.5g/ container; Preferably, described particle is the 0.05-0.2g/ container.
In another aspect of this invention, the purposes of described inditron is provided, is used to prepare the test kit that detects microorganism growth.
In another aspect of this invention, a kind of test kit that detects microorganism growth is provided, contains described inditron in the described test kit.
In another aspect of this invention, the preparation method of described inditron is provided, said method comprises:
(1) redox indicator is mixed with saccharoid, obtain the particle that surface attachment has redox indicator;
(2) particle that (1) is obtained places internal tank.
In another preference, step in the said method (1) comprising: redox indicator is mixed with saccharoid, obtain the particle that surface attachment has redox indicator.
In another preference, also comprise with the saccharoid blended: can carry out the material and/or the stablizer of proton transfer, the acquisition surface attachment has redox indicator and surface attachment to have can carry out the material of proton transfer and/or the particle of stablizer.
In another preference, the consumption of described redox indicator is the 0.05-10mg/g saccharoid; Be preferably the 0.1-5mg/g saccharoid; It more preferably is the 0.2-2mg/g saccharoid.
In another preference, when being used to mix, the final concentration of described stablizer is 0.001-0.1M, more preferably is 0.01-0.05M.
In another preference, when being used to mix, the said consumption that can carry out the material of proton transfer is the 1/100-1/1000 of redox indicator quality.
In another preference, said method also comprises: have the particle of redox indicator (selectively the surface also is attached with the material and/or the stablizer that can carry out proton transfer) to carry out drying surface attachment.
In another preference, described redox indicator (selectively also comprising the material and/or the stablizer that can carry out proton transfer) can be water or organic solvent with saccharoid blended medium.Described organic solvent such as methyl alcohol, ethanol etc.
In another aspect of this invention, a kind of detection method of microorganism (for the detection of non-diagnostic purpose) is provided, said method comprises:
(a) described inditron is provided;
(b) testing sample and microbiological culture media are joined in the inditron of (a), cultivate; With
(c) confirm whether have mikrobe and amount in the testing sample according to the colour developing situation of inditron.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown a kind of synoptic diagram of preferable growth inditron (particle of not packing into).Wherein, 1 is pipe shaft; 2 is spiral cover; 3 are the bottle end (being round bottom at the bottom of the illustrated tubes); 4 are bottle an end (at the bottom of the illustrated tubes be the pipe that narrows at the bottom of).
Fig. 2 has shown that the growth inditron is used for common quick growth pathogenic bacterium (intestinal bacteria, Pseudomonas aeruginosa, Klebsiella pneumonia, streptococcus aureus) and detects.It is thus clear that it is obvious to contain the colour developing indication particulate inditron colour developing (redness) of useful TTC preparation; Develop the color more shallow, obvious inadequately and do not contain the inditron of indicating particle only to contain TTC that develops the color.
Fig. 3 has shown that the bacterial growth inditron is used for the cultivation of mycobacterium tuberculosis H37Rv (13 days result of different bacterium amount growths).Wherein, 10
-7Mg is equivalent to contain 1-10 (bar) bacterium, and cultivation results colony number calculates therewith and conforms among the figure.
Fig. 4 has shown part smear positive spit sample mycobacterium tuberculosis separation and Culture result, is characteristic with red colony behind the mycobacterium growth, need not smear staining, lies in to observe red bacterium colony on the inverted microscope and be tubercule bacillus strand growth characteristics structure.
Embodiment
The inventor is through extensive studies; Be surprised to find that the particle that will carry redox indicator places suitable inditron; Can form aseptic, stable, as a to seal environment, greatly improve the efficient that detects mikrobe, indicating effect is good.Accomplished the present invention on this basis.
As used herein, described " containing ", " having " or " comprising " comprised " comprising ", " mainly by ... constitute (compositions) ", " basically by ... constitute " and " by ... formation "; " mainly by ... constitute ", " basically by ... constitute " belong to the subordinate concept of " containing ", " having " or " comprising " with " by ... formation ".
The inditron of microorganism growth
The invention provides a kind of inditron of indicator microoraganism growth, described inditron comprises: a container; And the particle that places the described indicator microoraganism growth of this internal tank.
Container of the present invention is suitable for loading the particle of described indicator microoraganism growth, and can add sample (or appearance liquid, or nutrient solution) therein.Preferably, described container is aseptic, exsiccant container.Preferably, described container is transparent, is convenient to observe coupling reaction like this.Preferably, described container is the round bottom test tube (like Fig. 1) of band spiral cover, and round bottom or pipe narrow at the end and can make the colour developing particle focus on the bottom to be convenient to color observation more accurately.The diameter of inditron is with 12-18mm, and high 80-120mm is advisable; Optimum diameter 16mm, high 90mm, material are preferably glass or plastics (PC, PET).Described particle can be fixed in container bottom or inwall, or places container with the on-fixed mode.
Described particle mainly is made up of 50-600 purpose saccharoid and the redox indicator that is attached to this saccharoid surface.
The size of saccharoid is correlated with for detecting effect, and too big saccharoid surface-area is too little, and too little saccharoid settling velocity is too slow, is unfavorable for improving the efficient of detection.Therefore, the inventor studies the back repeatedly and finds that the size of described saccharoid is suitable at the 50-600 order.Preferably, the size of described saccharoid is the 100-400 order; It more preferably is the 150-300 order; Be 200 orders best.
The shape of described saccharoid can be diversified, for example can be selected from (but being not limited to): sphere, elliposoidal, cube shaped, cuboid, cylindricality, taper or irregularly shaped etc.; Preferably structural, the size of porous surface evenly, this helps increasing the contact area with sample to be detected.
The material for preparing described saccharoid is preferably silicon-dioxide (SiO
2).The method for preparing 50-600 purpose silicon-dioxide (or hydrated SiO 2) saccharoid is well known to those skilled in the art.50-600 purpose silicon-dioxide (or hydrated SiO 2) also can commercially be bought.
In addition, the saccharoid beyond the silica dioxide granule also is an available, cellulose grain for example, pvdf particle etc.Silica dioxide granule preferably.
Whether and amount the existence that the organic reagent (being redox indicator) that utilization of the present invention has a redox property comes indicator microoraganism.Described redox indicator can change in specific electrodes current potential generation obvious color, and its oxidized form and reduced form have distinct colors.
Described redox indicator can be diversified, as long as its oxidized form and reduced form have distinct colors.For example described redox indicator is selected from (but being not limited to): tetrazolium, methylene blue (methylene blue) is born reddish black (Resauzurin) or its combination.
Described tetrazolium is one type of redox agent.For example, described tetrazolium can be selected from: 2,3, and 5-triphenyltetrazolium chloride (2,3; 5-triphenyltetrazolium chloride, TTC), 3-(4 ', 5 '-dimethyl--2-thiazolyl)-2,4-phenylbenzene tetrazole bromide; Tetrazolium bromide, the thiazole bromide blue tetrazolium, tetrazolium bromide, Thiazolyl blue, 3-(4; 5-dimethyl--2-thiazole)-2,5-phenylbenzene bromination tetrazole, 3-(4,5-dimethyl--2-thiazolyl)-2,5-phenylbenzene tetrazole bromide; Bromination-3-(4,5-dimethyl--2-thiazolyl)-2,5-phenylbenzene tetrazole, 3-(4,5-dimethyl--2-thiazolyl)-2; 5-phenylbenzene-2H-bromo tetrazolium (MTT), and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-tetrazolium chlorine (2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-tetrazoliumchloride, INT), tetranitro tetrazole blue (2,2 '; 5,5 '-tetra-(p-nitrophenyl)-3,3 '-(3-dimethoxy-4-diphenylene)-ditetrazoliumchloride, TNBT), the nitrogen ditetrazolium chloride (2,2 '-di-(p-nitrophenyl)-5; 5 '-diphenyl-3,3 ' dimethoxy-4,4 '-diphenylene)-dit etrazolium chloride, NBT), 2; 2 '-p-diphenylene-3,3 ', 5,5 '-tetraphenylditetrazolium chloride (neotetrazolium chloride) is (NTC); OTC, PTC, MTC, WST etc.
Described to bear reddish black be a kind of redox agent commonly used, and its oxidized form presents blueness usually, and reductibility presents redness usually.
Described methylene blue is a kind of redox agent commonly used, and its oxidized form presents blueness usually, and reductibility appears colourless usually.
As a kind of selectable mode of the present invention, also be attached with stablizer on the surface of described saccharoid.Described stablizer plays auxiliary effect, optimizes coupling reaction through stablizing redox indicator, improves the accuracy that detects.The material that any ability is stablized redox indicator all is an available.Preferably, described stablizer is that acidic substance are selected from (but being not limited to): boric acid, Hydrocerol A or succsinic acid or its combination.
As a kind of selectable mode of the present invention, also be attached with the material that can carry out proton transfer on the surface of described saccharoid.This kind material plays auxiliary effect, promotes the generation of coupling reaction through the efficient that improves proton transfer.Any material that can bring into play protolysis all is an available.Preferably, the described material that can carry out proton transfer is selected from (but being not limited to): and the azophenlyene Methylsulfate (Phenazinemethosulfate, PMS), the azophenlyene sulfovinate (Phenazine ethosulfate, PES) or its combination.
The particle of described indicator microoraganism growth can exist with the dispersive form, after having added mikrobe appearance liquid contact to be measured, can be suspended in dispersedly in kind liquid, or fall to container bottom.In addition, can the particle of indicator microoraganism growth be processed ball-type, cylinder shape or positive square through tackiness agent as required, stick on the pipe bottom.Perhaps, can be capable of using as required the particle characteristics of indicator microoraganism growth, wait the catching image recognition of devices by scanning, cooperate to cultivate and set up cultivation reporting system automatically.
The particle of described indicator microoraganism growth can be prepared as follows: with redox indicator (selectively; Also comprise the material and/or the stablizer that can carry out proton transfer) mix with saccharoid; Acquisition is attached with the particle of (selectively, also being attached with the material and/or the stablizer that can carry out proton transfer) redox indicator.Described redox indicator, the material that can carry out proton transfer and/or stablizer add quadrat method and order by merging is arbitrarily, the present invention has no particular limits.
Described redox indicator (selectively also comprising the material and/or the stablizer that can carry out proton transfer) can water or organic solvent with saccharoid blended medium.Described organic solvent such as methyl alcohol, ethanol etc.The usage quantity of water or organic solvent is an amount minimum when guaranteeing to mix, and strengthens the amount of solvent effect is not had influence, only can prolong time of drying.The adjusting of usage quantity or control are that those skilled in the art rule of thumb are easy to learn.
Preferably, said method also comprises: the particle that will be attached with redox indicator (selectively also being attached with the material and/or the stablizer that can carry out proton transfer) carries out drying.Thereby help making redox indicator stably to be present in the saccharoid surface, also help particulate and be in store for.Drying can be carried out in baking oven.
The particle of the indicator microoraganism growth that above method obtains can be used for preparing the reagent or the inditron of indicator microoraganism growth.
As a kind of embodiment of the present invention, tetrazole salt redox indicator is adsorbed on the silica gel particle, be prepared into stable hypotoxic colour developing indication particle.
Indication particulate consumption will enough develop the color in the growth inditron.Generally can according to the sample of container and adding (or appearance liquid, or nutrient solution) what and decide, for example 0.02-0.5g/ manages (2ml-5m1).
As a preferred implementation of the present invention; A kind of preparation method of inditron of concrete microorganism growth is provided; Comprise: (1) with the material (boric acid, Hydrocerol A or succsinic acid) of a certain amount of tetrazolium (among TTC, MTT, INT, TNBT, NBT, OTC, STC, NTC, PTC, the MTC etc. a kind of) and a certain amount of stable tetrazolium and a certain amount of can carry out Substance P MS or the PES of proton transfer and in advance 2 hours cooled silica gel particles of 130 ℃ of activation mix rearmounted oven drying; Processing colour developing indication particle, tetrazolium etc. is adsorbed on the silica gel particle surface.(2) above-mentioned colour developing indication particle is installed in empty, that sterilized, the exsiccant culture tube by a certain amount of branch, sealing is made into the growth inditron.
The described colour developing indication particulate growth inditron that contains can be used as a kind of general reagent use; Before inoculation or the use; As long as sample (or appearance liquid, or nutrient solution) is joined in the growth inditron, colour developing indication particle will sink to the bottom of substratum; Cultivate inoculation, sealing back, as long as there is the biological growth particle will develop the color or form red colony.Add substratum can be according to cultivating biological kind different choice, as adding the sensitivity testing that certain drugs can be carried out medicine to this medicine in the substratum.
The present invention also provides a kind of detection method of microorganism (for the detection of non-diagnostic purpose), and said method comprises: described inditron (a) is provided; (b) testing sample and microbiological culture media are joined in the inditron of (1), cultivate; (c) confirm whether have mikrobe and amount in the testing sample according to the colour developing situation of inditron.
The present invention also provides a kind of test kit that detects mikrobe, contains described inditron in the described test kit.Preferable, also can comprise substratum, water or organic solvent, the working instructions etc. of culturing micro-organisms in the described test kit.
Major advantage of the present invention is:
(1) stability strengthens: the inventor creatively will develop the color and indicate particle to be placed in aseptic, the exsiccant container; The growth inditron is as a kind of general container; Adopt drying means to develop the color and indicate particulate to store and transportation, guaranteed before using, indicate particle to remain on dryness always; Avoided the influence of humidity, be difficult for the degraded variable color redox indicator (like tetrazolium); Stability is measured the result and shown: 4 ℃ of refrigerators, normal temperature keep in Dark Place a year and a half, its proterties function no change; These stable characteristics make to produce in batches becomes possibility.
(2) versatility increases: contain colour developing indication particulate growth inditron and can be used as a kind of general reagent use; Before inoculation or the use; As long as substratum is joined in the bacterial growth inditron; Colour developing indication particle will sink to the bottom of substratum, and cultivate inoculation, sealing back, as long as there is the biological growth particle will develop the color or form red colony.Add substratum can be according to cultivating biological kind different choice, as adding the sensitivity testing that certain drugs can be carried out medicine to this medicine in the substratum.
(3) sensitivity improves: the colony that the colour developing inditron shows is concentrated, and color is very bright-coloured, bright, distinguishes easily and judges.
(5) reduce mikrobe and operate infectious risk: for separation, the cultivation of mikrobe such as mycobacterium tuberculosis; If the mycobacterium tuberculosis well-grown will form specific cord structures; General through routine smear, dyeing, under simple microscope, observe cord structures then and judge or identify whether be mycobacterium tuberculosis.The unexpected discovery be, mycobacterium tuberculosis contains above that growth can form the colony that takes on a red color in the colour developing indication particulate inditron, and this colony can be known on inverted microscope and sees red strand feature structure.So just can exempt the trouble (mycobacterium tuberculosis has very strong infectivity) of evaluations such as opening bottle, smear, dyeing.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Only if definition separately, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any with the institute similar content of putting down in writing or the equalization method and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
The particulate blank pipe of not packing into is as shown in Figure 1.
A kind of blank pipe is the situation of round bottom for the pipe end, like left figure among Fig. 1.Wherein, 1 is pipe shaft; 2 is spiral cover; 3 is the bottle end.
The situation that another kind of blank pipe narrowed for the pipe end is schemed like Fig. 1 right-of-center in political views.Wherein, 1 is pipe shaft; 2 is spiral cover; 4 is the bottle end.
1. reagent preparation
(1) takes by weighing 200 order silica gel (available from Industrial Co., Ltd. of last marine nation) 30g and put 120 ℃ of activation of baking oven 2 hours, the sealing postcooling.
(2) take by weighing TTC 40mg and be dissolved in (1mg/ml) in the 40ml zero(ppm) water, packing after the sterile filtration is put 4 ℃ and is kept in Dark Place.
(3) take by weighing PMS 40mg and be dissolved in (1mg/ml) in the 40ml zero(ppm) water, packing after the sterile filtration is put 4 ℃ and is kept in Dark Place.
(4) preparation 0.1M citric acid solution, packing after the sterile filtration, 4 ℃ of preservations.
2. get 0.1M citric acid solution 8ml under the gnotobasis and join in 40ml 1mg/ml tetrazolium (TTC) solution, add 0.2ml 1mg/ml PMS again, pour into after the mixing in the silica gel particle after the 30g activation, be stirred to evenly with the glass rod after the sterilization.
3. mixture is put 80 ℃ of oven dryings, the centre can be stirred under gnotobasis, can flow until particle, and elevated temperature to 108 ℃ baking 1 hour, the sealing postcooling is put 4 ℃ of preservations.
4. above-mentioned colour developing indication particle is divided by a certain amount of (0.075g) to install in sky, that sterilized, the exsiccant pipe, spiral cover is sealed to the growth inditron.
Pathogenic bacterium comprise that (Gram-negative (Gram-), Pseudomonas aeruginosa (Pseudomonas aeruginosa Gram-), Klebsiella pneumonia (Gram-), (Gram-positive (Gram+) is seen table 1 to streptococcus aureus to intestinal bacteria.
The preparation of MH broth culture: water intaking 100ml, take by weighing Carnis Bovis seu Bubali cream 0.3g, peptone 1g, NaCl 0.5g adding, after dissolving, heating transfers pH7.0-7.2, high pressure steam sterilization.
Under the gnotobasis, MH broth culture branch is filled in 4 growth inditrons mark by the 2mL/ pipe; In addition, add 4 by the 2mL/ pipe and do not add colour developing indication particulate inditron, add same amount TTC (100ug/ pipe) to these 4 inditrons again.
Inoculation, cultivate: respectively with cultivate on the blood agar plate bacterial strain mill bacterium than turbid to 1 Maxwell unit (1McFarland=1mg/mL), by 1: 10 (saline water) gradient dilution to 10
-6Mg/mL.Respectively from 10
-6Respectively getting 100uL in the mg/mL dilution tube is inoculated in three different growth inditrons.
The rearmounted 37 ℃ of incubators of inoculation are cultivated, and observe the growth result of cultivating after 24 hours.
Table 1
| Mikrobe | Bacterial strain | Gramstaining | Bacterial strain number |
| E.coli | Intestinal bacteria | Gram- | ATCC?25922 |
| Pseudomonas?aeruginosa | Pseudomonas aeruginosa (Pseudomonas aeruginosa) | Gram- | ATCC?29213 |
| Klebsiella?pneumoniae | Klebsiella pneumonia | Gram- | Strain isolated 3069 |
| Staphyloccocus?aureus | Streptococcus aureus | Gram+ | ATCC29213 |
The result is as shown in Figure 2.Show that colour developing indication particle can be used for showing the growth of most of bacterium, and the red colony that shows is concentrated, the colony color is vivid.
The cultivation that embodiment 4, growth inditron are used for mycobacterium tuberculosis type strain H37Rv (Mycobacteriumtuberculosis H37Rv, ATCC95054 Gram+) detects
1. substratum:
BBL
TMThe MGIT substratum, BBL
TMThe OADC substratum, BBL
TMThe PANTA substratum is available from U.S. Becton Dickinson (BD) company).
Take out above substratum respectively from container,, three kinds of substratum are carried out mixed preparing, obtain to be used to cultivate the substratum of mycobacterium tuberculosis according to the specification sheets requirement that BD company provides, subsequent use.
2. under the gnotobasis, the substratum branch of the cultivation mycobacterium tuberculosis of above-mentioned preparation is filled in the growth inditron mark by the 2mL/ pipe.
3. inoculation, cultivation: the H37Rv bacterial strain mill bacterium after will cultivating to 1 Maxwell unit (1McFarland=1mg/mL), dilutes 10 by 1: 10 (saline water) gradient than turbid
-1To 10
-6Mg/mL.Respectively from 10
-2Mg/mL, 10
-4Mg/mL, 10
-6Respectively getting 100uL in the mg/mL dilution tube is inoculated in three different growth inditrons.
The rearmounted 37 ℃ of incubators of inoculation are cultivated, and observe growth result after 13 days.
Even the result sees Fig. 3 and show that very low inoculation bacterium amount also can grow, and red colony number coincide with inoculum size, and the bacterium in the time of can estimating to inoculate according to the colony number is measured.
Embodiment 5, growth inditron are used for 92 parts of mycobacterium phlegm smear positive sample mycobacterium separation and Culture
1. substratum:
BBL
TMThe MGIT substratum, BBL
TMThe OADC substratum, BBL
TMThe PANTA substratum is available from U.S. Becton Dickinson (BD) company).
Take out above substratum respectively from container,, three kinds of substratum are carried out mixed preparing, obtain to be used to cultivate the substratum of mycobacterium tuberculosis according to the specification sheets requirement that BD company provides, subsequent use.
2. under the gnotobasis, the substratum branch of the cultivation mycobacterium tuberculosis of above-mentioned preparation is filled in the growth inditron mark by the 2mL/ pipe.
3. sputum is handled: adopt N-acetyl-L-cysteine sodium hydroxide (NALC-NaOH) method, the NALC-NaOH solution with the expectoration adding equivalent of gathering stirs with turbine mixer.
The pH value is proofreaied and correct: with above-mentioned mixing solutions, left standstill at normal temperatures 15 minutes, add the phosphoric acid buffer (pH6.8) of the cold sterilization of 10 times of amounts then; Behind the mixing; At 3000 * g centrifugal 20 minutes, deposition suspended with the same buffer of 1mL, got 100uL and was inoculated in the inditron of growing.Postvaccinal substratum is put 36 ± 1 ℃ of incubators and is cultivated.Inoculating BD960 mycobacterium growth inditron (MGIT) simultaneously contrasts as fast culture.
92 parts of samples have 79 parts to cultivate the positive as a result, and the result is consistent with BD960 mycobacterium fast culture reagent, and the average sentence read result time is only than the late 1-2 of BD960 days.
Fig. 4 left side is 92 parts of mycobacterium phlegm smear male phlegm separation and Culture results; Mycobacterium tuberculosis growth back is a characteristic with red colony; Need not smear staining; Lie in and observe the distinctive strand growth structure of tubercule bacillus that red bacterium colony appears on the inverted microscope, Fig. 4 is right for partly indicating tubulose growth structure figure.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. the inditron of indicator microoraganism growth is characterized in that described inditron comprises:
One container; And the particle that places this internal tank, described particle comprises: 50-600 purpose saccharoid and the redox indicator that is attached to this saccharoid surface.
2. inditron as claimed in claim 1 is characterized in that, described particle surface also is attached with the material and/or the stablizer that can carry out proton transfer.
3. inditron as claimed in claim 1 is characterized in that, described redox indicator is selected from: tetrazolium, and methylene blue is born reddish black or its combination; And/or
The described material that can carry out proton transfer is selected from: azophenlyene Methylsulfate, azophenlyene sulfovinate or its combination; And/or
Described stablizer is mineral acid or organic acid; Preferably be selected from: boric acid, Hydrocerol A or succsinic acid or its combination.
4. inditron as claimed in claim 1 is characterized in that described saccharoid is a silica gel particle.
5. inditron as claimed in claim 1 is characterized in that, described inditron also comprises: the lid of a salable said container.
6. inditron as claimed in claim 1 is characterized in that, is round bottom or arcuate bottom at the bottom of the pipe of described inditron; Perhaps, narrow at the bottom of the pipe of described inditron.
7. the purposes of the arbitrary described inditron of claim 1-6 is used to prepare the test kit that detects microorganism growth.
8. a test kit that detects microorganism growth is characterized in that, contains the arbitrary described inditron of claim 1-6 in the described test kit.
9. the preparation method of the described inditron of claim 1 is characterized in that, said method comprises:
(1) redox indicator is mixed with saccharoid, obtain the particle that surface attachment has redox indicator;
(2) particle that (1) is obtained places internal tank.
10. one kind is detected method of microorganism, it is characterized in that said method comprises:
(a) claim 1-6 is provided arbitrary described inditron;
(b) testing sample and microbiological culture media are joined in the inditron of (a), cultivate; With
(c) confirm whether have mikrobe and amount in the testing sample according to the colour developing situation of inditron.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010102427107A CN102344887A (en) | 2010-08-02 | 2010-08-02 | Indicator tube for indicating microbe growth and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102427107A CN102344887A (en) | 2010-08-02 | 2010-08-02 | Indicator tube for indicating microbe growth and application thereof |
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| Publication Number | Publication Date |
|---|---|
| CN102344887A true CN102344887A (en) | 2012-02-08 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102427107A Pending CN102344887A (en) | 2010-08-02 | 2010-08-02 | Indicator tube for indicating microbe growth and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104673662A (en) * | 2013-12-03 | 2015-06-03 | 深圳市艾瑞生物科技有限公司 | Culture bottle and preparation method and application thereof |
| CN106755287A (en) * | 2017-02-07 | 2017-05-31 | 盎亿泰地质微生物技术（北京）有限公司 | A kind of microbial is to content detection method of counting |
| CN111500670A (en) * | 2020-04-20 | 2020-08-07 | 杭州伽玛生物科技有限公司 | High-throughput drug sensitivity detection kit and use method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673662A (en) * | 2013-12-03 | 2015-06-03 | 深圳市艾瑞生物科技有限公司 | Culture bottle and preparation method and application thereof |
| CN106755287A (en) * | 2017-02-07 | 2017-05-31 | 盎亿泰地质微生物技术（北京）有限公司 | A kind of microbial is to content detection method of counting |
| CN106755287B (en) * | 2017-02-07 | 2020-05-08 | 盎亿泰地质微生物技术（北京）有限公司 | Method for detecting and counting relative content of microorganisms |
| US11505817B2 (en) | 2017-02-07 | 2022-11-22 | Advanced Energy & Environmental Technologies, Inc. | Method for detecting and counting relative content of microorganism |
| CN111500670A (en) * | 2020-04-20 | 2020-08-07 | 杭州伽玛生物科技有限公司 | High-throughput drug sensitivity detection kit and use method and application thereof |
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Application publication date: 20120208 |