CN105255760A - In-situ culture method of atmospheric fine particle microorganisms - Google Patents
In-situ culture method of atmospheric fine particle microorganisms Download PDFInfo
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- CN105255760A CN105255760A CN201510689593.1A CN201510689593A CN105255760A CN 105255760 A CN105255760 A CN 105255760A CN 201510689593 A CN201510689593 A CN 201510689593A CN 105255760 A CN105255760 A CN 105255760A
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Abstract
The invention discloses an in-situ culture method of atmospheric fine particle microorganisms. The in-situ culture method of the atmospheric fine particle microorganisms comprises steps as follows: 1), atmospheric fine particles are collected by a filter membrane, and the filter membrane containing the atmospheric fine particles is obtained; 2), the filter membrane containing the atmospheric fine particles is flatly spread on a sterile LB solid medium and uniformly pressed by a coating rod, and the medium inoculated with the atmospheric fine particle microorganisms is obtained; 3), the medium inoculated with the atmospheric fine particle microorganisms is cultured under the sterile condition, and the in-situ cultured atmospheric fine particle microorganisms are obtained. The experiment proves that the in-situ culture method of the atmospheric fine particle microorganisms can be used for in-situ culture of the atmospheric fine particle microorganisms.
Description
Technical field
The present invention relates to the Culture in situ method of Fine Particles microorganism in biological technical field.
Background technology
Current China environmental problem is comparatively outstanding, and environmental pollution threatens human health, and particularly atmosphere polluting problem is on the rise, and haze is locked Liancheng event and taken place frequently.Fine Particles (PM) particle diameter in haze less (< 10 μm), can be sucked by human body, is deposited on the position such as respiratory tract, alveolar thus diseases induced.The diameter of particulate matter is less, and the position entering respiratory tract is darker.The particulate matter of 10 μm of diameters is deposited on the upper respiratory tract usually, the deep of the entered respiratory tract of 5 μm of diameters, less than 2 μm 100% can be deep into bronchiole and alveolar.Fine Particles can be divided into 3 classes according to its aerodynamic diameter (d): coarseparticulate (courseparticle, d<10um, PM
10), fine particle (fineparticle, d<2.5 μm, PM
2.5), superfine particulate matter (ultrafineparticle, UFPs, d<0.1 μm, PM
0.1).Along with Fine Particles physics and chemistry characteristic research be tending towards ripe, the microbe composition in Fine Particles receives more concern.
Filter film type Fine Particles sampling thief gathers property and the more advantage such as small particle size collection property by its high sampling efficiency, high sample size, many particle diameters, is widely used.The Fine Particles of different-grain diameter can be collected, as PM by changing different sampling cutting heads
10and PM
2.5.But the Fine Particles obtained by the above-mentioned method of sampling is attached on atmospheric sampling filter membrane, classical culture protocols (as coating method) is utilized to carry out microorganism culturing experimental procedure to it loaded down with trivial details, complicated operation.At present still not cultural method effectively easily, for microbial drug tolerance research in the Fine Particles that carries out using Filter film type Fine Particles samplers sample to obtain.
In Fine Particles the research of microbial drug tolerance adopt more directly by Fine Particles the method collected on substratum or in solution cultivate, microorganism in the Fine Particles of different-grain diameter cannot be cultivated by this method respectively, or same sample can only be used for single culture experiment.
Summary of the invention
Technical problem to be solved by this invention is how Culture in situ Fine Particles microorganism.
For solving the problems of the technologies described above, the present invention provide firstly the Culture in situ method of Fine Particles microorganism.
The Culture in situ method of Fine Particles microorganism provided by the present invention, comprises following 1)-3):
1) gather Fine Particles with filter membrane, obtain the filter membrane containing Fine Particles;
2) the described filter membrane containing Fine Particles is laid on aseptic LB solid medium, obtains the substratum inoculating Fine Particles microorganism;
3) there is the substratum of Fine Particles microorganism aseptically to cultivate described inoculation, obtain the Fine Particles microorganism of Culture in situ.
In aforesaid method, described sampling membrane is quartz filter.Described quartz filter specifically can be Munktell Products, and catalog number is 420065.Described quartz filter is aseptic quartz filter.Described aseptic quartz filter can be calcined and obtain for 5-6 hour at 500-600 DEG C.
In aforesaid method, described Fine Particles can be the Atmospheric particulates of air equivalent diameter≤10 μm.
In aforesaid method, described the described filter membrane containing Fine Particles is laid in LB solid medium can be the sampling face of the described filter membrane containing Fine Particles is laid on LB solid medium.
In aforesaid method, described cultivation can be carried out in constant incubator.
In aforesaid method, the temperature of described cultivation can be 37 DEG C.
In aforesaid method, the time of described cultivation can be 48 hours.
Experiment proves, utilize the Culture in situ method of Fine Particles microorganism of the present invention, can turn out different single bacterium colonies, bacterium colony is more, and the method is simple to operate, quick, PM
2.5colony number can reach 44 bacterium colonies, PM
10colony number can reach 98 bacterium colonies; Sampling membrane in experiment after sampling repeatedly can utilize and then repeatedly can cultivate with a collection of Fine Particles microorganism.And utilize the coating cultural method of Fine Particles microorganism, by with Culture in situ method the same terms of Fine Particles microorganism under obtain containing PM
2.5and PM
10the colony number of atmospheric sampling filter membrane be respectively 10 and 14, the method complicated operation, the colony number obtained is few, is respectively 22.7% and 14.3% of the Culture in situ method of Fine Particles microorganism.Experiment proves, the Culture in situ method of Fine Particles microorganism of the present invention can be used for Culture in situ Fine Particles microorganism, and for the research of Fine Particles Bacterial diversity.
Accompanying drawing explanation
Fig. 1 is the quartz filter gathering Fine Particles.Left figure (figure C) is for gathering Fine Particles PM
2.5quartz filter, middle figure (figure A) is for gathering Fine Particles PM
10quartz filter, right figure (Blank) is blank.
Fig. 2 is the picture that the filter membrane containing Fine Particles is laid on aseptic LB solid medium.
Fig. 3 is postvaccinal LB substratum.Left figure (figure A) has PM for inoculation
2.5lB substratum, right figure (figure B) is blank.
Fig. 4 is the substratum that the growth utilizing the Culture in situ method of Fine Particles microorganism to obtain has bacterium colony.C and A is the substratum that the growth utilizing the Culture in situ method of Fine Particles microorganism to obtain has bacterium colony, and C is PM
10, A figure is PM
2.5, B is blank.
Fig. 5 is the substratum that growth that the coating cultural method of Fine Particles microorganism obtains has bacterium colony.(figure is c) PM to left figure
10the growth that obtains of coating cultural method have the substratum of bacterium colony, (figure is a) PM to right figure
2.5the growth that obtains of coating cultural method have the substratum of bacterium colony.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The Fine Particles gathered in following embodiment is PM
2.5(equivalent aerodynamic diameter is less than or equal to 2.5 μm).
Quartz filter in following embodiment is Mulktell Products, and catalog number is 420065.Quartz filter before collection Fine Particles, need be calcined, obtains aseptic quartz filter in retort furnace; Calcination condition is 500-600 DEG C; Calcination time is 5-6 hour.
The Culture in situ of embodiment 1, Fine Particles microorganism
The Culture in situ method of Fine Particles microorganism, comprises following 1)-3):
1) gather Fine Particles with atmospheric sampling quartz filter, obtain the sampling membrane containing Fine Particles;
2) sampling membrane containing Fine Particles is laid on aseptic LB solid medium, obtains the substratum inoculating Fine Particles microorganism;
3) there is the substratum of Fine Particles microorganism aseptically to cultivate inoculation, obtain the Fine Particles microorganism of Culture in situ.
In triplicate, that repeats experiment arranges three parallel laboratory tests at every turn, and concrete operation step is as follows in experiment:
1, answer 2034 type air heavy metal sampling instruments by PM with the Lao with aseptic atmo sampling membrane (quartz filter)
2.5particulate matter is collected on atmospheric sampling filter membrane, and the sampling time is 23h, obtains containing PM
2.5atmospheric sampling filter membrane (Fig. 1);
2, aseptic LB solid medium is prepared;
3, by step 1 containing PM
2.5the sampling face of atmospheric sampling filter membrane be laid on the aseptic LB solid medium of step 2, with spreading rod evenly and lightly pressing containing PM
2.5the sampling membrane of particulate matter, contact completely with media surface (Fig. 2) to sampling membrane, sampling membrane (the sampling membrane that preservation aseptic nipper takes out is taken out again with aseptic nipper, sampling membrane by after the inoculation once of its called after), obtain postvaccinal LB substratum (Fig. 3);
4, the postvaccinal LB substratum of step 3 is aseptically cultivated 48h in 37 DEG C of constant incubators, obtain Culture in situ PM
2.5the bacterial colony (in Fig. 4 A) of Atmospheric particulates.
Will containing PM according to the method for step 1-4
2.5atmospheric sampling filter membrane replace with the sampling membrane after inoculation once, other steps are all constant, obtain secondary Culture in situ PM
2.5the bacterial colony (in Fig. 4 B) of Atmospheric particulates.
2034 type air heavy metal sampling instruments are answered by Lao to be adjusted to PM
10sampling pattern, answers 2034 type air heavy metal sampling instruments by PM with the Lao with aseptic atmo sampling membrane (quartz filter)
2.5particulate matter is collected on atmospheric sampling filter membrane, and the method according to step 1-4 will containing PM
2.5atmospheric sampling filter membrane replace with and do not gather PM
2.5the atmospheric sampling filter membrane of particulate matter, other steps are all constant, obtain Culture in situ PM
10the bacterial colony (in Fig. 4 C) of Atmospheric particulates.
Quartz filter that aseptic atmo is sampled be placed in Lao answer obtain in 2034 type air heavy metal sampling instruments do not gather PM
2.5the atmospheric sampling filter membrane of particulate matter, will containing PM according to the method for step 1-4 as blank (Blank)
2.5atmospheric sampling filter membrane replace with and do not gather PM
2.5the atmospheric sampling filter membrane of particulate matter, other steps are all constant, obtain blank cultures.
Result shows, and by the Culture in situ method of above-mentioned Fine Particles microorganism, can turn out different single bacterium colonies (part bacterium colony as shown in Figure 5), bacterium colony is more, and the method is simple to operate, quick; Sampling time be 23h containing PM
2.5atmospheric sampling filter membrane on average can obtain 44 bacterium colonies, secondary Culture in situ PM
2.5the bacterial colony of Atmospheric particulates also can obtain 28 bacterium colonies; Sampling time be 23h containing PM
10atmospheric sampling filter membrane on average can obtain 98 bacterium colonies, secondary Culture in situ PM
10the bacterial colony of Atmospheric particulates also can obtain 71 bacterium colonies.Sampling membrane in experiment after sampling can repeatedly utilize can repeatedly be cultivated with a collection of Fine Particles microorganism.
The coating cultural method of comparative example 1, Fine Particles microorganism
The coating cultural method of Fine Particles microorganism is conventional Fine Particles method for culturing microbes, and concrete operation step is as follows:
1, (PBS damping fluid is sterile phosphate buffer in 50mL centrifuge tube, to add 40mLPBS damping fluid, the pH of PBS damping fluid is 7.2-7.4, PBS damping fluid dilutes ten times by Solarbio Products and obtains, and products catalogue is P1022-500), embodiment 1 step 1 adopted PM
2.5sampling membrane (sampling membrane used is less than or equal to 9 × 10cm
2fold, containing PM
2.5one side inside, not containing PM
2.5one side outside, then put it in 50mL centrifuge tube, the tube wall of this sampling membrane and centrifuge tube is in a certain angle, filter membrane can not be attached on the tube wall of centrifuge tube, PBS damping fluid (filter membrane is immersed in PBS damping fluid completely) is added at 4 DEG C, under 200 × g centrifugal 1 hour in this centrifuge tube, abandon liquid, obtain the centrifuge tube of the sampling membrane after PBS buffer solution for cleaning is housed;
2, to step 1 PBS buffer solution for cleaning is housed after sampling membrane centrifuge tube in add PBS damping fluid (filter membrane is immersed in PBS damping fluid completely), then in supersonic cleaning machine (the KQ-300D type numerical control ultrasonic cleaner of Kunshan Ultrasonic Instruments Co., Ltd.), ultrasonic cleaning is carried out, obtain the ultrasonic mixture of filter membrane-PBS, ultrasound condition is 10 DEG C, power 40%, and ultrasonic time is 15 minutes; By ultrasonic for filter membrane-PBS mixture 4 DEG C, continue centrifugal 1 hour under 200 × g, abandon liquid, obtain the centrifuge tube of the sampling membrane after ultrasonic cleaning is housed;
3, PBS damping fluid (filter membrane is immersed in PBS damping fluid completely) is added at 4 DEG C, under 200 × g centrifugal 1 hour to the centrifuge tube that the sampling membrane after ultrasonic cleaning is housed of step 2, abandon liquid, obtain the centrifuge tube of the sampling membrane after PBS damping fluid secondary cleaning is housed;
4, to step 3 PBS damping fluid secondary cleaning is housed after sampling membrane centrifuge tube in add above-mentioned PBS damping fluid (filter membrane is immersed in PBS damping fluid completely), manually this centrifuge tube of concussion, makes particulate matter suspend, discards filter membrane, obtain PM
2.5particle suspension liquid body;
5, aseptic LB solid medium is prepared;
6, the PM of 100 μ l steps 5 is got
25particle suspension liquid body, on sterile LB medium, uses spreading rod even spread, obtains postvaccinal LB substratum;
7, the postvaccinal LB substratum of step 6 is aseptically cultivated 48h in 37 DEG C of constant incubators, obtain coating and cultivate PM
2.5the bacterial colony (in Fig. 5 right figure) of Atmospheric particulates.
According to step 1-7, PM will be adopted
2.5sampling membrane replace to and adopted PM
10sampling membrane, other steps are constant, obtain coating cultivate PM
10the bacterial colony (in Fig. 5 left figure) of Atmospheric particulates.
Result shows, by with embodiment 1 the same terms under obtain containing PM
2.5and PM
10atmospheric sampling filter membrane on average can obtain 10 and 14 bacterium colonies by the coating cultural method of above-mentioned Fine Particles microorganism, be respectively 22.7% and 14.3% of the Culture in situ method of Fine Particles microorganism, and the method complicated operation, the colony number obtained is few.
Claims (6)
1. the Culture in situ method of Fine Particles microorganism, comprises following 1)-3):
1) gather Fine Particles with filter membrane, obtain the filter membrane containing Fine Particles;
2) the described filter membrane containing Fine Particles is laid on aseptic LB solid medium, obtains the substratum inoculating Fine Particles microorganism;
3) there is the substratum of Fine Particles microorganism aseptically to cultivate described inoculation, obtain the Fine Particles microorganism of Culture in situ.
2. method according to claim 1, is characterized in that: described filter membrane is quartz filter.
3. method according to claim 1 and 2, is characterized in that: described being laid on LB solid medium by the described filter membrane containing Fine Particles is be laid on LB solid medium in the described sampling face containing the filter membrane of Fine Particles.
4., according to described method arbitrary in claim 1-3, it is characterized in that: described cultivation is carried out in constant incubator.
5., according to described method arbitrary in claim 1-4, it is characterized in that: the temperature of described cultivation is 37 DEG C.
6., according to described method arbitrary in claim 1-5, it is characterized in that: the time of described cultivation is 48 hours.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109827814A (en) * | 2019-03-11 | 2019-05-31 | 南京大学 | A kind of sampling film preparation of novel air particulate matter agar and exempt from solvent extraction cell process for exposing |
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2015
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109827814A (en) * | 2019-03-11 | 2019-05-31 | 南京大学 | A kind of sampling film preparation of novel air particulate matter agar and exempt from solvent extraction cell process for exposing |
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Application publication date: 20160120 |