CN101130808A - Fast-culturing susceptibility and micro-detection method for mycobacteria and device thereof - Google Patents
Fast-culturing susceptibility and micro-detection method for mycobacteria and device thereof Download PDFInfo
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Abstract
The present invention relates to a micro detection method capable of quickly culturing mycobacterium to make drug sensitivity test and drug identificiation. Said method includes the following steps: (1), treating speciment, inoculating said treated specimen in culture medium, making culture, taking out the grown acid-fast mycobacterium from biological safety cabinet and preparing bacterial suspension with defined concentration; (2), in a biological safety cabinet opening upper cover of special-purpose micro culture plate, every well interior of said culture plate has previously-added culture medium and correspondent drug, adding 0.1ml of bacterial suspension with defined concentration into every well, closing upper cover and sealing; (3), placing every plate into a sealed wet box, and placing said sealed wet box into a constant temperature incubator to make culture; and (4), taking out culture plate, utilizing optical amplification system to collect bacterial growth image and comparing said bacterial growth image with negative and positive contrast well images, if said bacterial growth image is similar to the positive contrast well image, it is drug resistance, otherwise it is sensitivity.
Description
Technical field
The present invention relates to a kind of to the cultivation of microorganism and the method and the isolated plant of medical test.Especially mycobacterium fast-culturing susceptibility and identify the trace detection method and device.
Background technology
The 4th national tuberculosis epidemiological random sampling survey, 59 points are carried out total man's mouth infection rate investigation result: the census de facts is 365,097 people, active tuberculosis morbidity 3,67/,100,000, bacterium sun pulmonary tuberculosis morbidity rate are 1,60/,100,000, the tuberculosis epidemic situation in the whole nation is still very serious.In 27 kinds of emphasis transmissible diseases that the Ministry of Health announces, the morbidity number of tuberculosis occupies second, and death toll occupies the 3rd, all comes before the SARS.
Laboratory examination lungy is the main means of clinical diagnosis lungy and epidemiological survey, and wherein bacteriology checking is the gold standard of diagnosis of tuberculosis.Current tuberculotherapy is still based on the antitubercular agent chemotherapy, but at present to one or more antitubercular agents while drug-fast bacterial strain (many resistant organisms) showed increased, also have some to rely on bacterium and (promptly certain medicine is had dependency, when this medicine exists in addition growth better), therefore it is responsive detecting which medicine early, and which resistance is to instruct treatment ten often important.
The pathogenic agent of tuberculosis is antiacid mycobacterium.Most of mycobacteriums, particularly retarding of growing mycobacterium flora, mycobacterium tuberculosis for example, mycobacterium bovis BCG etc., it is very slow to grow.Mycobacterium is cultivated still traditional method (hereinafter to be referred as " rules " method), i.e. absolute concentration method and the scaling method (the national Clinical Laboratory working specification third edition 2,006 916～917) of adopting of drug sensitive test more at present.On tradition improvement sieve (L-J) family name solid medium, cultivate one's ability through 4～8 weeks and grow bacterium colony, could obtain drug sensitivity tests through 4 weeks again, can not satisfy the needs of clinical chemotherapy.Therefore acid-fast bacilli is cultivated the development that susceptibility is checked, mainly concentrates on the research of short-time test.
Chinese scholars all strives to find and cultivates fast and medicament sensitivity testing method in recent years.BACTEC 460
TBQuick mycobacterium automatic detection device, can be ahead of time more than 10 days with traditional incubation time, susceptibility time average 7 days (Lin Jianxiong, the clinical application of the automatic rapid detection tubercule bacillus of Zheng Chong brightness BACTEC460-TB and estimate international medical and health Leader 2001V N994), but this method is owing to use
14C isotropic substance palmitinic acid is made matrix, and radioactive substance causes environmental pollution, after using for many years, now by many developed countries and China's forbidding.
Emphatically the on-radiation method is studied both at home and abroad over nearly 20 years, has been delivered many this respect method articles:
(1) biloluminescence method: ATP is the index of bacterium energy metabolism, and biological half-life is short, and after bacterial growth inhibition or death, ATP content promptly obviously descends in the born of the same parents, thereby can be used as the sign of viable bacteria.After Mycobacterium tuberculosis was cultivated for some time, ATP in the extracting bacterium added bioluminescent reagents then, carries out detection by quantitative by photometer.Can obtain the result in 5-7 days, can't distinguish pathogenic bacterium and non-pathogenic bacteria but exist simultaneously, shortcoming such as the ATP extracting is loaded down with trivial details gets bacterium sample extractive process easily to pollute after the cultivation, need further to improve just can be used for clinical.
(2) phage bioluminescent detection method: but adopt bioluminescence technique to detect the mycobacteriophage phage40 of expressing luciferase, the luminous reaction and the drug sensitive test of different bacterium are measured.In containing the substratum of antitubercular agent, the non-resistance tubercule bacillus of the luminous strength ratio of resistance tubercule bacillus is strong, and its strength difference has the significance meaning.The drug sensitive test result of phage40 is consistent with conventional Russell medium method coincidence rate, can obtain the result in 72h.(Chen Zhifei detects mycobacterium tuberculosis resistance China laboratory medicine magazine 2000V23N3 144-147 with recombinant phage for Lv Bin, Xu Shunqing)
(3) flow cytometer method: the etheric acid fluorescein is a kind of nonpolar fluorescent substance, can see through cell walls and the cytolemma of mycobacterium by the mode of active transport and passive diffusion.Non-specific lipase can the rapid external FDA of hydrolysis be the free fluorescein in the mycobacterium kytoplasm of living, and dead bacterium reduces in a large number because of active lipase, and a little less than the effect of hydrolysis FDA, free fluorescein produces seldom.Flow cytometer can detect the mycobacterium of band fluorescence, be used for the viable bacteria diagnosis of Mycobacterium tuberculosis, 24h draws drug sensitive test result (Norden Ma, Kurzynsik TA, Bownds SE, etal.Rapid susceptibility testing of Mycobacterium tuberculosis (the mycobacterium H37Ra of flow cytometer rapid detection band fluorescence) by Flow Cytometry[J] .J.ClinMicrobiol, 1995,33 (5): 1231-1237.).Fluoresced by the hydrolysis of bacterium lactonase owing to FDA also can enter other bacteriums, influence the specificity of detected result.The etheric acid fluorescein can natural hydrolysis, causes false positive.It is open operation that culture sample imports the flow cytometer process, is easy to pollute.Flow cytometer device costliness in addition, the technical requirements height, thereby be difficult in clinical expansion.
(4) mensuration of drug resistant gene: PCR, probe hybridization, dna sequencing equimolecular biological method have been used for the direct detection of mycobacterium at present, have greatly shortened Diagnostic Time.Dna sequencing is the gold standard of detection in Gene Mutation, can determine the position and the character of suddenling change.But concerning each strain bacterial strain, measure all known mutable sites of various antitubercular agents and need repeatedly reaction, expense is very expensive, therefore can't be used for clinical.DNA chip detection principle be with multiple probe stationary on substrate, hybridize with the DNA or the RNA of sample to be tested then, obtain information.Since this method simultaneously with a large amount of probe stationary on upholder, so once can detect and analyze to a large amount of sequences of sample.Very tempting in theory, but still do not have DNA chip supply applicable to clinical examination.The gene test aspect, it must be noted that be not the drug resistant gene of each medicine have only a kind of or only with a kind of sudden change, resistance mechanism is relevant, although several antitubercular agents commonly used have all found at least one gene relevant with resistance, but be successfully applied to the detection of having only of the clinical drug sensitive test ropB gene relevant at present with the Rifampin resistance, itself and traditional drug sensitive test as a result coincidence rate more than 80%, and the bacterial strain of anti-Rifampin the more than 95% is simultaneously to other antitubercular agent resistance, thereby the primary dcreening operation that can be used as MDR-TB is tested (Cheng Shaoji, Yan Biya, the cold SSCP rapid detection of horse small island PCR-tubercule bacillus rpoB transgenation tuberculosis and breast tumor 1996V N3 117-121).
In sum, various new methods are all quick than traditional method, but also have many weak points, therefore also.No matter that a kind of method, only towards quick, accurate, sensitive, antifouling, special and economic direction develops and perfect, just is expected to replace traditional method at clinical application.
The more fast method of clinical at home use has following several at present:
(1) the BACTEC MGIT960 of Becton Dickinson company instrument: at home and abroad use, BACTEC-TB960 utilizes oxysensible fluorescence ruthenium mixture as fluorescent probe, detects the content of oxygen in the substratum.When bacterial growth is good, oxygen level descends in the substratum, get final product fluorescence excitation, fluorescence strengthens expression bacterial growth or resistance (Wang Wei, Li Hongmin, Wang Ansheng BAC TEC-M GIT960 fast-culturing susceptibility be national defence consumptive disease magazine the 25th the 6th phase 379 of volume of December in 2003 to the application of pulmonary tuberculosis diagnosis and treatment with in estimating).But its instrument and special-purpose culture tube cost an arm and a leg (instrument is about 1,000,000 yuan, and each test pencil is about 70 yuan), the necessary dependence on import of reagent is difficult to popularize in basic unit at home especially.Only 4～5 kinds of the anti-tubercle bacillus drugs that provides in addition.
(2) the BacT ALERT 3D culture systems of Aksu company: BacT ALERT 3D instrument is to utilize the color reaction device to monitor the CO2 that discharges in the mycobacterium process of growth, along with the rising of CO2 concentration, and H in the substratum
+Concentration also directly raises, thereby the color that makes inductor block is by blackish green golden yellow (the Thorpe TC that becomes, Wilson ML, Turner JE, et al.Bact/Alert:an automated colorimetric microbial detection system J.Clin Microbiol, 1990,28 (7): 1608-1612; Liao passes evaluation clinical lung section magazine the 10th the 1st phase 115 of volume of January in 2005 of high ten thousand phlegm mycobacterium fast culture of beautiful Jiang Ke jade-like stone and drug sensitive test).
(3) the ESP system of DIFCO company: be to utilize pressure inductor, detect in the sealing culturing bottle, produce CO in the metabolism of culturing process bacterial growth
2, H
2, N
2With consumption O
2Cause the change of gaseous tension in the culturing bottle.At present, abroad by the full-automatic fast culture II of system of ESP of Ddifco company development, can carry out mycobacterium tuberculosis fast and cultivate and drug sensitive test.They all can obtain drug sensitivity tests at 7-12 days, but need the special instruments and equipment of import, and the complete dependence on import of agents useful for same, cost an arm and a leg, and are difficult to promote on the basis.
(4) phage splitting method: mycobacteriophage is merely able to infect the mycobacterium of corresponding work, and breeds in thalline, makes the indicator cellular lysate, plaque occurs.By observing having or not of plaque, can judge drug-resistance of bacteria.This method and other method relatively have following advantage: fast, average 48h obtains drug sensitivity tests; Accurately, reach 90% with traditional method comparison consistence; Simply, need not special instruments and equipment (Hu Zhongyi, Pang Maoyin, Jin Anjia etc. use phage splitting method Rapid identification Mycobacterium tuberculosis, Chinese tuberculosis and breathe magazine, 2001,24:611-613; McMerney R, TB:the return of the phage, A review of fifty years of mycobaterphageresearch, Int J Tuberc Lucg Dis, 1999,3:179-184; Huanghai Sea honor, Ma Yu, the application of phage in Mycobacterium tuberculosis research, Chinese tuberculosis and breathing magazine .2002,25 (8): 797-799).But mycobacteriophage infects mycobacterium has specificity, only can be used for Mycobacterium tuberculosis, insensitive to other tuberculosis or the antiacid mycobacterium of non-tuberculosis, makes and detects in this way and can not detect.Identify that in addition medicine is very limited, can't detect most antibacterial medicines, only there is Rifampin to use at present, and cost an arm and a leg, the reagent valency of each test is 70 yuan, even if enlarged the susceptibility kind in the future, if do 10 kinds of medicines commonly used, the light reagent cost will 700 yuan, and therefore this method lacks practical value at present.The speed that above method has is fast inadequately as the 3D method, and the susceptibility time average takes 12 days.Though the speed that has is fast as the phage method, because of the medicine that can do can not enter the clinical practice stage very little.All these methods are all very expensive, and for domestic most of people can not bear, particularly therefore the tuberculosis poor areas of the western region occurred frequently can't promote in basic unit.Therefore it is very necessary to set up quick, easy, efficient, an antifouling economic again drug sensitive test method.
Summary of the invention
Goal of the invention: the present invention seeks to propose mycobacterium fast-culturing susceptibility and evaluation and surpass thousand times of continuous trace detection methods of amplification and device; Especially by computer control, steplessly surpass thousand times of amplification, trace is cultivated and is line-of-sighted observation and supper-fast cultivation susceptibility of mycobacterium and identification systems that instant image acquisition report is formed continuously.The antiacid mycobacterium that rapid detection goes out from the tuberculosis patient separation and Culture is to the susceptibility of various antibacterials, for clinical treatment provides foundation.Set up a kind of fast, simple, directly perceived, economical, safety is antifouling and more near the cultivation susceptibility method of human internal environment's state.
Technical scheme: mycobacterium fast-culturing susceptibility and evaluation trace detection method:
1. after the sample disposal, be inoculated in substratum, through cultivating, the antiacid mycobacterium that will grow in the Biosafety cupboard is taken out, and makes the bacteria suspension of normality;
2. in the Biosafety cupboard, the special-purpose microtest plate loam cake that is provided with is opened, each hole has added substratum and relative medicine in advance in the plate, other has positive control hole (contain substratum and do not have medicine) and negative control hole (no substratum does not have medicine), the bacteria suspension that in each hole, adds the 0.1ml normality, loam cake is built, and lid and bottom are asked seal; Below all carry out according to cultivating rules;
3. each plate is put into the wet box of sealing, placed in 37 ± 2 ℃ of constant incubators and cultivate;
4. every day or take out culture plate every other day, be placed on the live bacteria direct-view amplification system and observe; From the viewing window of each bottom, hole of special-purpose culture plate,, observe the bacterial multiplication situation continuously in the bottom imaging.By optical amplification system, gather the bacterial growth image, and compare with negative and positive control wells image, the similar resistance that is judged to the positive control hole, on the contrary be responsive.
Special-purpose observing device of the present invention is: the culture hole of microtest plate is the tapered stage body of cylinder, and diameter only has 1 ± 0.3mm at the bottom of the hole, has the smooth and slide glass sample light transmission of minute surface, as the view port of gathering image.Behind application of sample, by natural subsidence, make bacterium concentrate on view port at the bottom of the hole, observation efficiency in the time of can improving bacterial multiplication.There is an outstanding sealing-ring corresponding each top, hole of plate lid, covers the back and embeds in the aperture, prevents that effectively liquid is excessive in the hole.
Image acquisition amplification detection: constitute by opticmicroscope or with optics amplification CCD image acquistion system, by camera lens image is carried out micro-amplification, amplify and directly to observe through the CCD image acquistion system from watch-dog, for example can directly observe the bacterial multiplication state continuously from the microtest plate bottom, magnification can be carried out step-less adjustment.High-visible.Because the direction of observation of design can carry out from the culture plate bottom, therefore the breeding that can observe bacterium continuously under the live body culture condition.
Beneficial effect of the present invention: fast: the bacterium in cultivating can be amplified 1900～4000 times by surpassing thousand times of amplification systems continuously, can be early stage in culturing process, observe the propagation of single bacterium and the propagation form of small bacterioflora, therefore can judge pharmaceutically-active result in early days.And classic methods is to grow macroscopic bacterium colony on bacterium through a large amount of breedings, and the ability judged result is so " rules " regulation wanted for 4 weeks just reported the result.In addition, improvement (using the medicine that adds appropriate amount in the improvement 7H9 substratum) by substratum, make it be more suitable for the growth of antiacid mycobacterium, compare with other substratum, accelerate the speed of growth, therefore can obtain in advance drug sensitivity tests (Shi Xudong, Liu Zhenghua, Wu Xiaoyuan etc. mycobacterium tuberculosis cultivation and drug sensitive test and flora are identified the research of rapid method method. Chinese laboratory medicine magazine 2,005 28 (8) 790-792).
This law is measured the clinical strains of 80 examples, drug sensitive detection time average 5.2 days, and 71.3% in 5 days, and 90.3% obtained the result in 7 days.All (take 12 days soon than present any cultivation susceptibility method as 3D method susceptibility time average; BACTEC-MGIT960 fast-culturing susceptibility time average takes 6.5 days).
Simply: in each micro-culture hole of microtest plate, added the substratum that contains certain dose (can make freeze-dried reagent and make commercialization) that configures in advance, directly add the bacteria suspension that comes from first cultivation, place 37 ℃ of constant incubators, every other day or put live bacteria direct-view every day and surpass continuously on thousand times of amplification systems and observe, the quantity of the bacterial growth that shows according to watch-dog and the form of small bacterioflora, with the positive control boring ratio than result of determination, gather visual store recording by computer, the printable picture and text report of result.Method is simple, and all have certain empirical Clinical Laboratory personnel, can learn operation through all trainings.And the large-scale instrument complex structure of import needs higher technology and foreign language level.
Intuitively: surpass thousand times of amplification systems continuously by the live bacteria direct-view, can observe directly the growth conditions of bacterium under drug effect, observation is under airtight animation, can judge according to the quantity of bacterium individuality and the form of small bacterioflora, this bacterial strain is sensitivity, resistance or dependence (promptly this bacterial strain is grown in the presence of this medicine better) to this medicine, and can judge whether mould or some living contaminantses according to form.Though method also belongs to the method for line-of-sighting observation, it is the bacterium colony that detects by an unaided eye " rules ", can't see each bacterium, so the time that needs is long, and can not see the fine variation of bacterium in process of growth; And additive method (as BACTEC MGIT960 instrument, ESP system, BacT ALERT3D instrument etc.), all be that the indirect biological phenomena to measure biology is index (as the consumption of oxygen, the pressure change that produces gas and a carbon dioxide generating etc.), might not be relevant fully with antiacid mycobacterium growth, false positive often appears.
Economical: the live bacteria direct-view of use surpasses thousand times of amplification systems continuously and special-purpose microtest plate is to make up and make with domestic raw materials fully, and cost is very low, every five yuan of special-purpose microtest plates (contain 40 and detect orifice plate).The substratum that micro plate uses seldom, each detects only 0.1ml of orifice plate (test), (only be well-established law 1/25～1/40).Therefore therefore the cost price of the every test of this law monobasic has only saved a large amount of reagent costs.
Safety is antifouling: the breezing plate that will install substratum and relative medicine places in the Biosafety cupboard, open and after loam cake adds bacteria suspension lid is covered, being placed on 37 ℃ of incubators with the scotch tape sealing cultivates, no longer uncap when observation is checked afterwards, until the result reports whole culture plate autoclave sterilization in back or microwave disinfection.And some rapid method (as phage method and flow cytometer method) need be uncapped, taking-up, tube and open mensuration repeatedly, has increased Pollution risk.
More near the human internal environment: use liquid nutrient medium, bacterium is grown at liquid internal, with the solid medium that " rules " are used, bacterium is the growth phase ratio under the environment that the surface is direct with air contacts of substratum, more approaches the human internal environment.Because tubercule bacillus is to parasitize in the cell, so the result who cultivates in liquid is more near clinical state.
Be particularly suitable for the MIC of antibacterials and mix drug sensitive test: no matter be MIC or mixing drug sensitive test, it all needs the pipe of a plurality of different concns or the pipe of a plurality of different pharmaceutical and concentration collocation to each medicine, no matter on the cost or in operation, all can not in Clinical Laboratory, implement at other rapid methods.This law is owing to use porous microtest plate, and the substratum consumption seldom is easy to carry out the collocation of doubling dilution and medicine and mixes, and cost is low easy and simple to handle, so can realize on the clinical meaning it being MIC or mixing drug sensitive test.
Description of drawings
Fig. 1 is the negative control display image
Fig. 2 is the positive control display image
Fig. 3 is the responsive display image in vazadrine
Fig. 4 is a Rifampin resistance display image
Fig. 3 is a Tibutol resistance display image
Fig. 4 is the responsive display image of Levofloxacin
Fig. 5 is a Streptomycin sulphate resistance display image
Fig. 8 is the responsive display image of amikacin
Fig. 9 is that the present invention cultivates active somatic cell (bacterium) and observation testing process figure is amplified in direct-view
Figure 10 is the bacterium microtest plate modular construction synoptic diagram of (or claiming microtest plate),
Embodiment
1. after patient's sample (phlegm, ascites pleural fluid, cerebrospinal fluid, urine, just or irrigating solution etc.) is handled by Ministry of Health's " rules " prescriptive procedure, be inoculated in substratum, through cultivating (L-J method or other rapid methods), the antiacid mycobacterium that will grow in the Biosafety cupboard is taken out, and makes the bacteria suspension of normality.
2. in the Biosafety cupboard, special-purpose microtest plate loam cake is opened, each hole has added substratum and relative medicine in advance in the plate, other has positive control hole (contain substratum and do not have medicine) and negative control hole (no substratum does not have medicine), the bacteria suspension that in each hole, adds the 0.1ml normality, loam cake is built, will be sealed between lid and bottom with scotch tape.
3. each plate is put into the wet box of sealing, placed in 37 ℃ of constant incubators and cultivate.
4. every day or take out culture plate (not opening lid) every other day, be placed on the live bacteria direct-view and surpass continuously on thousand times of amplification systems and observe.From the viewing window of each bottom, hole of special-purpose culture plate, by surpassing thousand times of amplification systems, gather the bacterial growth image, and compare with negative and positive control wells image, the similar resistance that is judged to the positive control hole, on the contrary be responsive.
Material and equipment:
(1) improvement Michaelis (Middleblook) 7H-9 liquid nutrient medium.
(2) special-purpose microtest plate: every pore volume 3.5ml has sample and concentrates at the bottom of the optical aperture and closed cover.After this plate aseptically process, in each hole, add the substratum that contains various different antibacterials in advance respectively.
(3) the continuous direct-view of live bacteria surpasses thousand times of amplification systems in the cultivation.This system can amplify 1000～4000 times (magnification is stepless adjustable) with the bacterium in cultivating and be presented on the watch-dog, and the result gathers image by computer control and stores and printable picture and text report.
(4) Biohazard Safety Equipment (one-level or secondary), aseptic imbibition head, aseptic dropper, sterile distilled water, bacterial concentration standard opacity tube etc.
(5) bacterial classification source:
The mycobacterium clinical strains: from outpatient service and inpatient's sample, through after the pre-treatment, inoculation culture is separated the back and is obtained by " rules ".
Concrete cultural method:
(1) in Biohazard Safety Equipment, with the clinical samples isolated strains, take out, add the sterile distilled water dilution through grinding from substratum, make the proper concn bacteria suspension, every hole 0.1ml.
(2) special-purpose microtest plate loam cake is opened, each hole has added substratum and relative medicine in advance in the plate, other has positive control hole (contain substratum and do not have medicine) and negative control hole (no substratum does not have medicine), the bacteria suspension that in each hole, adds the 0.1ml normality, loam cake is built, will be sealed between lid and bottom with scotch tape.
(3) each plate is put into the wet box of sealing, placed in 37 ℃ of constant incubators and cultivate.
(4) result observes: every interval 24h or 48h, special-purpose culture plate is taken out from 37 ℃ of constant temperature culture, bacterium continuously direct-view surpass under thousand times of amplification systems and observe once, from each bottom, hole of special-purpose culture plate, by surpassing thousand times of amplification systems, gather the bacterial growth image, and compare with yin, yang control wells image, be judged to resistance with positive control hole growth population and small bacterioflora plesiomorphism, otherwise be responsive.And, make picture and text report with the acquisition and recording observations.
The bacterium microtest plate:
Microtest plate has cylinder taper trace culture hole, and diameter only has 1mm at the bottom of the hole, has the smooth and slide glass sample light transmission of minute surface, as the view port of gathering image.There is an outstanding sealing-ring corresponding each top, hole of plate lid, covers the back and embeds in the aperture, prevents that effectively liquid is excessive in the hole.See that Figure 10 microtest plate tool wants cylinder taper trace culture hole, the culture hole of culture plate is the tapered stage body of cylinder, by natural subsidence, makes bacterium concentrate on view port at the bottom of the hole, observation efficiency in the time of can improving bacterial multiplication behind application of sample.The culture hole of microtest plate 1 is that cylinder adds a tapered stage body 2, is pure flat 3 at the bottom of the hole at the bottom of being tapered.Figure 10 is a unit of microtest plate, generally is to be arranged with many row's multiple rows on the plate, and the plate of microtest plate covers 4 and is provided with an outstanding sealing-ring 5.
Amplification detection instrument directly perceived (can optics be amplified in more than 700 times and can observe) can be accomplished the stepless thousand times of amplifications that surpass, amplifying the CCD image acquistion system by light source that places micro-culture hole top and optics constitutes, after amplifying, can directly observe the bacterial multiplication state continuously by microtest plate bottom object lens imaging optical path from the microtest plate bottom, but the magnification step-less adjustment.Make non-painted living specimen be stereoscopic image, high-visible.Because the direction of observation of design is to carry out from the culture plate bottom, therefore the breeding that can observe bacterium continuously under the live body culture condition.
Claims (5)
1. the trace detection method of mycobacterium fast-culturing susceptibility and evaluation,
1. after the sample disposal, be inoculated in substratum, through cultivating, the antiacid mycobacterium that will grow in the Biosafety cupboard is taken out, and makes the bacteria suspension of normality;
2. in the Biosafety cupboard, the special-purpose microtest plate loam cake that is provided with is opened, each hole has added substratum and relative medicine in advance in the plate, other has positive control hole (contain substratum and do not have medicine) and negative control hole (no substratum does not have medicine), the bacteria suspension that in each hole, adds the 0.1ml normality, loam cake is built, and will be sealed between lid and bottom;
3. each plate is put into the wet box of sealing, placed in 37 ± 2 ℃ of constant incubators and cultivate;
It is characterized in that 4. every day or take out culture plate every other day, be placed on the live bacteria direct-view amplification system and observe; From the viewing window of each bottom, hole of special-purpose culture plate, by optical amplification system, gather the bacterial growth image, and compare with negative and positive control wells image, the similar resistance that is judged to the positive control hole, on the contrary be responsive.
2. the trace detection method of mycobacterium fast-culturing susceptibility according to claim 1 and evaluation, it is characterized in that adopting microtest plate to cultivate sample, the culture hole of described microtest plate is the tapered stage body of cylinder, behind application of sample, pass through natural subsidence, make bacterium concentrate on view port at the bottom of the hole, in the bottom imaging, observe the bacterial multiplication situation continuously.
3. the trace detection device of mycobacterium fast-culturing susceptibility and evaluation: adopting optical imagery amplification system and culture systems to constitute, it is characterized in that the culture hole of the microtest plate (1) of culture systems is the tapered stage body of cylinder (2), is pure flat (3) at the bottom of the hole.
4. the trace detection device of mycobacterium fast-culturing susceptibility according to claim 3 and evaluation: the plate that it is characterized in that microtest plate covers (4) and is provided with an outstanding sealing-ring (5).
5. the trace detection device of mycobacterium fast-culturing susceptibility according to claim 3 and evaluation: it is characterized in that diameter 1 ± 0.3mm at the bottom of the hole.
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