CN103045518A - Method for cultivating and detecting mycoplasma pneumoniae - Google Patents
Method for cultivating and detecting mycoplasma pneumoniae Download PDFInfo
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- CN103045518A CN103045518A CN2012105879552A CN201210587955A CN103045518A CN 103045518 A CN103045518 A CN 103045518A CN 2012105879552 A CN2012105879552 A CN 2012105879552A CN 201210587955 A CN201210587955 A CN 201210587955A CN 103045518 A CN103045518 A CN 103045518A
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Abstract
The invention relates to a method for cultivating and detecting the mycoplasma pneumonia and belongs to the technical field of microbial culture. Devices of the method include a culture bottle I (100), a bottle cap (200) provided with a through hole (201) in the center and a culture bottle II (300); the culture bottle I (100) and the culture bottle II (300) are connected at two ends of the bottle cap (200); and a gasket (500), a filter membrane (600) and a filter membrane fixing unit (700) are further arranged in the bottle cap (200). According to the method, sputum samples are digested and revived through a digestion culture solution in the culture bottle I (100), and achieve initial multiplication, perform multiplication and enrichment through a selection bacteria enrichment solution in the culture bottle II (300), and the preliminary identification of the mycoplasma pneumonia is achieved; and finally the mycoplasma pneumonia is vaccinated to a solid medium of the culture bottle I (100) for re-multiplication, therefore, culture and identification of the mycoplasma pneumonia are completed. The devices of the method are designed aimed at the mycoplasma pneumonia, so that the mycoplasma pneumonia is rapid and convenient to culture, the result accuracy is high, and needs of clinicalsputum sample culture and detection can be met.
Description
Technical field
The present invention relates to a kind of cultivation and detection method of mycoplasma pneumoniae, belong to the microorganism culturing technical field.
Background technology
Mycoplasma pneumoniae (M. pneumonia) is one of important pathogen microorganism of acute human respiratory tract infection, mainly by respiratory tract and droplet transmission, can cause school-ager and teenager's lower respiratory infection, such as severe acute respiratory syndrome, trachitis, bronchitis etc.; Also can cause the extrapulmonary complication of other system, such as meningitis, brain stem inflammation, myocarditis, pericarditis, ephritis etc.Mycoplasma pneumoniae is that a class lacks cell walls, is different from cell and viral prokaryotic micro-organisms.It is of a great variety, widely distributed, and its size generally between 0.2-0.3um, can be passed through general sterilization filter.
The clinical symptom of mycoplasma pneumoniae infection has atypism, and the difficult respiratory tract infection that causes with other reasons is distinguished; Mycoplasma pneumoniae usually is adsorbed, is wrapped in the protein-colloid of thickness in the clinical sputum sample basis, and growth is restricted, and often is mixed with a large amount of different types of miscellaneous bacterias in the sputum sample basis, can't be directly used in liquid culture clinically; And sputum sample is originally directly slower in the solid medium growth, and growing bacterium colony needs 3-4 days time, and positive rate is not high; In addition, use impaired mycoplasma in the antibiotic clinical sputum sample basis, needed the process of a recovery, could Fast Growth.These all become the obstacle of clinical pneumonia detection of mycoplasma, therefore detect fast, accurately and efficiently mycoplasma the prevention of clinical disease and treatment tool are of great significance.
The mycoplasma pneumoniae detection method of commonly using both at home and abroad at present has: detection of specific antibody technology, Southern Blot, PCR method, isolated culture, metabolic inhibition test etc.The detection of specific antibody technology is prone to cross reaction, and occurs in specific IgM 1 week after infection, and 3～4 weeks reached the peak, and specific IgG occurs more late, generally about 6 weeks.Southern Blot requires very high to experimental technique, be difficult for generally carrying out.False positive and false negative easily appear in the PCR method, are ordered to forbid for Clinical Laboratory by health ministry.Metabolic inhibition test need add the growth that specific antibody suppresses mycoplasma, and clinical application is few.Generally speaking, aforesaid method not only detection time long, complex operation, and need some accurate plant and instrument needs the professional and technical personnel to test, and uses inconvenient.
Summary of the invention
The object of the invention is to overcome above-mentioned the deficiencies in the prior art, provide a kind of for the mycoplasma pneumoniae characteristics design, cultivate mycoplasma pneumoniae efficient and convenient, identify that the mycoplasma pneumoniae accuracy rate is high, satisfy this cultivations of clinical sputum sample and detection needs, effectively cultivation and the detection method of recovery mycoplasma pneumoniae.
The object of the present invention is achieved like this:A kind of cultivation of mycoplasma pneumoniae and detection method, it comprises the steps:
Step 1: clinical sputum sample originally is inoculated in the culturing bottle I that has the digestion nutrient solution;
Step 2: assemble successively bottle cap and the culturing bottle II that is provided with sealing-ring I, sealing-ring II, pad, filter membrane, filter membrane stationary installation on the culturing bottle I, build the bottom II;
Step 3: said apparatus is placed on the shaking table at upper state in lower, culturing bottle II with the culturing bottle I, under 37 degree constant temperatures, cultivated 12～24 hours;
Step 4: said apparatus is inverted, and namely the culturing bottle I descending, is carried out negative pressure leaching by the suction filtration mouth II of culturing bottle II in upper, culturing bottle II, makes bacterium liquid flow to the culturing bottle II by the culturing bottle I;
Step 5: the bottom I by the culturing bottle I adds enrichment culture liquid in the culturing bottle II, cultivates 12～24 hours under 37 degree constant temperatures;
Step 6: by naked eyes and or observation of use instrument culturing bottle II in nutrient solution muddy degree, colour-change, have or not fluorescence phenomenon, or add reagent and further observe.
In step 6, the nutrient solution in the described culturing bottle II is accredited as mycoplasma pneumoniae and is positive, and proceeds following operation:
Step 7: the accessory in culturing bottle I, bottle cap and the bottle cap that more renews, the cultivation support that will have again solid medium is inserted in the culturing bottle I, outside the just bottle wall facing to the culturing bottle I of culture plate, builds the bottom I;
Step 8: the above-mentioned device that assembles is inverted, and namely the culturing bottle I upper, is carried out negative pressure leaching by suction filtration mouth I in lower, culturing bottle II, makes bacterium liquid flow into the culturing bottle I, the submerged culture support of bacterium liquid, and be inoculated on the solid medium in the culture plate;
Step 9: said apparatus is inverted again, and namely the culturing bottle I lower, is carried out negative pressure leaching by suction filtration mouth II in upper, culturing bottle II, makes bacterium liquid flow into the culturing bottle II;
Step 10: said apparatus is placed in the incubator, and under the temperature that is fit to mycoplasma pneumoniae, cultivated 24～48 hours;
Step 11: by naked eyes and or the observation of use instrument said apparatus in cultivate on the support colonial morphology in the culture plate.
In step 4 and step 9, when carrying out negative pressure leaching, described bottle cap I is unscrewed.
In step 8, when carrying out negative pressure leaching, described bottle cap II is unscrewed.
The invention has the beneficial effects as follows:
Apparatus of the present invention comprise culturing bottle I, lid central bottle cap, culturing bottle II with through hole, described bottle cap inside arranges internal thread I, internal thread II and internal thread III culturing bottle I, culturing bottle II is connected with bottle cap, inject the digestion nutrient solution in the culturing bottle I, inject enrichment culture liquid in the culturing bottle II, place in the culture plate in the culturing bottle I and cultivate and the solid medium that detected.The method that the mycoplasma pneumoniae of sputum sample in this cultivated by substep: the i.e. digestion of sputum sample herbal classic digestion nutrient solution, recovery, and finish preliminary propagation, preliminary evaluation, enrichment culture medium is bred, enrichment by selecting again, be inoculated at last in the solid medium of the difference in functionality that contains abundant nutrition and breed again, thereby finish cultivation and the discriminating of mycoplasma pneumoniae.
The cultivation of a kind of mycoplasma pneumoniae of the present invention and proofing unit, but the clinical sputum sample of effective digestion is originally, and the recovery mycoplasma pneumoniae suppresses, filters and remove miscellaneous bacteria; Adopt simultaneously the method for solid-liquid double-phase, bacterium, separation and Culture are selected, increased to mycoplasma pneumoniae; Easy and simple to handle, accuracy rate is high, can satisfy this cultivation of clinical sputum sample and the needs that detect.
Description of drawings
Fig. 1 is the cultivation of a kind of mycoplasma pneumoniae of the present invention and the schematic diagram of proofing unit.
Fig. 2 is the schematic diagram of the amplification of the profile of local I among Fig. 1.
Fig. 3 is the schematic diagram of the amplification of the profile of bottle cap among Fig. 2.
Fig. 4 is the schematic perspective view of bottle cap among Fig. 2.
Fig. 5 is the schematic perspective view of filter membrane stationary installation among Fig. 2.
Fig. 6 is for cultivating the schematic perspective view of support embodiment one.
Fig. 7 is for cultivating the schematic perspective view of support embodiment two.
Fig. 8 is the schematic diagram of the profile of Fig. 7.
Wherein:
Culturing bottle I 100
Bottom I 101
Suction filtration mouth I 102
Outside screw I 103
Bottle cap 200
Through hole 201
Internal thread I 211
Internal thread II 212
Internal thread III 213
Sealing groove I 221
Sealing groove II 222
Culturing bottle II 300
Bottom II 301
Suction filtration mouth II 302
Outside screw II 303
Sealing-ring I 401
Sealing-ring II 402
Filter membrane stationary installation 700
Cross-hole 701
Outside screw II 702
Cultivate support 800
Embodiment
Referring to Fig. 1 to Fig. 5, cultivation and the proofing unit of a kind of mycoplasma pneumoniae that the present invention uses comprise the culturing bottle II 300 that bottleneck is provided with outside screw II 303 with internal thread I 211, lid central authorities with bottle cap 200 and the bottleneck of through hole 201 with the culturing bottle I 100 of outside screw I 103 and sidewall.The inside top of described internal thread I 211 arranges sealing groove I 221, is provided with sealing-ring I 401 in the described sealing groove I 221, and the outside screw I 103 of described culturing bottle I 100 is connected with the internal thread I 211 of bottle cap 200.The bottleneck shoulder of described culturing bottle I 100 arranges suction filtration mouth I 102, and cultivates supports 800 interior settings of culturing bottle I 100, such as Fig. 6～shown in Figure 8.The bottom of described culturing bottle II 300 arranges bottom II 301, the bottleneck shoulder arranges suction filtration mouth II 303.
The sidewall reverse extending of described bottle cap 200 also sets gradually internal thread II 212 and internal thread III 213 from the bottom up in the inside sidewalls of bottle cap 200 reverse extendings, the interior diameter of described internal thread II 212 is less than the interior diameter of internal thread III 213, the interior diameter of through hole 201 is less than the interior diameter of internal thread II 212, the bottom inside of internal thread II 212 arranges spacer groove 223, the bottom inside of internal thread III 213 arranges sealing groove II 222, described sealing groove II 222 interior placement sealing-ring II 402.The internal thread III 213 of described bottle cap 200 is connected with the outside screw II 303 of culturing bottle II 300.
Set gradually pad 500, filter membrane 600, filter membrane stationary installation 700 in the described bottle cap 200, described filter membrane stationary installation 700 is offered the right cylinder that cross-hole 701, outer wall are provided with outside screw II 702 for central authorities, described pad 500 is arranged in the spacer groove 223, and the outside screw II 702 of filter membrane stationary installation 700 is connected with the internal thread II 212 of bottle cap 200.
The center of described culturing bottle I 100, bottle cap 200, culturing bottle II 300, sealing-ring I 401, sealing-ring II 402, pad 500, filter membrane 600, filter membrane stationary installation 700 and cultivation support 800 is all coaxial.
The cultivation of a kind of mycoplasma pneumoniae of the present invention and detection method mainly comprise three phases: one, digestion recovery.In the digestion nutrient solution with clinical this access of sputum sample culturing bottle I 100, cultivate 12～24 hours at 37 degree under the constant temperatures, until sputum sample in this sputum complete digestion of thickness be solution, mycoplasma pneumoniae obtains recovery, preliminary propagation.Two, enrichment propagation.With the bacterium liquid after the digestion recovery by the bottle cap 200 with millipore filtration 600, in sedimentation filtration or the negative pressure filtration selective enrichment nutrient solution in the culturing bottle II 300, under 37 degree constant temperatures, cultivated 12～24 hours, make mycoplasma pneumoniae obtain abundant enrichment, propagation.Three, separation and Culture.Bacterium liquid after enrichment, the propagation is inoculated on the solid medium in the new culturing bottle I 100, under 37 degree constant temperatures, cultivates 24～48 hours, until form clear, visible bacterium colony.
Cultivation and the detection method of a kind of mycoplasma pneumoniae of the present invention comprise the steps:
One, digestion recovery.
Step 1: clinical sputum sample originally is inoculated in the culturing bottle I 100 that has the digestion nutrient solution; Described digestion nutrient solution is selective enrichment, the digestion nutrient solution that contains abundant nutrition material, digestive ferment and antibacterial medicines, and this digestion nutrient solution has the effect of dissociating and disperseing the sputum sample basis, suppressing living contaminants, release and recovery mycoplasma pneumoniae.
Step 2: assemble successively bottle cap 200 and the culturing bottle II 300 that is provided with sealing-ring I 401, sealing-ring II 402, pad 500, filter membrane 600, filter membrane stationary installation 700 on the culturing bottle I 100, build bottom II 301.
Step 3: said apparatus is placed on the shaking table at upper state in lower, culturing bottle II 300 with culturing bottle I 100, under 37 degree constant temperatures, cultivated 12～24 hours.
Two, enrichment propagation.
Step 4: said apparatus is inverted, be that culturing bottle I 100 is being descended in upper, culturing bottle II 300, suction filtration mouth II 302 by culturing bottle II 300 is carried out negative pressure leaching, and filter membrane 600 intercepts miscellaneous bacteria, and makes the mycoplasma pneumoniae in the bacterium liquid flow to culturing bottle II 300 through filter membrane 600 by culturing bottle I 100.Unobstructed for bacterium liquid is flowed, can realize by unscrewing bottle cap I 101.
Step 5: the bottom I 101 by culturing bottle I 100 was cultivated 12～24 hours under 37 degree constant temperatures to the enrichment culture liquid of culturing bottle II 300 interior adding capacities; Described enrichment culture liquid is selective enrichment, the enrichment culture liquid that contains abundant nutrition material, antibacterial medicines and/or color indicator, and this enrichment culture liquid has the effect that suppresses living contaminants, enrichment mycoplasma pneumoniae, the growth of indication mycoplasma pneumoniae.
Step 6: by naked eyes and or observation of use instrument culturing bottle II 300 in nutrient solution muddy degree, colour-change, have or not fluorescence phenomenon, or add reagent and further observe.
Three, separation and Culture.
If in step 6, nutrient solution is accredited as mycoplasma pneumoniae and is positive in the described culturing bottle II 300, then proceeds following operation.
Step 7: the accessory in the culturing bottle I 100 that more renews, bottle cap 200 and the bottle cap 200, the cultivation support 800 that will have again solid medium is inserted in the culturing bottle I 100, outside the just bottle wall facing to culturing bottle I 100 of culture plate 801, builds bottom I 101.Described solid medium is one or more in isolation medium, selective medium, the biochemical differential medium; This class substratum includes but not limited to color developing culture medium, or a kind of substratum, and the specific compound that can and add subsequently after bacterium colony forms reacts, and presents the variation of some outward appearances, as: variable color, generation bubble etc.; Such substratum has separation, and the effect of mycoplasma pneumoniae is differentiated in screening.
Step 8: the above-mentioned device that assembles is inverted, be culturing bottle I 100 in lower, culturing bottle II 300 upper, carry out negative pressure leaching by suction filtration mouth I 102, make bacterium liquid flow into culturing bottle I 100, the submerged culture support 800 of bacterium liquid, and be inoculated on the solid medium in the culture plate 801.Filter membrane 600 works to intercept miscellaneous bacteria again.Unobstructed for bacterium liquid is flowed, can realize by unscrewing bottle cap bottom II 301.
Step 9: said apparatus is inverted again, and namely culturing bottle I 100 lower, is carried out negative pressure leaching by suction filtration mouth II 302 in upper, culturing bottle II 300, makes bacterium liquid flow into culturing bottle II 300.Unobstructed for bacterium liquid is flowed, can realize by unscrewing bottle cap I 101.
Step 10: said apparatus is placed in the incubator, and under the temperature that is fit to mycoplasma pneumoniae, cultivated 24～48 hours.
Step 11: by naked eyes and or the observation of use instrument said apparatus in cultivate on the support 800 colonial morphology in the culture plate 801.
Claims (4)
1. the cultivation of a mycoplasma pneumoniae and detection method, it comprises the steps:
Step 1: clinical sputum sample originally is inoculated in the culturing bottle I (100) that has the digestion nutrient solution;
Step 2: assemble successively bottle cap (200) and the culturing bottle II (300) that is provided with sealing-ring I (401), sealing-ring II (402), pad (500), filter membrane (600), filter membrane stationary installation (700) on the culturing bottle I (100), build bottom II (301);
Step 3: said apparatus is placed on the shaking table at upper state in lower, culturing bottle II (300) with culturing bottle I (100), under 37 degree constant temperatures, cultivated 12～24 hours;
Step 4: said apparatus is inverted, and namely culturing bottle I (100) descending, is carried out negative pressure leaching by the suction filtration mouth II (302) of culturing bottle II (300) in upper, culturing bottle II (300), makes bacterium liquid flow to culturing bottle II (300) by culturing bottle I (100);
Step 5: the bottom I (101) by culturing bottle I (100) adds enrichment culture liquid in culturing bottle II (300), cultivates 12～24 hours under 37 degree constant temperatures;
Step 6: by naked eyes and or observation of use instrument culturing bottle II (300) in nutrient solution muddy degree, colour-change, have or not fluorescence phenomenon, or add reagent and further observe.
2. the cultivation of a kind of mycoplasma pneumoniae according to claim 1 and detection method, it is characterized in that: in step 6, the nutrient solution in the described culturing bottle II (300) is accredited as mycoplasma pneumoniae and is positive, and proceeds following operation:
Step 7: the accessory in the culturing bottle I (100) that more renews, bottle cap (200) and the bottle cap (200), the cultivation support (800) that will have again solid medium is inserted in the culturing bottle I (100), outside the just bottle wall facing to culturing bottle I (100) of culture plate (801), build bottom I (101);
Step 8: the above-mentioned device that assembles is inverted, be culturing bottle I (100) in lower, culturing bottle II (300) upper, carry out negative pressure leaching by suction filtration mouth I (102), make bacterium liquid flow into culturing bottle I (100), the submerged culture support of bacterium liquid (800), and be inoculated on the interior solid medium of culture plate (801);
Step 9: said apparatus is inverted again, and namely culturing bottle I (100) lower, is carried out negative pressure leaching by suction filtration mouth II (302) in upper, culturing bottle II (300), makes bacterium liquid flow into culturing bottle II (300);
Step 10: said apparatus is placed in the incubator, and under the temperature that is fit to mycoplasma pneumoniae, cultivated 24～48 hours;
Step 11: by naked eyes and or the observation of use instrument said apparatus in cultivate colonial morphology in the upper culture plate (801) of support (800).
3. the cultivation of a kind of mycoplasma pneumoniae according to claim 2 and detection method is characterized in that: in step 4 and step 9, when carrying out negative pressure leaching, described bottle cap I (101) is unscrewed.
4. the cultivation of a kind of mycoplasma pneumoniae according to claim 2 and detection method is characterized in that: in step 8, when carrying out negative pressure leaching, described bottle cap II (301) is unscrewed.
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| CN2012105879552A CN103045518A (en) | 2012-12-31 | 2012-12-31 | Method for cultivating and detecting mycoplasma pneumoniae |
| CN201310741589.6A CN103937662B (en) | 2012-12-31 | 2013-12-30 | Microorganism seperation culture device and method thereof |
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| CN2012105879552A CN103045518A (en) | 2012-12-31 | 2012-12-31 | Method for cultivating and detecting mycoplasma pneumoniae |
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| CN201310741589.6A Active CN103937662B (en) | 2012-12-31 | 2013-12-30 | Microorganism seperation culture device and method thereof |
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| CN116075586A (en) * | 2020-07-08 | 2023-05-05 | 快速微型生物系统公司 | Filtration assemblies, cartridges, systems and methods for filtration and cell growth |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105349410A (en) * | 2015-11-30 | 2016-02-24 | 苏州钧隆塑胶有限公司 | Bidirectional bacterial culture bottle |
| CN106867897A (en) * | 2017-04-19 | 2017-06-20 | 北京倍肯创新诊断技术研究院有限责任公司 | A kind of Micro biological Tests pretreating device |
| CN107164222A (en) * | 2017-06-22 | 2017-09-15 | 成都远洋蓝猫科技有限公司 | Sample process box for Bacteria Detection |
| CN107287090A (en) * | 2017-06-22 | 2017-10-24 | 成都远洋蓝猫科技有限公司 | Sample preprocessing container |
| GB2580358B (en) * | 2019-01-04 | 2022-08-31 | Oribiotech Ltd | Cell processing device cell processing system and methods of use thereof |
| GB2580359B (en) * | 2019-01-04 | 2022-07-27 | Oribiotech Ltd | Cell processing platform cell processing system and methods of use thereof |
| CN110029057A (en) * | 2019-04-19 | 2019-07-19 | 烟台职业学院 | A kind of biotechnology cell culture apparatus |
| CN109913366B (en) * | 2019-05-08 | 2024-03-22 | 青海省畜牧兽医科学院 | Culture bottle and filamentous fungus's culture apparatus |
| CN110317715A (en) * | 2019-08-16 | 2019-10-11 | 贵州大学 | A kind of mixed fermentation device for laboratory liquid state fermentation |
| CN112717513B (en) * | 2020-12-23 | 2023-03-03 | 广东产品质量监督检验研究院（国家质量技术监督局广州电气安全检验所、广东省试验认证研究院、华安实验室） | Centrifuging tube suction filter device |
| CN112940914A (en) * | 2021-01-29 | 2021-06-11 | 赵彬 | Experimental device for promote differentiation of epidermal stem cells and growth |
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| US7291267B2 (en) * | 2004-01-30 | 2007-11-06 | Ljc Technologies, L.L.C. | Molecular separator |
| WO2010010355A2 (en) * | 2008-07-25 | 2010-01-28 | Smith & Nephew Plc | Controller for separation apparatus |
| CN201942543U (en) * | 2011-01-27 | 2011-08-24 | 天年生物（中国）有限公司 | Internal pressure-type ultrafiltration membrane water filter |
| CN102816688B (en) * | 2012-09-14 | 2013-11-20 | 江苏嘉语生物医药技术有限公司 | Microorganism culture detection device in oxygen-controllable environment and detection method using same |
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| CN116075586A (en) * | 2020-07-08 | 2023-05-05 | 快速微型生物系统公司 | Filtration assemblies, cartridges, systems and methods for filtration and cell growth |
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| CN103937662A (en) | 2014-07-23 |
| CN103937662B (en) | 2016-08-24 |
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Application publication date: 20130417 |