CN100430486C - Quick determination for microbe munity - Google Patents
Quick determination for microbe munity Download PDFInfo
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- CN100430486C CN100430486C CNB011032812A CN01103281A CN100430486C CN 100430486 C CN100430486 C CN 100430486C CN B011032812 A CNB011032812 A CN B011032812A CN 01103281 A CN01103281 A CN 01103281A CN 100430486 C CN100430486 C CN 100430486C
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Abstract
The present invention relates to a method for quickly determining microbial susceptibility. Microbes to be determined are inoculated in a viscous liquid culture medium containing a certain substance of which the concentration is to be determined; within the culture time, the cell number and the cell aggregation mode are checked; when the cell number is increased and micro colonies are formed, the state represents that the microbial population increases and is not sensitive to substances to be determined, such as antivitamin and antibiotic; the minimal inhibitory concentration of the substance to be determined shows the cell number is not increased and no micro colonies are formed.
Description
The present invention relates to the method for a kind of rapid determination microorganism to various material susceptibilities.
Since nineteen twenty, existing several different methods is suggested and is used to check that microorganism is to the susceptibility such as materials such as antimicrobial element/microbiotic.But, have only two kinds of methods to be widely accepted and become the standard method of clinical practice.These two kinds of methods are dilution susceptibility test and scraps of paper diffusion test (seeing that L.M.Prescott etc. writes pause in U.S. Persian " microbiology " third edition 660-662 page or leaf of publication of Wm.C.Brown press in 1996).
In the test of dilution susceptibility, the microorganism to be measured of preferred number is inoculated in and contains in the antimicrobial element of a series of concentration/antibiotic meat soup culture tube, checks that then these are by the microbial population growth conditions of inoculated tube.Do not show in the pipe of microorganism growth that at all that pipe that antimicrobial element/antibiotic concentration is minimum is promptly represented minimum inhibition concentration.The microorganism growth situation is to determine by the turbidity of measuring culture.This mensuration needs can show in 16 to 20 hours the variation of significance usually.
In disk diffusion method, microorganism to be measured is inoculated on the agar plate, places one then thereon and contains antimicrobial element/antibiotic round scraps of paper.An antimicrobial element/antibiotic antimicrobial element/antibiotic the concentration gradient that diffuses to form: maximum concentration is arranged in the scraps of paper, and minimum concentration is positioned at the leading edge of diffusion.Containing effective antimicrobial element/antibiotic place, microorganism can not grow and demonstrate a transparent circle around the scraps of paper.Outside this circle, because antimicrobial element/antibiotic concentration is lower than minimum inhibition concentration, microorganism growth and form lawn.Need considerable time owing to form macroscopic lawn, the susceptibility of microorganism just can be reported after cultivating 16-18 hour usually.
In disk diffusion method, antimicrobial element/the antibiotic concentration that is positioned at the transparent circle edge is not to be drawn by disk diffusion method itself, but need dilute the susceptibility test in addition, to draw the typical curve and the form of transparent circle and minimum inhibition concentration relation, Cha Tu or table look-up and draw this concentration then.Because the transparent circle size is subjected to the factor affecting such as situation of antimicrobial element/antibiotic initial concentration, solvability and velocity of diffusion and agar plate, as estimating minimum inhibition concentration from disk diffusion method exactly, typical curve or criteria table should be benchmark to criticize antimicrobial element/microbiotic scraps of paper together and to criticize agar plate together.Therefore, although disk diffusion method is implemented easily than the test of dilution susceptibility, it depends on back one method could obtain complete results.
The test of all conventional microorganism susceptibilities all requires sample is cultivated on agar plate in advance to observe microorganism growth and Identifying micro-organisms, carries out secondary cultivation enriched microorganism colony then and is used to inoculate the meat soup pipe of dilution susceptibility experiment or the agar plate of scraps of paper diffusion test with the microorganism that obtains enough preferred numbers.Add the time that these preparation works are required, complete microorganism susceptibility mensuration takes two to three days usually and just can finish.Conventional microorganism susceptibility assay method uses Petri dish and test tube to cultivate.Therefore, need with a large amount of substratum and a large amount of test substances.In addition, cultivate and store instrument and the space that these devices also will be bigger.
Recently, the method for some improvement has been suggested the rapid determination that is used for the microorganism susceptibility.For example:
No. 5789173, United States Patent (USP), microorganism susceptibility rapid assay methods.
No. 5770373, United States Patent (USP) uses the oligonucleotide probe at the ribosome-RNA(rRNA) precursor that the resistance mycobacterium is carried out quick and responsive detection.
No. 5738989, United States Patent (USP) uses the rrna nucleic acid hybridization to measure the susceptibility of microorganism combating microorganisms material.
No. 5738988, United States Patent (USP) is checked microorganism and antimicrobial material unknown in the sample with the rrna probe hybridization.
No. 5712095, United States Patent (USP) uses the oligonucleotide probe at the ribosome-RNA(rRNA) precursor to carry out quick and responsive detection to chemical sproof mycobacterium is arranged.
No. 5948633, United States Patent (USP) is measured the action of microorganisms of compound to cultured continuously.
The sample that No. 5789173 described fast microbiological susceptibility tests of United States Patent (USP) depend on Short-term Culture carries out the DNA amplification to reach the purpose of early stage judgement microbiotic efficient.This method does not need sample is cultivated in advance.By to the DNA after amplifying detect rather than to the detecting of meat soup pipe turbidity or dull and stereotyped inhibition zone, this method has shortened the required time of report minimum inhibition concentration (MIC).It is said that this method whole process can be as short as 12 hours.
United States Patent (USP) is described a kind of quick and responsive method that detects the resistance mycobacterium that is specifically designed to for No. 5770373.By use specially designed can with mycobacterium ribosome-RNA(rRNA) precursor (pre-RNA) bonded oligonucleotide probe, this method can be checked out existence and the upgrowth situation of mycobacterium.Inspection about mycobacterium Pre-RNA is introduced in an early stage invention (United States Patent (USP) 5712095).
No. 5738989, United States Patent (USP) is described a kind of methods of using the rrna nucleic acid hybridization to determine the sensitivity testing of microorganism combating microorganisms material, and this method only need prepare can the bonded probe to the sequence of a certain special subunit of rrna nucleic acid of some selected microorganism.
The method that United States Patent (USP) is described a kind of detection compound to the action of microorganisms of cultured continuously No. 5948633.In this method, nutrition and test compounds are added in the growth room with the flow velocity of controlling respectively, make the concentration of test compounds increase gradually.Test compounds is treated the survey action of microorganisms and is learnt by the mensuration that convection current goes out the cell density of growth room's (chemostat).When microorganism cells density did not increase, compound concentrations was the minimum inhibition concentration to this microorganism to be measured.
Microorganism susceptibility measuring method elapsed time, manpower and the material of standard.Though existing modification method has overcome some shortcomings of standard method, still has such or such defective.
No. 5789173 described extra complex steps of methods needs of United States Patent (USP): the reporter molecules that amplifies DNA, usefulness ad hoc report molecular marker DNA, quantitative assay institute mark with polymerase chain reaction,PCR.This method needs extra instrument, as thermal cycling machine and fluorescent meter.This method also will have certain knowledge to be used for DNA and to amplify reaction to design suitable primer the DNA of the microorganism measured.
No. 5770373, United States Patent (USP) (together with No. 5712095, United States Patent (USP)) and United States Patent (USP) all require the instrument of specially designed probe and corresponding detection probes for No. 5738989.These methods only are fit to the microorganism of a certain selected scope because the specificity of probe is different.If radio isotope is used to label probe, also need special instrument, license and safety training.
No. 5948633 described microorganism susceptibility detection methods of United States Patent (USP) can be measured the minimum inhibition concentration of a certain compound to microorganism in split hair caccuracy ground in single test.But in order to find this minimum inhibition concentration in same test, the population density of microorganism must repeatedly be measured at the different time that increases test compounds concentration.This means that each cultured continuously microorganism susceptibility experiment must the operation long period.This method needs special culture apparatus and a large amount of nutritional medium and testing compounds.Final this method still depends on ordinary method such as microscopic counting, electronics grain count and wandering cells instrument to the mensuration of culture cell density.
Therefore, although existing a lot of make great efforts flower in other method of invention to measure the microorganism susceptibility, obtain to accept in clinical labororatory without any new invention.Conventional microorganism susceptibility method of testing still is the used standard method of everyday practice.
The objective of the invention is to overcome the deficiency of above-mentioned ordinary method and modification method and create a kind of quick, responsive, special, simple, economic, easy microorganism susceptibility assay method of row.
The objective of the invention is to reach by following measure:
(a) use the thick liquid substratum to carry out the cultivation of microorganism.The use of thick liquid substratum makes microbe inoculation, adds the substratum composition and adds test substance more convenient.Also more help the mixing between nutrition, test substances and the microorganism.The more important thing is that it makes offspring from same ancestors microorganism to stay together and forms bacterium colony.
(b) bacterium colony form early stage, i.e. the microcolony phase, just carry out the observation of microorganism to the test substance susceptibility.Microcolony is meant and only contains several cells and have only the bacterium colony that just can see by microscope, rather than the macroscopic macrocolony of indication usually.Theoretically, can need be contained up to a million cells by the macrocolony that naked eyes are seen.And unicellularly breed into up to a million cells and need carry out promptly lacking tens reproductive cycles and cultivate for a long time by one.In contrast to this, the formation of confirmation microcolony only needs the cultivation of one or two reproductive cycle just to come out.
Compare with other modification method with ordinary method, the present invention has the following advantages:
(a) greatly shorten the report microorganism needed time of susceptibility.
(b) can get rid of pre-incubated dependence.
(c) allow the sample that contains unknown mixing microorganisms is carried out the susceptibility test.
(d) allow to use different microbial inoculant concentration.
(e) reduce nutrition exhaustion and product inhibition influence to test result.
(f) allow the large quantities of samples of fast processing and numerous test substance.
(g) give extremely sensitive and special test result.
(h) provide the minimum inhibition concentration relevant with the original position microorganism concn.
(i) greatly reduce the Master Cost that the microorganism susceptibility is tested.
(j) save a large amount of cultivation and storage space.
(k) General Instrument that uses routine operation process and most laboratory all to have.
(l) can realize in various degree automatization.
(m) be convenient to combine with other experiment.
(n) be convenient to on-the-spot inoculation.
(o) be convenient to cultivate in the process of planting cultivation passing on.
(p) be convenient to sort out and the classical collection test material.
(q) reduce the contact that lab assistant is treated the micrometer biology.
(r) reducing test substance such as antimicrobial element/microbiotic is released among the environment.
Above advantage will further be set forth in conjunction with following implementation process.
Concrete enforcement of the present invention can be undertaken by following steps:
(a) the thick liquid substratum that adds the definite composition composition is in the cultivation vessel of sky,
(b) add certain density test substance such as antimicrobial element/microbiotic in the cultivation vessel that substratum is housed and mix,
(c) sample that contains microorganism is suspected in inoculation,
(d) cultivate the culture of inoculating the sixth of the twelve Earthly Branches under proper condition,
(e) check the cell density and the cell aggregation form of culture at suitable incubation time,
(f) situation about forming by cell count increase and decrease and microcolony is determined the susceptibility of microorganism to the survey material.
The preparation of thick liquid substratum can by add a kind of thick substances such as polyethylene than pyrrolidone, methyl fibres sugars, gelatin in substratum.The time that adds thick substances can add test substance before or after the substratum.
Using the thick liquid substratum to carry out the susceptibility test has overcome meat soup and cultivates peaceful plate and cultivate both weakness and kept strong point separately.Compare with solid medium, the thick liquid substratum makes fully mixing of nutrition and test substance.It makes also that inoculation sample microorganism to be measured is more easy and forms the initial distribution of uniform microbial population.This will greatly reduce because the deviation of the microorganism count aspect that the concentration difference causes to influence that microorganism growth caused with owing to sampling error.Therefore it will increase the accuracy and the tolerance range of susceptibility test.The result that microbial population is grown in the liquid nutrient medium of routine forms how single celled suspension.In contrast to this, the thick liquid substratum allows to form microcolony from ancester cell, and the detection microcolony is compared more simple with inspection broth culture turbidity, and it can carry out the original position inspection under the situation of not taking culture.Simultaneously, this inspection is also more responsive and accurate.Because microcolony can carry out once or can detect during twice reproductive cycle, and the formation of microcolony necessarily shows the growth of microbial population.
Use the thick liquid substratum to carry out the inoculation that susceptibility is tested convenient various types of samples.If the sample that inoculation is liquid, an available syringe extract sample and exactly a certain amount of sample are injected each and cultivate in the vessel.An aseptic inoculating needle or transfering loop are used with the culture mixing in inoculation back.As inoculating solid-state sample, sample should be smashed earlier to being cultivated the size that vessel hold, and fully mixing with the correct concentration distribution that shows microorganism at type specimen.
Using the thick liquid substratum to carry out the convenient and various subsequent experimental inspections of susceptibility test combines.This be because in the liquid nutrient medium substance in solid medium substance more easy and mixing is more even.The present invention can combine with multiple microorganism identification reacting phase and reach the microorganism susceptibility and the purpose of finishing in same experiment is identified in classification.
The thickness substratum of fluidization can be produced in enormous quantities easily.Test substance also can add needed the time in the cultivation.These superiority can make the personnel that carry out the test of microorganism susceptibility free from cumbersome manual preparation substratum, and shorten the total time of finishing test.Use the substratum of same quality and test substance also will reduce between the laboratory and the difference on experimental implementation between the laboratory technician, thereby make experimental result have more comparability.
Conventional microorganism susceptibility experiment needs pre-the cultivation to obtain pure growth as inoculum usually.The result that the susceptibility of contained microorganism in the sample is tested needs to compare with the typical curve of pure growth and judge.The present invention can directly check existing microorganism in the type specimen.No matter the kind of this microorganism how, its susceptibility to test substance is judged in the variation of the image by record cell and cell aggregation form.Therefore, the present invention has the efficient of height, can check all suspicious pathogenic bacteria combating microorganisms element/antibiotic susceptibility contained in the sample.In fact, perhaps the shape information of the microorganism of being write down can provide preliminary nation to help to the evaluation of microorganism.
Conventional microorganism susceptibility experiment needs the standardized inoculum of high density, to form lawn on the flat board or to form higher turbidity to obtain reliable and consistent inspection in meat soup.High sensitive that the present invention had and high resolution make and can carry out under the original concentration of microorganism in sample the inspection of susceptibility.
Owing to the early stage microbe colony of observing is grown and can be carried out in the microcosmic scope, the present invention is with the requirement of huge amount ground minimizing to microorganism sample, microbiological culture media and test substance.Because the present invention can use the vessel more much smaller than ordinary method to cultivate and observe, the present invention has also reduced the consumption of vessel material aspect and dramatically to the demand of cultivation and storage space and plant and instrument.
Microminiaturized microorganism susceptibility test is convenient to substratum and test substance are made the sealing fit that installs in advance.This fit can be carried into various scenes such as doctor's office, Surgical Operating Room or other outlying few doctor's place fully and carry out the scene inoculation.This fit can be done so for a short time, to such an extent as to just can cultivate the way of delivering to testing laboratory from the scene putting portable incubator after the inoculation into.These extra benefits will make the time of finishing the susceptibility experiment more shorten.
Microminiaturized microorganism susceptibility test reduces huge amount ground the discharging of microorganism, test substance and other refuse and nuisance.This helps the protection of environment on the one hand, also reduces the spending for the treatment of refuse refuse on the one hand.As test substance is antibiotic, reduces its discharging to environment and also helps alleviating serious day by day microorganism resistance diffusion.As microorganism to be measured is deleterious pathogenic micro-organism, and the microminiaturization of test and sealingization will reduce operator's infection chance effectively.
To the inspection of the form of the density of cell in the culture and cell aggregation, available various forms of optical microscopies.The culture apparatus of closing can be placed on the microscopical Stage microscope, light passes the cultivation well, obtains to cultivate the image of well content at different focussing planes.For the exactness that microscopic image is checked, should check a plurality of different areas of culture, the mean number of the number of cell density and microcolony can be more near its true number in culture like this.
On the TV screen or computer screen that microscopic image can be presented on microscopical eyepiece, link with microscope.The record of microscopic image is different information carrier such as light sensation film, tape, disk or CDs.To the identification and the judgement of microscopic image, can manually carry out also can carrying out automatically.This identification and judgement can be carried out in experimental observation, can carry out by the image by record of search after experimental observation.This identification and judgement can be carried out at the scene, can carry out according to the picture information that the internet obtains at the root away from the experimental observation scene.It is this that to give the supervisory personnel fully free with the schedule of rationally arranging work to the microscopical inspection of leaving of depositing image.Simultaneously, owing to check and can repeat, even can proofread mutually by different supervisory personnel, the objectivity of inspection and accuracy will increase.But use has the aggregated forms that the computer software pair cell number of artificial intelligence calculated and reported cell in the culture automatically.Use such image analysis software greatly to reduce manpower consumption of the present invention and make the more effective and easy implementation of the present invention.
Claims (8)
1. method of measuring the microorganism susceptibility may further comprise the steps:
(a) add thick substances in liquid nutrient medium, form the thick liquid substratum, described thick substances is selected from polyethylene and adjoins pyrrolidone, methyl fibres sugars or gelatin;
(b) in above-mentioned substratum, add test substance,
(c) inoculation contains the sample of microorganism,
(d) cultivate vaccinated microorganism,
(e) check microbial cell density and aggregated forms,
(f) judge the susceptibility of microorganism to test substance.
2. according to the described method of claim 1, wherein test substance is selected from antimicrobial element.
3. according to the described method of claim 1, wherein test substance is selected from microbiotic.
4. according to the described method of claim 1, the wherein effectively motion of restriction micro-organisms of thick liquid substratum.
5. according to the described method of claim 1, check that wherein the method for microbial cell density and aggregated forms is selected from the MIcrosope image inspection.
6. according to the described method of claim 5, wherein the MIcrosope image inspection is selected from and checks the image that is presented on microscope ocular, check to be presented on the TV that links to each other with microscope or the image on the computer monitor, or the image of record of search on information carrier.
7. according to the described method of claim 1, judge that wherein microorganism is selected from artificial congnition judgement or machine recognition judgement to the means of test substance susceptibility.
8. according to the described method of claim 7, wherein machine recognition is judged and is comprised and use robot calculator pattern recognition analysis software to judge.
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|---|---|---|---|
| CNB011032812A CN100430486C (en) | 2001-01-20 | 2001-01-20 | Quick determination for microbe munity |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB011032812A CN100430486C (en) | 2001-01-20 | 2001-01-20 | Quick determination for microbe munity |
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| CN1333374A CN1333374A (en) | 2002-01-30 |
| CN100430486C true CN100430486C (en) | 2008-11-05 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20160069786A1 (en) * | 2013-04-19 | 2016-03-10 | Koninklijke Philips N.V. | An optical system and a method for real-time analysis of a liquid sample |
| JP6619544B2 (en) | 2014-03-19 | 2019-12-11 | 株式会社日立ハイテクノロジーズ | Inspection device |
| CN105259146A (en) * | 2015-11-03 | 2016-01-20 | 南开大学 | Method and system for quantitatively detecting narcotics |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5789173A (en) * | 1996-07-29 | 1998-08-04 | Academia Sinica | Methods for rapid antimicrobial susceptibility testing |
| US5948633A (en) * | 1997-08-07 | 1999-09-07 | Disney; Loren | Method for determining the effect of a chemical compound on microorganisms growing in continous culture |
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2001
- 2001-01-20 CN CNB011032812A patent/CN100430486C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5789173A (en) * | 1996-07-29 | 1998-08-04 | Academia Sinica | Methods for rapid antimicrobial susceptibility testing |
| US5948633A (en) * | 1997-08-07 | 1999-09-07 | Disney; Loren | Method for determining the effect of a chemical compound on microorganisms growing in continous culture |
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| CN1333374A (en) | 2002-01-30 |
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