CN102262110A - Color-developing grain for indicating microbial growth and preparation method thereof - Google Patents
Color-developing grain for indicating microbial growth and preparation method thereof Download PDFInfo
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- CN102262110A CN102262110A CN2010101881997A CN201010188199A CN102262110A CN 102262110 A CN102262110 A CN 102262110A CN 2010101881997 A CN2010101881997 A CN 2010101881997A CN 201010188199 A CN201010188199 A CN 201010188199A CN 102262110 A CN102262110 A CN 102262110A
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Abstract
The invention relates to a color-developing grain for indicating microbial growth and a preparation method thereof. The indicating grain provided by the invention has the advantages of good stability, high sensitivity and low toxicity to microbes.
Description
Technical field
The invention belongs to the microorganism detection field; More specifically, the present invention relates to a kind of colour developing particle and preparation method of indicator microoraganism growth.
Background technology
Clinically pathogenic bacteria to obtain the matrix of energy mainly be carbohydrate, oxidation or glycolysis by sugar release energy, and with form (ADP, the ATP) storage power of energy-rich phosphate bond.The type of bacterium living beings oxidation is divided into breathes and fermentation.In the bioid process, the hydrogen that the nutrients of bacterium (as sugar) is taken off through the dehydrogenasa effect needs the transmission transhipment through hydrogen carrier (as cozymase, codehydrogenase, flavoprotein etc.) in the middle of a series of, 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 (as nitrate, sulfate).Be the fermentation that is called of hydrogen acceptor with various organism in the bio-oxidation, most of pathogens 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 succinate dehydrogenase and the ectogenic MTT on the living cells mitochondria, be reduced into water-fast bluish violet crystal be deposited in the cell (as shown in the formula), and dead cell does not have this function.Zi Se Za crystal in the DMSO energy dissolved cell, enzyme-linked immunosorbent assay instrument is measured absorbance value at 490nm wavelength place then, just can reflect the quantity of living cells indirectly.
In a word, all can the produce power metabolism when living cells, bacterial growth, institute's energy requirement in the metabolism, the overwhelming majority obtains by biological oxidation, the serial redox reaction that bio-oxidation is promptly taken place in the biological cell under the effect of enzyme.Redox reaction taking place the hydrogen proton transfer will take place, therefore, can claim redox indicator (Redox indicator) to detect again by the dehydrogenasa hydrogen acceptor.
Redox indicator is a class dehydrogenasa hydrogen acceptor, and essence is the organic reagent that a class self has redox property, can change in specific electrodes current potential generation obvious color, and its oxidized form has different colors with reduced form.
Redox indicator commonly used comprises: 2,3,5-triphenyltetrazolium chloride (2,3,5-triphenyltetrazolium chloride, TTC), 3-(4 ', 5 '-dimethyl-2-thiazolyl)-2,4-diphenyl tetrazole bromide, tetrazolium bromide, thiazole bromide blue tetrazolium, tetrazolium bromide, Thiazolyl blue, 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl bromination tetrazole, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazole bromide, bromination-3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazole, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-bromo tetrazolium (MTT), and 2-(4-iodophenyl)-3-(4-nitrobenzophenone)-5-phenyl-tetrazolium chlorine (2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-tetrazolium chloride, INT), tetranitro tetrazole indigo plant (2,2 ', 5,5 '-tetra-(p-nitrophenyl)-3,3 '-(3-dimethoxy-4-diphenylene)-ditetrazoliumchloride, TNBT), the nitrogen tetrazolium blue (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 class redox indicator is the detection reducing substances of sensitivity very, itself be reduced into the Shen Se Za class material that to distinguish easily under the visible light, in the cultivation of biochemistry detection, cellular incubation and conventional bacterium, evaluation, susceptibility, obtained using widely.
But there is following shortcoming in the tetrazole material:
1, tetrazolium instability, comparatively responsive to temperature, humidity and light, very easily degraded especially easily generates coloured product in aqueous solution, 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, the fresh preparation of general recommendations tetrazolium solution 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 shelf-life, evaluation, susceptibility reagent or the product.
2, in fact, the tetrazole material is either large or small all toxic to bacterial growth, studies show that, tetrazolium TTC content is at 0.005% o'clock, the red indicating effect the best of Gram-negative bacteria, and also less to bacteria effect, but to most of gram-positive bacterias (as mycobacterium etc.), present stronger inhibiting 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 microorganism is exceeded standard is protection food.
3, can not join indication its growth, especially Much's bacillus in the nutrient culture media of some living slowly bacterium (as mycobacterium, fungi etc.) in advance.
Should use very extensively in cultivation, evaluation, the susceptibility of cellular incubation and quick growth bacterium by tetrazole, but on using, mycobacterium especially Much's bacillus is restricted, reason is: the nutritional condition that the growth of (1) Much's bacillus 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) Much's bacillus growth extremely slow (cycle of a breeding generation needs 18-20 hour), energetic supersession 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 nutrient culture media is easy to degraded, has reduced the indication ability of tetrazolium.
Therefore, usual way is at present, tetrazolium is not joined in the nutrient culture media earlier, but earlier Much's bacillus is carried out a period of time the cultivation of (general 7-10 days), isometric to certain bacterium amount (this amount bore hole can have been seen substantially), add tetrazolium this moment again, colour developing after 2-4 hour, determining the life or death of bacterium, or the bacterium amount is carried out quantitatively (measuring the optical density absorption value).
At 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 microorganism.
In a first aspect of the present invention, a kind of particle of indicator microoraganism growth is provided, described particle comprises: 50-600 purpose shot-like particle and the redox indicator that is attached to this shot-like particle surface.
In a preference, the size of described shot-like particle is the 100-400 order; It more preferably is the 150-300 order; Be 200 orders best.
In another preference, the shape of described shot-like particle 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 has different colors with reduced form.
In another preference, described particle surface also is attached with the material and/or the stabilizing agent (preferably for stablizing the reagent of reductant-oxidant) 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 stabilizing agent is mineral acid or organic acid (not comprising hydrofluorite), preferably is selected from (but being not limited to): boric acid, citric acid or succinic acid or its combination.
In another preference, described tetrazolium is selected from: 2,3,5-triphenyltetrazolium chloride (2,3,5-triphenyltetrazolium chloride, TTC), 3-(4 ', 5 '-dimethyl-2-thiazolyl)-2,4-diphenyl tetrazole bromide, tetrazolium bromide, thiazole bromide blue tetrazolium, tetrazolium bromide, Thiazolyl blue, 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl bromination tetrazole, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazole bromide, bromination-3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazole, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-bromo tetrazolium (MTT), 2-(4-iodophenyl)-3-(4-nitrobenzophenone)-5-phenyl-tetrazolium chlorine (2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-tetrazolium chloride, INT), tetranitro tetrazole indigo plant (2,2 ', 5,5 '-tetra-(p-nitrophenyl)-3,3 '-(3-dimethoxy-4-diphenylene)-ditetrazoliumchloride, TNBT), nitrogen tetrazolium blue (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) (NTC), OTC, PTC, MTC, WST etc.
In another preference, described shot-like particle is a silica gel particle.
In another aspect of this invention, provide the preparation method of described particle, described method comprises: redox indicator is mixed with shot-like particle, obtain the particle that surface attachment has redox indicator.
In another preference, that mixes with shot-like particle also comprises: can carry out the material and/or the stabilizing agent 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 stabilizing agent.
In another preference, the consumption of described redox indicator is the 0.05-10mg/g shot-like particle; Be preferably the 0.1-5mg/g shot-like particle; It more preferably is the 0.2-2mg/g shot-like particle.
In another preference, when being used to mix, the final concentration of described stabilizing agent is 0.001-0.1M, more preferably is 0.01-0.05M.
In another preference, when being used to mix, the described consumption that can carry out the material of proton transfer is the 1/100-1/1000 of redox indicator quality.
In another preference, described method also comprises: have the particle of redox indicator (selectively the surface also is attached with the material and/or the stabilizing agent that can carry out proton transfer) to carry out drying surface attachment.
In another preference, the medium that described redox indicator (selectively also comprising the material and/or the stabilizing agent that can carry out proton transfer) mixes with shot-like particle can be water or organic solvent.Described organic solvent such as methyl alcohol, ethanol etc.
In another aspect of this invention, provide the purposes of described particle, be used to prepare the reagent of indicator microoraganism growth.
In another aspect of this invention, provide a kind of kit that detects microorganism, contain the particle of described indicator microoraganism growth in the described kit.
In another aspect of this invention, provide a kind of detection method of microorganism, described method comprises:
(1) testing sample and microbiological culture media are mixed with the particle of described indicator microoraganism growth, cultivate; With
(2) determine whether have microorganism and amount in the testing sample according to the colour developing situation of particle.
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 that growth indication particle is used for common quick growth pathogenic bacteria (Escherichia coli, pseudomonas aeruginosa, Friedlander, staphylococcus aureus) and detects.As seen the indication particle that contains the colour developing indication particle of useful TTC preparation develops the color (redness) obviously; Develop the color more shallow, obvious inadequately and do not contain the indication particle of indicating particle only to contain TTC that develops the color.
Fig. 2 has shown that the indication particle is used for the cultivation of Much's bacillus 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.
Embodiment
The inventor is through extensive studies, is surprised to find that redox indicator can greatly be improved the efficient that detects microorganism after attached to the shot-like particle surface, and indicating effect is good.Finished 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 " and " by ... formation " belong to the subordinate concept of " containing ", " having " or " comprising ".
The particle of indicator microoraganism growth
The invention provides the particle (colour developing indication particle) of a kind of indicator microoraganism growth, described particle mainly is made of 50-600 purpose shot-like particle and the redox indicator that is attached to this shot-like particle surface.
The size of shot-like particle is correlated with for detecting effect, and too big shot-like particle surface area is too little, and too little shot-like particle 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 shot-like particle is suitable at the 50-600 order.Preferably, the size of described shot-like particle is the 100-400 order; It more preferably is the 150-300 order; Be 200 orders best.
The shape of described shot-like particle 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 shot-like particle is preferably silicon dioxide (SiO
2).The method for preparing 50-600 purpose silicon dioxide (or hydrated SiO 2) shot-like particle 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 shot-like particle beyond the silica dioxide granule also is available, cellulose grain for example, Kynoar 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 has different colors with reduced form.
Described redox indicator can be diversified, as long as its oxidized form has different colors with reduced form.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 a class reductant-oxidant.For example, described tetrazolium can be selected from: 2,3,5-triphenyltetrazolium chloride (2,3,5-triphenyltetrazolium chloride, TTC), 3-(4 ', 5 '-dimethyl-2-thiazolyl)-2,4-diphenyl tetrazole bromide, tetrazolium bromide, thiazole bromide blue tetrazolium, tetrazolium bromide, Thiazolyl blue, 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl bromination tetrazole, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazole bromide, bromination-3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazole, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-bromo tetrazolium (MTT), 2-(4-iodophenyl)-3-(4-nitrobenzophenone)-5-phenyl-tetrazolium chlorine (2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-tetrazoliumchloride, INT), tetranitro tetrazole indigo plant (2,2 ', 5,5 '-tetra-(p-nitrophenyl)-3,3 '-(3-dimethoxy-4-diphenylene)-ditetrazoliumchloride, TNBT), nitrogen tetrazolium blue (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) (NTC), OTC, PTC, MTC, WST etc.
Described to bear reddish black be a kind of reductant-oxidant commonly used, and its oxidized form presents blueness usually, and reductibility presents redness usually.
Described methylene blue is a kind of reductant-oxidant commonly used, and its oxidized form presents blueness usually, and reductibility presents colourless usually.
As a kind of selectable mode of the present invention, also be attached with stabilizing agent on the surface of described shot-like particle.Described stabilizing agent plays auxiliary effect, optimizes chromogenic reaction by stablizing redox indicator, improves the accuracy that detects.The material that any energy is stablized redox indicator all is available.Preferably, described stabilizing agent is that acidic materials are selected from (but being not limited to): boric acid, citric acid or succinic 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 shot-like particle.This kind material plays auxiliary effect, promotes the generation of chromogenic reaction by the efficient that improves proton transfer.Any material that can bring into play protolysis all is 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 form of disperseing, and after having added microorganism sample liquid contact to be measured, can be suspended in dispersedly in the sample liquid, or fall to container bottom.In addition, the particle of indicator microoraganism growth can be made ball-type, column type or positive square by bonding agent as required, stick on container bottom.Perhaps, can utilize the particle characteristics of indicator microoraganism growth as required, wait the catching image recognition of devices, cooperate to cultivate to set up and cultivate reporting system automatically by scanning.
The present invention also provides the preparation method of the particle of indicator microoraganism growth, described method comprises: with redox indicator (selectively, also comprise the material and/or the stabilizing agent that can carry out proton transfer) mix with shot-like particle, acquisition is attached with the particle of (selectively, also being attached with the material and/or the stabilizing agent that can carry out proton transfer) redox indicator.Described redox indicator, the material that can carry out proton transfer and/or stabilizing agent add quadrat method and order by merging is arbitrarily, the present invention has no particular limits.
The medium that described redox indicator (selectively also comprising the material and/or the stabilizing agent that can carry out proton transfer) mixes with shot-like particle can water or organic solvent.Described organic solvent such as methyl alcohol, ethanol etc.The use amount of water or organic solvent is the amount of minimum when guaranteeing to mix, and strengthens the amount of solvent effect is not had influence, only can prolong drying time.The adjusting of use amount or control are that those skilled in the art rule of thumb are easy to learn.
Preferably, described method also comprises: the particle that will be attached with redox indicator (selectively also being attached with the material and/or the stabilizing agent that can carry out proton transfer) carries out drying.Thereby help making redox indicator stably to be present in the shot-like particle surface, also help being in store for of particle.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 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.
As a kind of preferred implementation of the present invention; a kind of preparation method of indication particle of concrete indicator microoraganism growth is provided; comprise: (1) is with a certain amount of tetrazolium (TTC; MTT; INT; TNBT; NBT; OTC; NTC; PTC; a kind of among the MTC etc.) with the material (boric acid of a certain amount of stable tetrazolium; citric acid or succinic acid) and a certain amount of Substance P MS that can carry out proton transfer or PES and in advance 2 hours cooled silica gel particles of 130 ℃ of activation mix rearmounted oven drying; making colour developing indication particle, tetrazolium etc. is adsorbed on the silica gel particle surface.
Described colour developing indication particle can be used as a kind of general reagent and uses, and before inoculation or the use, as long as sample (or sample liquid) and nutrient culture media are mixed with described particle, cultivates, as long as there is the biological growth particle will develop the color or form red colony.Add nutrient culture media can be according to cultivating biological kind different choice.Therefore, the present invention also provides a kind of detection method of microorganism (for the detection of non-diagnostic purpose), and described method comprises: testing sample and microbiological culture media are mixed with the particle of described indicator microoraganism growth, cultivate; With determine whether have microorganism and amount in the testing sample according to the colour developing situation of particle.
The present invention also provides a kind of kit that detects microorganism, contains described indication particle in the described kit.Preferable, also can comprise nutrient culture media, water or organic solvent, the operation instructions etc. of cultivating microorganism in the described kit.
Major advantage of the present invention is:
(1) stability strengthens: the inventor is surprised to find that, the colour developing indication particle of the present invention's preparation, and the reductant-oxidant of its absorption is highly stable to temperature, and the character that autoclaving (121 ℃ 15 minutes), 108 ℃ can not cause to be reduced colour developing in 2 hours changes; Susceptibility to light has also reduced.
(2) sensitivity improves: the color that colour developing indication particle shows is very bright-coloured, bright, distinguishes easily and judges.
(3) toxicity reduces: this colour developing indication particle has reduced the toxicity of redox indicator (as tetrazolium) to microorganism (as bacterium) growth, even Much's bacillus to the difficulty cultivation, even low-down inoculation bacterium amount also can well be grown, amount compares in the nutrient culture media with directly being put into, the consumption of redox indicator can be brought up to ten times and do not influence growth, and these hypotoxicity characteristics enlarge range of application.Can be used for the detection of some poky bacterium fungies, mould, mycobacterium.
Embodiment 1, colour developing indication preparation of granules
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 cooling of sealing back.
(2) take by weighing TTC 40mg and be dissolved in (1mg/ml) in the 40ml distilled water, packing after the aseptic 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 distilled water, packing after the aseptic filtration is put 4 ℃ and is kept in Dark Place.
(4) preparation 0.1M citric acid solution, packing after the aseptic 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 30g activates, be stirred to evenly with the glass rod after the sterilization.
3. potpourri is put 80 ℃ of oven dryings, the centre can be stirred under gnotobasis, can flow until particle, and rising temperature to 108 ℃ baking 1 hour, 4 ℃ of preservations are put in the cooling of sealing back.
Embodiment 2, growth indication particle are used for common quick growth pathogenic bacteria and detect
Pathogenic bacteria comprise that (Gram-negative (Gram-), pseudomonas aeruginosa (Pseudomonas aeruginosa Gram-), Friedlander (Gram-), (Gram-positive (Gram+) sees Table 1 to staphylococcus aureus to Escherichia coli.
The preparation of MH broth bouillon: water intaking 100ml, take by weighing beef extract 0.3g, peptone 1g, NaCl 0.5g adding, after dissolving, heating transfers pH7.0-7.2, high pressure steam sterilization.
Under the gnotobasis, by the 2mL/ bottle MH broth bouillon branch is filled in 4 axenic cultivation bottles, every bottle adds about 50mg colour developing indication particle, mark; In addition, add 4 culture flasks that do not add colour developing indication particle, in these 4 culture flasks, add same amount TTC (100ug/ bottle) again by the 2mL/ bottle.
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 (physiological saline) gradient dilution to 10
-6Mg/mL.Respectively from 10
-6Respectively getting 100uL in the mg/mL dilution is inoculated in three different grown cultures bottles.
The rearmounted 37 ℃ of incubators of inoculation are cultivated, and observe the growth result of cultivating after 24 hours.
Table 1
| Microorganism | Bacterial strain | Gram | Bacterial strain number |
| E.coli | Escherichia coli | Gram- | ATCC?25922 |
| Pseudomonas?aeruginosa | Pseudomonas aeruginosa (Pseudomonas aeruginosa) | Gram- | ATCC?29213 |
| Klebsiella?pneumoniae | Friedlander | Gram- | Separated strain 3069 |
| Staphyloccocus?aureus | Staphylococcus aureus | Gram+ | ATCC29213 |
The result as shown in Figure 1.Show that colour developing indication particle can be used for showing the growth of most of bacterium, and the color that shows is vivid.
Embodiment 3, growth indication particle are used for Much's bacillus type strain H37Rv, and (Mycobacteriumtuberculosis H37Rv, cultivation ATCC95054Gram+) detects
1. nutrient culture media:
BBL
TMThe MGIT nutrient culture media, BBL
TMThe OADC nutrient culture media, BBL
TMThe PANTA nutrient culture media is available from U.S. Becton Dickinson (BD) company).
Take out above nutrient culture media respectively from container,, three kinds of nutrient culture media are carried out mixed preparing, obtain to be used to cultivate the nutrient culture media of Much's bacillus according to the instructions requirement that BD company provides, standby.
2. under the gnotobasis, be filled in the grown cultures bottle by the nutrient culture media branch of 2mL/ bottle with the cultivation Much's bacillus of above-mentioned preparation, every bottle adds about 50mg colour developing indication particle, mark.
3. inoculation, cultivation: the H37Rv bacterial strain mill bacterium after will cultivating to 1 Maxwell unit (1McFarland=1mg/mL), dilutes 10 by 1: 10 (physiological saline) gradient than turbid
-1To 10
-6Mg/mL.Respectively from 10
-2Mg/mL, 10
-4Mg/mL, 10
-6Respectively getting 100uL in the mg/mL dilution is inoculated in three different grown cultures bottles.
The rearmounted 37 ℃ of incubators of inoculation are cultivated, and observe growth result after 13 days.
The results are shown in Figure 2.Also can grow even show very low inoculation bacterium amount, and red colony number coincide with inoculum concentration, the bacterium in the time of can estimating to inoculate according to the colony number is measured.
Embodiment 4, add or do not add the preparation and the comparison of the colour developing indication particle of electron transport substance PMS
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 cooling of sealing back.
(2) take by weighing TTC 40mg and be dissolved in (1mg/ml) in the 40ml distilled water, packing after the aseptic 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 distilled water, packing after the aseptic filtration is put 4 ℃ and is kept in Dark Place.
(4) preparation 0.1M citric acid solution, packing after the aseptic filtration, 4 ℃ of preservations.
(5) get 0.1M citric acid solution 8ml under the gnotobasis and join in 40ml 1mg/ml tetrazolium (TTC) solution, after the mixing, be divided into two parts of each 24ml, a 0.1ml 1mg/ml PMS (BGITP) that adds, portion does not add PMS (BGITC).
Pour into after the mixing in the silica gel particle after 15g activates, be stirred to evenly with the glass rod after the sterilization.
(6) potpourri is put 80 ℃ of oven dryings, the centre can be stirred under gnotobasis, can flow until particle, and rising temperature to 108 ℃ baking 1 hour, 4 ℃ of preservations are put in the cooling of sealing back.
2. nutrient culture media:
BBL
TMThe MGIT nutrient culture media, BBL
TMThe OADC nutrient culture media, BBL
TMThe PANTA nutrient culture media is available from U.S. Becton Dickinson (BD) company).
Take out above nutrient culture media respectively from container,, three kinds of nutrient culture media are carried out mixed preparing, obtain to be used to cultivate the nutrient culture media of Much's bacillus according to the instructions requirement that BD company provides, standby.
3. under the gnotobasis, be filled in the grown cultures bottle by the nutrient culture media branch of 2mL/ bottle with the cultivation Much's bacillus of above-mentioned preparation, every bottle adds about 50mg colour developing indication particle, mark.
4. inoculation, cultivation: the H37Rv bacterial strain mill bacterium after will cultivating to 1 Maxwell unit (1McFarland=1mg/mL), dilutes 10 by 1: 10 (physiological saline) gradient than turbid
-2Mg/mL.Respectively from 10
-2Respectively getting 100uL in mg/mL, the dilution is inoculated in two kinds of different grown cultures bottles (BGITP, BGITC).
The rearmounted 37 ℃ of incubators of inoculation are cultivated.
BGITP just can see the red colony of growth in back 6 days in inoculation as a result, and BGITC inoculates the red colony of seeing growth in back 9 days.
Presentation of results, the colour developing particle does not contain electron transport substance PMS and can develop the color yet, and applying electronic transmitter substance PMS colour developing is faster.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. the particle of an indicator microoraganism growth is characterized in that described particle comprises: 50-600 purpose shot-like particle and the redox indicator that is attached to this shot-like particle surface.
2. particle as claimed in claim 1 is characterized in that, described particle surface also is attached with the material and/or the stabilizing agent that can carry out proton transfer.
3. particle as claimed in claim 1 or 2 is characterized in that, described redox indicator is selected from: tetrazolium, methylene blue is born reddish black or its combination.
4. particle as claimed in claim 1 or 2 is characterized in that, the described material that can carry out proton transfer is selected from: azophenlyene Methylsulfate, azophenlyene sulfovinate or its combination; And/or
Described stabilizing agent is mineral acid or organic acid; Preferably be selected from: boric acid, citric acid or succinic acid or its combination.
5. particle as claimed in claim 1 is characterized in that described shot-like particle is a silica gel particle.
6. the preparation method of the described particle of claim 1 is characterized in that, described method comprises: redox indicator is mixed with shot-like particle, obtain the particle that surface attachment has redox indicator.
7. method as claimed in claim 6, it is characterized in that, that mixes with shot-like particle also comprises: can carry out the material and/or the stabilizing agent 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 stabilizing agent.
8. the purposes of the arbitrary described particle of claim 1-5 is used to prepare the reagent of indicator microoraganism growth.
9. a kit that detects microorganism is characterized in that, contains the particle of the described indicator microoraganism growth of claim 1 in the described kit.
10. one kind is detected method of microorganism, it is characterized in that described method comprises:
(1) testing sample and microbiological culture media are mixed with the particle of the described indicator microoraganism growth of claim 1, cultivate; With
(2) determine whether have microorganism and amount in the testing sample according to the colour developing situation of particle.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN2010101881997A CN102262110A (en) | 2010-05-28 | 2010-05-28 | Color-developing grain for indicating microbial growth and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010101881997A CN102262110A (en) | 2010-05-28 | 2010-05-28 | Color-developing grain for indicating microbial growth and preparation method thereof |
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| CN102262110A true CN102262110A (en) | 2011-11-30 |
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| CN2010101881997A Pending CN102262110A (en) | 2010-05-28 | 2010-05-28 | Color-developing grain for indicating microbial growth and preparation method thereof |
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| CN (1) | CN102262110A (en) |
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| CN102344887A (en) * | 2010-08-02 | 2012-02-08 | 新疆维吾尔自治区胸科医院 | Indicator tube for indicating microbe growth and application thereof |
| CN102559844A (en) * | 2012-01-09 | 2012-07-11 | 上海交通大学 | Preparation method of detecting system for minimal inhibitory concentration of mycobacteria |
| CN102721683A (en) * | 2012-05-25 | 2012-10-10 | 中国水产科学研究院黄海水产研究所 | Method for rapidly detecting salt and sugar content in dry sea cucumber product |
| CN106480160A (en) * | 2015-09-01 | 2017-03-08 | 郑兆珉 | Method for manufacturing microorganism detection device, microorganism detection method, microorganism detection kit, and device |
| CN109212015A (en) * | 2018-09-18 | 2019-01-15 | 泰山医学院 | A kind of material surface oxidation-reduction quality detection method |
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344887A (en) * | 2010-08-02 | 2012-02-08 | 新疆维吾尔自治区胸科医院 | Indicator tube for indicating microbe growth and application thereof |
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| CN106480160A (en) * | 2015-09-01 | 2017-03-08 | 郑兆珉 | Method for manufacturing microorganism detection device, microorganism detection method, microorganism detection kit, and device |
| CN109212015A (en) * | 2018-09-18 | 2019-01-15 | 泰山医学院 | A kind of material surface oxidation-reduction quality detection method |
| CN109212015B (en) * | 2018-09-18 | 2021-02-23 | 山东第一医科大学(山东省医学科学院) | Method for detecting surface oxidation-reduction property of substance |
| 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: 20111130 |