CN102242074B - Screen method for facultative anaerobe - Google Patents
Screen method for facultative anaerobe Download PDFInfo
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- CN102242074B CN102242074B CN201110090585.7A CN201110090585A CN102242074B CN 102242074 B CN102242074 B CN 102242074B CN 201110090585 A CN201110090585 A CN 201110090585A CN 102242074 B CN102242074 B CN 102242074B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
- C12M21/04—Bioreactors or fermenters specially adapted for specific uses for producing gas, e.g. biogas
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
Abstract
The invention relates to a method for screening facultative anaerobe in an environmental sample, and an apparatus for implementing the method. With the method and the apparatus, isolation of the facultative anaerobe can be realized effectively, and shortages of the facultative anaerobe isolation method and the facultative anaerobe isolation apparatus in the prior art are overcome.
Description
Technical field
The present invention relates to bacteria screening field.More specifically, the present invention relates to screening method and the device of facultative anaerobe.
Background technology
Bacterium is divided into aerobic bacteria, anerobe, facultative anaerobe etc.Facultative anaerobe (facultative anaerobe) claims again the anaerobe of holding concurrently, and holds concurrently and dislikes gas bacterium, facultative aerobe.The microorganism of equal energy growth and breeding in aerobic or oxygen-free environment.At aerobic (O
2) or anoxia condition under, can obtain energy by different mode of oxidizing.With respect to the triage techniques of aerobic bacteria or anerobe, the triage techniques of facultative anaerobe is perfect not enough, effective, ripe.Traditional amphimicrobian bacterial screening method exists more drawback, as screen strictly anaerobic bacterium and facultative anaerobe in the flora obtaining and mix (or strict aerobic bacteria and facultative anaerobe mix), the defect such as the bacterial classification class that filters out is less.
Screening to facultative anaerobe, mainly contains following methods traditionally:
(1) respectively aerobic bacteria and anerobe are identified, filtered out facultative anaerobe;
(2) enrichment culture under aerobic condition, isolated bacterium is cultivated by anaerobic bacteria culture technology again, and that can grow is facultative anaerobe;
(3) enrichment culture under anaerobic, isolated bacterium is cultivated by aerobic culture technique again, and that can grow is facultative anaerobe.
Because facultative anaerobe can survive in oxygen free condition, can survive in aerobic conditions again, so their wide accommodations to redox potential in environment (Eh).Just because of this specific character of facultative anaerobe, for the variation range great Qie Yi anaerobic district of dissolved oxygen, be main environment, the effect of facultative anaerobe is also just even more important than the effect of strict aerobic bacteria or strictly anaerobic bacterium.And filter out comprehensively and effectively facultative anaerobe (group) from environmental sample, be to understand basis and the prerequisite that facultative anaerobe act on and applies in environment, so filter out comprehensively and effectively facultative anaerobe (group), for understanding facultative anaerobe, in environment, act on and apply and have great significance.
Summary of the invention
The present invention utilizes intermittently the gradient oxygen technology of exposing to the sun, a kind of screening rate and accuracy rate all novel method and the device of very high facultative anaerobe screening are provided, the method can realize the separated of effective facultative anaerobe with device, has also overcome the deficiency existing in existing facultative anaerobe separation method and device simultaneously.
In first aspect, the invention provides a kind of method that facultative anaerobe in sample is screened, said method comprising the steps of:
(1) under oxygen free condition, described sample anaerobic is cultivated to the first anaerobic time;
(2) then under aerobic conditions, described sample aerobic is cultivated to the first aerobic time;
(3) above step (1) and (2) are repeated at least 1 cycle again, wherein to repeat the time that middle anaerobic incubation time and anaerobic last time cultivate identical or different at every turn, and aerobic incubation time is longer than last time aerobic incubation time.
In one embodiment, the anaerobic incubation time in the method for first aspect present invention can be about 8-72 hour, and preferred about 12-14 hour is for example approximately 24 hours.
In another embodiment, the first aerobic time of cultivating of the aerobic in the method for first aspect present invention is about 0.5-24 hour, is approximately preferably 0.5-12 hour, for example, be approximately 0.5 hour.Follow-up aerobic incubation time is longer than last time aerobic incubation time.For example, in one embodiment, the follow-up aerobic aerobic incubation time in cultivating is all approximately 2 times of aerobic incubation times last time.
In yet another embodiment, the follow-up aerobic aerobic incubation time in cultivating is all approximately 2 times of aerobic incubation times last time.
In another embodiment, in the method for first aspect present invention step (3) to repeat be at least for example 2,3,4,5,6,7,8,9,10,11,12,13,14,15,20,30 or the more cycle.
In a specific embodiment, repeating in the step in the method for first aspect present invention (3) is 8 cycles, anaerobic incubation time is all approximately 24 hours, and aerobic cultivated for the first aerobic time and follow-up aerobic incubation time is respectively 0.5,1,2,4,8,12,24,48 and 72 hour.
In another specific embodiment, repeating in the step in the method for first aspect present invention (3) is 5 cycles, anaerobic incubation time is all approximately 24 hours, and aerobic cultivated for the first aerobic time and follow-up aerobic incubation time is respectively 4,8,12,24,48 and 72 hours.
In another specific embodiment, repeating in the step in the method for first aspect present invention (3) is 4 cycles, anaerobic incubation time is all approximately 24 hours, and aerobic cultivated for the first aerobic time and follow-up aerobic incubation time is respectively 8,12,24,48 and 72 hours.
In second aspect, the invention provides a kind of method that facultative anaerobe in sample is screened, described method is in comprising first aspect present invention step, further comprising the steps of:
(4) step (1)-(3) are obtained to environmental sample culture and carry out separation, obtain the single strain of amphimicrobian flora.
In one embodiment, the separation in second aspect present invention step (4) can be undertaken by methods such as coating, line separation.
The expose to the sun facultative anaerobe screening novel method of oxygen technique improvement of gradient intermittently of utilizing of the present invention has overcome in the amphimicrobian flora that traditional method filters out and has mixed the less deficiency that waits of strict aerobic bacteria and strictly anaerobic bacterium and facultative anaerobe kind, compare with existing amphimicrobian bacterial screening method, have the following advantages:
(1) the instrument consumptive material that present method is used is relatively simple, simple to operation;
(2) present method is by gradient, intermittently to expose to the sun that anaerobism in same device that oxygen technology increases aerobic incubation time is gradually cultivated and the enrichment culture at aerobic cultivation interval obtains amphimicrobian flora, resulting flora is facultative anaerobe completely, does not have strictly anaerobic bacterium or strict aerobic bacteria to mix;
(3) present method is to increase gradually aerobic incubation time by the gradient oxygen technology of intermittently exposing to the sun, facultative anaerobe has an adaptive process to the time of the oxygen that exposes to the sun, can be because of the oxygen that exposes to the sun for a long time suddenly, and cause some facultative anaerobe not grow, also just can be more comprehensively and effectively filter out the facultative anaerobe in sample, the kind of the facultative anaerobe filtering out is more.
In the third aspect, the invention provides the device of implementing amphimicrobian bacterial screening method of the present invention, described device comprises:
(1) for packing substratum into and cultivating the container of testing sample;
(2) for sealing the gas tight seal thing of described container;
(3) vacuum extractor being connected by sealing pipeline with described container;
(4) rare gas element is filled with to the inflation mechanism in described device; And
(5) for sealing the breathable sealing film of described container.
In a specific embodiment, facultative anaerobe screening plant of the present invention comprises:
(1) for packing substratum into and cultivating the triangular flask of testing sample;
(2) for sealing the airtight rubber plug of described triangular flask;
(3) vacuum pump that described container connects by sealing pipeline;
(4) rare gas element is filled with to the syringe in described device; And
(5), for sealing the breathable sealing film of described container, for example its ventilation aperture is 0.22 μ m.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, specific embodiment of the invention scheme result is described in detail:
The flora number of strict aerobic bacteria, strictly anaerobic bacterium and facultative anaerobe in the Wetland Sediments of lakeside, Taihu Lake when Fig. 1 screens initial and end.The result that Fig. 1 shows is to adopt to utilize the expose to the sun amphimicrobian bacterial screening method of oxygen new technology improvement of gradient intermittently, screens flora number initial in facultative anaerobe process and strict aerobic bacteria, strictly anaerobic bacterium and the facultative anaerobe in settling while finishing from the Wetland Sediments of lakeside, Taihu Lake.As can be seen from Figure through intermittence gradient expose to the sun after oxygen method screening, strict aerobic bacteria in settling and the flora number of strictly anaerobic bacterium are almost nil, and the flora number of facultative anaerobe is with respect to the not too large variation of flora number of the facultative anaerobe in embryo deposit thing, illustrate and utilize the oxygen method of exposing to the sun of gradient intermittently not have strict aerobic bacteria or strictly anaerobic bacterium to mix from take in the amphimicrobian flora filtering out the environmental sample that facultative anaerobe is dominant microflora, and in screening process, amphimicrobian flora can well adapt to the oxygen time of exposing to the sun, the situation that some facultative anaerobe that generation causes because of the oxygen that exposes to the sun for a long time does not grow, be that the gradient oxygen method of intermittently exposing to the sun can filter out the amphimicrobian flora in the Wetland Sediments of lakeside comprehensively and effectively.
Fig. 2 screens the initial and flora number of strict aerobic bacteria, strictly anaerobic bacterium and facultative anaerobe in fluvial deposit while finishing. and the result that Fig. 2 shows is to adopt to utilize the expose to the sun amphimicrobian bacterial screening method of oxygen new technology improvement of gradient intermittently, screens flora number initial in facultative anaerobe process and strict aerobic bacteria, strictly anaerobic bacterium and the facultative anaerobe in settling while finishing from fluvial deposit.As seen from the figure through intermittence the gradient flora number of the strict aerobic bacteria in settling and strictly anaerobic bacterium that exposes to the sun after oxygen method screening almost nil, and the flora number of facultative anaerobe slightly rises with respect to the flora number of the facultative anaerobe in embryo deposit thing, illustrate and utilize intermittently gradient to expose to the sun oxygen method from strict aerobic bacteria, in the amphimicrobian flora filtering out in the essentially identical environmental sample of flora number of strictly anaerobic bacterium and facultative anaerobe, do not have strict aerobic bacteria or strictly anaerobic bacterium to mix, and in screening process, amphimicrobian flora can well adapt to the oxygen time of exposing to the sun, the situation that some facultative anaerobe that generation causes because of the oxygen that exposes to the sun for a long time does not grow, intermittently the gradient oxygen method of exposing to the sun can filter out the amphimicrobian flora in fluvial deposit comprehensively and effectively.
Fig. 3 screens the initial and flora number of strict aerobic bacteria, strictly anaerobic bacterium and facultative anaerobe in the Wetland Sediments of lakeside, Taihu Lake while finishing. and the result that Fig. 3 shows is that to adopt 4 hours be utilization the expose to the sun amphimicrobian bacterial screening method of oxygen new technology improvement of gradient intermittently of the first aerobic incubation time, screens flora number initial in facultative anaerobe process and strict aerobic bacteria, strictly anaerobic bacterium and the facultative anaerobe in settling while finishing from settling.As seen from the figure through intermittence the gradient flora number of the strict aerobic bacteria in settling and strictly anaerobic bacterium that exposes to the sun after oxygen method screening almost nil, and the flora number of facultative anaerobe has increased with respect to the flora number of the facultative anaerobe in embryo deposit thing, illustrate and utilize intermittently gradient to expose to the sun oxygen method from strict aerobic bacteria, in the amphimicrobian flora filtering out in the essentially identical environmental sample of flora number of strictly anaerobic bacterium and facultative anaerobe, do not have strict aerobic bacteria or strictly anaerobic bacterium to mix, and in screening process, amphimicrobian flora can well adapt to the oxygen time of exposing to the sun, the situation that some facultative anaerobe that generation causes because of the oxygen that exposes to the sun for a long time does not grow, intermittently the gradient oxygen method of exposing to the sun can filter out the amphimicrobian flora in fluvial deposit comprehensively and effectively.
Fig. 4 screens the initial and flora number of strict aerobic bacteria, strictly anaerobic bacterium and facultative anaerobe in the Wetland Sediments of lakeside, Taihu Lake while finishing. and the result that Fig. 4 shows is that to adopt 8 hours be utilization the expose to the sun amphimicrobian bacterial screening method of oxygen new technology improvement of gradient intermittently of the first aerobic incubation time, screens flora number initial in facultative anaerobe process and strict aerobic bacteria, strictly anaerobic bacterium and the facultative anaerobe in settling while finishing from settling.As seen from the figure through intermittence the gradient flora number of the strict aerobic bacteria in settling and strictly anaerobic bacterium that exposes to the sun after oxygen method screening almost nil, and the flora number of facultative anaerobe has increased with respect to the flora number of the facultative anaerobe in embryo deposit thing, illustrate and utilize intermittently gradient to expose to the sun oxygen method from strict aerobic bacteria, in the amphimicrobian flora filtering out in the essentially identical environmental sample of flora number of strictly anaerobic bacterium and facultative anaerobe, do not have strict aerobic bacteria or strictly anaerobic bacterium to mix, and in screening process, amphimicrobian flora can well adapt to the oxygen time of exposing to the sun, the situation that some facultative anaerobe that generation causes because of the oxygen that exposes to the sun for a long time does not grow, intermittently the gradient oxygen method of exposing to the sun can filter out the amphimicrobian flora in fluvial deposit comprehensively and effectively.
Embodiment
Propose following embodiment, to make those of ordinary skills understand better the present invention, described embodiment, only for exemplary purpose, is not intended to limit the scope of the present disclosure.
In first aspect, the invention provides a kind of method that facultative anaerobe in sample is screened, said method comprising the steps of:
(1) under oxygen free condition, described sample anaerobic is cultivated to the first anaerobic time;
(2) then under aerobic conditions, described sample aerobic is cultivated to the first aerobic time;
(3) above step (1) and (2) are repeated at least 1 cycle again, wherein to repeat the time that middle anaerobic incubation time and anaerobic last time cultivate identical or different at every turn, and aerobic incubation time is longer than last time aerobic incubation time.
In second aspect, the invention provides a kind of method that facultative anaerobe in sample is screened, described method is in comprising first aspect present invention step, further comprising the steps of:
(4) step (1)-(3) are obtained to environmental sample culture and carry out separation, obtain the single strain of amphimicrobian flora.
In the method for the invention, anaerobic incubation time can be about 8-72 hour, time is longer, the strict aerobic bacteria mixing in the flora screening is fewer, but overlong time can make complicated operation, increase unnecessary incubation time, also likely in long-term cultivation, the impact because of conditions such as nutrition makes some bacterium death etc.Anaerobic incubation time can be preferably about 12-48 hour, for example, be approximately 24 hours.
In the method for the invention, it can be about 0.5-24 hour that aerobic cultivated for the first aerobic time, and the time is longer, and whole operation is simpler, the screening time cycle is shorter, but overlong time likely can make some facultative anaerobe dead because adapting to the oxygen that exposes to the sun for a long time suddenly.Aerobic incubation time can be preferably about 0.5-12 hour, for example, be approximately 0.5 hour.
In the method for the invention, at least 1 cycle that repeated of step (3) just can be realized object of the present invention, but the strictly anaerobic bacterium and the strict aerobic bacteria that in the amphimicrobian flora that repeat cycle number of times filters out more, mix are fewer, but spending repeatedly the repeat cycle can increase operation complexity, cost, screening and culturing time, and also likely in long-term cultivation, the impact because of conditions such as nutrition makes some bacterium death etc.So, general 1-30 cycle of preferred repetition, more preferably 5-20 cycle, for example 8 cycles.
In the method for the invention, the aerobic incubation time at every turn repeating is longer than last time aerobic incubation time, and for example aerobic incubation time is all approximately 2 times of aerobic incubation times last time.
The preferably 72 hours time that last aerobic is cultivated, because the bacterium that can grow under condition at aerobic 72 hours also can grow under the condition of longer aerobic time.
The time that aerobic between aerobic cultivation for the first time and last aerobic cultivation is cultivated, between the above two time, can be uniformly distributed or uneven distribution between it, for example, can be to be uniformly distributed or exponential distribution.
In a preferred embodiment, in the method for the invention, repeating in step (3) is 8 cycles, and anaerobic incubation time is all approximately 24 hours, and aerobic cultivated for the first aerobic time and follow-up aerobic incubation time is respectively approximately 0.5,1,2,4,8,12,24,48 and 72 hour.
Separation in the method steps (4) of second aspect present invention can be undertaken by the method for single bacterium colony separation well known by persons skilled in the art, such as being undertaken by methods such as coating, line separation.
In the method for the invention, bacteria culture medium usually used in this field can be used for implementing the present invention, those skilled in the art can select applicable substratum as required, for example, when screening can be degraded the facultative anaerobe of kerosene, can use kerosene substratum: take kerosene as Carbon and nitrogen sources, inorganic salt solution (NH for example
4nO
3, Na
2hPO
4, KH
2pO
4, MgSO
47H
2o, MgCl
26H
2o, NaCl, FeSO
47H
2o, CaCl
2, ZnCl
2).For example, the substratum using in the inventive method is except can providing the required carbohydrate of general bacterial growth, nitrogenous substances, inorganic salt (comprising trace element) etc., also preferably provide required electron acceptor(EA) in facultative anaerobe anaerobic respiration process, as common NO
3 -, SO
4 2-, Fe (III), Mn (IV) etc.In substratum, preferably do not contain the nutrition that makes aerobic bacteria or the growth of anaerobic bacterium rapid, high volume.For example, the substratum using in method of the present invention can be taken from natural surroundings, for example lake bed sediment or river sediments; In some embodiments, also optionally add as required other components, for example, add multiple salt (Na for example
2cO
3, CaCl
26H
2o, MnSO
47H
2o, FeSO
4, Na
2hPO
4, KH
2pO
4, MgSO
47H
2o etc.).
In the method for the invention, the temperature of cultivation can be the optimum temps of general bacterial growth, for example 25 ℃-30 ℃, for guaranteeing in screening process, environment homogeneous in culture vessel, preferably should keep it in rocking state in culturing process, for example, under rotating speed 100-200rpm, rock.
In a specific embodiment, amphimicrobian bacterial screening method of the present invention comprises the steps:
(1) by will with substratum divide and be filled to a container, and sterilizing, in aseptic technique environment, adds testing sample in described container by a certain percentage, and seals described container with the gas tight seal thing of sterilizing;
(2) after air in described container being extracted out with vacuum pump, then by rare gas element N for example
2be filled with in described device.In order to reach sufficient anaerobic environment, can be so by described inert gas replacement three times for the air in container, the container anaerobism of having replaced is cultivated 24 hours;
(3) anaerobism was cultivated after 24 hours, in aseptic technique environment, changed described container sealing thing into aseptic breathable sealing film.Aerobic cultivation 0.5 hour.Sealed membrane with before sterilizing 30 minutes under 121 ℃, the condition of 0.11MPa.
(4) repeating step (2) anaerobism was cultivated after 24 hours again, the aerobic cultivation of repeating step (3) 1 hour, and so repeating step (2) and step (3) were to aerobic cultivation 72 hours, in whole process, anaerobism incubation time is all 24 hours, and aerobic incubation time is respectively 0.5 hour, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours;
(5) optionally, will amphimicrobian flora in environmental sample can be obtained by above step, by methods such as coating, line separation, the wherein facultative anaerobe of individual plant can be obtained.
In a specific embodiment, amphimicrobian bacterial screening method of the present invention, comprises the steps:
(1) by will with substratum divide and be filled to ground triangular flask, under 121 ℃, the condition of 0.11MPa, sterilizing is 30 minutes.Until the temperature of substratum, be down to after room temperature, in aseptic technique environment, by a certain percentage environmental sample added in triangular flask, and use through 75% alcohol wipe, the airtight rubber plug of ultraviolet sterilization screw thread and seal triangular flask;
(2) with the vacuum pump that syringe needle is housed, the air in triangular flask is drawn to rubber plug and caves in inwards, then from being full of N
2gas pouch in, with the syringe of syringe needle by N
2be filled with in triangular flask, so by the air N in triangular flask
2replace three times to reach anaerobic environment, the triangular flask of having replaced is placed in 30 ℃, the shaking table of 200rpm and is cultivated 24 hours;
(3) anaerobism was cultivated after 24 hours, in aseptic technique environment, it is the breathable sealing film of 0.22 μ m filter membrane that screw thread rubber plug is changed into aperture, is placed in 30 ℃, the shaking table of 200rpm aerobic cultivation 0.5 hour, sealed membrane with before sterilizing 30 minutes under 121 ℃, the condition of 0.11MPa;
(4) repeating step (2) anaerobism was cultivated after 24 hours again, the aerobic cultivation of repeating step (3) 1 hour, and so repeating step (2) and step (3) were to aerobic cultivation 72 hours, in whole process, anaerobism incubation time is all 24 hours, and aerobic incubation time is respectively 0.5 hour, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours;
(5) optionally, by above step, amphimicrobian flora in environmental sample can be obtained, by methods such as coating, line separation, the wherein facultative anaerobe of individual plant can be obtained.
In the above-described embodiment, describedly utilize the airtight rubber plug of screw thread that gradient intermittently exposes to the sun in the facultative anaerobe screening plant of oxygen technique improvement can not autoclave sterilization, can adopt other sterilization methods, for example, first use 75% alcohol disinfecting, then use ultraviolet sterilization 30 minutes.And other all utensils of sterilizing that need can carry out autoclave sterilization, for example sterilizing 30 minutes under 121 ℃, the condition of 0.11MPa.
In one embodiment, anaerobic environment in amphimicrobian bacterial screening method of the present invention can reach as the method for nitrogen by for example vacuumizing and filling, for example with the airtight rubber plug of screw thread, seal ground triangular flask, and with the vacuum pump that syringe needle is housed, the air in triangular flask is drawn to rubber plug and caves in inwards, then from being full of N
2gas pouch in, with the syringe of syringe needle by N
2be filled with in triangular flask.So by the air N in triangular flask
2displacement repeatedly for example 3 times to reach anaerobic environment.
In one embodiment, in amphimicrobian bacterial screening method of the present invention, aerobic environment reaches by the following method: in aseptic technique environment, changing airtight rubber plug into aperture is that the breathable sealing film of 0.22 μ m filter membrane is to reach aerobic environment.
In amphimicrobian bacterial screening method of the present invention, described microbial culture is preferably carried out in shaking table, to guarantee that in culturing process, the environment in device is uniformly, for example, under 200rpm, rocks cultivation.
In a specific embodiment, facultative anaerobe screening plant of the present invention comprises:
(1) for packing substratum into and cultivating the container of testing sample;
(2) for sealing the gas tight seal thing of described container;
(3) vacuum extractor being connected by sealing pipeline with described container;
(4) rare gas element is filled with to the inflation mechanism in described device; And
(5) for sealing the breathable sealing film of described container.
In said apparatus, described container can be any inorganic container that can be used for cultivating, for example, by glass, made, or be made of plastics, such as test tube, culture dish, flask etc.For sealing the gas tight seal thing of described container, it can be for example rubbery stopper of stopper.Described vacuum extractor can be any vacuum extractor as known in the art, for example vacuum pump.Described inflation mechanism can be any inflation mechanism known in the art, for example syringe.For sealing the breather hole of the breathable sealing film of described container, must be less than the size of air common bacteria, the aperture of for example ventilating is 0.22 μ m, this ventilative (aperture is 0.22 μ m) sealed membrane can obtain by purchase, for example, can buy from Princeton, Shanghai biotechnology Development Co., Ltd.
In a more particular embodiment, a kind of facultative anaerobe screening plant comprises:
(1) for packing substratum into and cultivating the triangular flask of testing sample;
(2) for sealing the airtight rubber plug of described triangular flask;
(3) vacuum pump that described container connects by sealing pipeline;
(4) rare gas element is filled with to the syringe in described device; And
(5), for sealing the breathable sealing film of described container, for example its ventilation aperture is 0.22 μ m.
Embodiment 1:
Below in conjunction with specific embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example is only for the present invention is described, and should not be considered as limiting scope of the present invention.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
In the present embodiment, utilize the expose to the sun amphimicrobian bacterial screening method of oxygen new technology improvement of gradient intermittently, from lakeside, Taihu Lake Wetland Sediments, (on 04 10th, 2010, gather from lakeside, Taihu Lake wetland (120 ° 03 ' 282 of longitude E; 31 ° 24 ' 361 of latitude N), in settling, add volume ratio be the settling overlying water in same place of 50% left and right with simulation local environment, in 10 days, use) in filter out amphimicrobian flora, comprise the steps:
(1) by following composition, with distilled water, configure minimal medium (g/L): Na
2cO
30.1, CaCl
26H
2o 0.01, MnSO
47H
2o 0.02, FeSO
40.01, Na
2hPO
40.05, KH
2pO
40.03, MgSO
47H
2o 0.02.All inorganic salt are all purchased from traditional Chinese medicines group chemical reagent Beijing company limited above.
(2) 100ml minimal medium is divided to the 250ml ground triangular flask that is filled to 24#, under 121 ℃, the condition of 0.11MPa, sterilizing is 30 minutes.Until the temperature of substratum, be down to after room temperature, in aseptic technique environment, add lakeside, 100g Taihu Lake Wetland Sediments to triangular flask, and use through 75% alcohol wipe, the ultraviolet sterilization airtight rubber plug of 24# screw thread of 30 minutes and seal triangular flask.In the shaking table of 200rpm, mix 30 minutes.Get 1ml sample and measure the flora number (as mentioned below) of aerobic bacteria, anerobe, facultative anaerobe in settling.
(3) with the vacuum pump (GM-0.33A is risen in Tianjin) that 1ml syringe needle is housed, the air in triangular flask is drawn to rubber plug and caves in inwards, then from being full of N
2gas pouch in, with the 50ml disposable syringe (purchased from Shanghai Zhi Yu Medical Devices Co., Ltd.) of 1ml syringe needle by N
2be filled with in triangular flask.So by the air N in triangular flask
2replace three times to reach anaerobic environment.The triangular flask of having replaced is placed in 30 ℃, the shaking table of 200rpm and is cultivated 24 hours.
(4) anaerobism was cultivated after 24 hours, and in aseptic technique environment, it is that 24mm, aperture are the breathable sealing film (Princeton, Shanghai biotechnology Development Co., Ltd) of 0.22 μ m filter membrane that screw thread rubber plug is changed into diameter.
Be placed in 30 ℃, the shaking table of 200rpm aerobic cultivation 0.5 hour.Sealed membrane was placed under 121 ℃, the condition of 0.11MPa sterilizing 30 minutes before use.
(5) repeating step (3) anaerobism was cultivated after 24 hours again, the aerobic cultivation of repeating step (4) 1 hour, and so repeating step (3) and step (4) were to aerobic cultivation 72 hours.In whole process, anaerobism incubation time is all 24 hours, and aerobic incubation time is respectively 0.5 hour, 1 hour, and 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours.Aerobic cultivation, after 72 hours, is got the flora number (as mentioned below) that 1ml sample is measured its aerobic bacteria, anerobe, facultative anaerobe.
(6) aerobic bacteria, anerobe and facultative anaerobe and flora number are measured by the following method: by MPN method, measured the flora number of aerobic bacteria, anerobe and facultative anaerobe in settling.Used medium is for containing 0.2g/L MgSO
47H
2the nutrient agar medium of O (preparing factory purchased from Beijing's bispin microbiological culture media).Substratum changes into after flat board, and each flat board is divided into 20 regions with four parallel lines and three vertical lines.1ml settling (measuring its quality and water ratio) adds in the sterilized physiological saline of 9ml (0.9%NaCl), as 10
-1extent of dilution, and dilute successively 10 times to 10
-8.Each extent of dilution is got 20 μ l to a zonule on flat board, does 5 Duplicate Samples.Aerobic bacteria is placed in biochemical cultivation case (granary LRH biochemical cultivation case) and cultivates, anerobe is placed in anaerobic box (the FA-12 anaerobic box that Hirayama manufacturing company produces) and cultivates, facultative anaerobe is first placed in anaerobic box and cultivates 24 hours, in biochemical cultivation case, cultivate after 72 hours anaerobism again and be cultured to same anerobe and count together, culture temperature is 30 ℃.Aerobic bacteria incubation time is a week, and anerobe, facultative anaerobe incubation time are one month, records each dilution growing state, and calculates bacterial count according to the MPN method of US EPA invention.
Below in conjunction with Fig. 1, the selection result is carried out to analytic explanation:
As can be seen from Figure 1, the flora number of strict aerobic bacteria, strictly anaerobic bacterium and facultative anaerobe in initial settling (Log (MPN)/g) is respectively 4.1,3.5 and 5.5, and utilize intermittently gradient to expose to the sun after oxygen method screens, in settling, the flora number of strictly anaerobic bacterium and strict aerobic bacteria is almost nil, in resulting amphimicrobian flora, does not have strictly anaerobic bacterium and strict aerobic bacteria to mix.And utilize intermittently gradient to expose to the sun after oxygen method screens, the flora number of facultative anaerobe (Log (MPN)/g) is 5.7, illustrate and utilize intermittently the gradient oxygen method of exposing to the sun can filter out comprehensively and effectively the amphimicrobian flora in settling, avoided not growing of some facultative anaerobe that traditional method causes because of the unexpected oxygen that exposes to the sun for a long time.
From the present embodiment, the present invention's utilization used intermittently the expose to the sun facultative anaerobe screening novel method of oxygen method improvement of gradient can filter out the amphimicrobian flora of take in the environmental sample that facultative anaerobe is dominant microflora comprehensively and effectively, and in the amphimicrobian flora filtering out, not strict aerobic or strictly anaerobic bacterium does not mix.
Embodiment 2:
In the present embodiment, utilize the expose to the sun amphimicrobian bacterial screening method of oxygen new technology improvement of gradient intermittently, from fluvial deposit (pick up from Beijing Xiaoqinghe River on 05 12nd, 2010, the preservation of sample and duration of service are identical with embodiment 1), filter out amphimicrobian flora, step is with embodiment 1.
Below in conjunction with Fig. 2, the selection result is carried out to analytic explanation:
As can be seen from Figure 2, the flora number of strict aerobic bacteria, strictly anaerobic bacterium and facultative anaerobe in initial settling (Log (MPN)/g) is respectively 3.9,4.1 and 4.3, and utilize intermittently gradient to expose to the sun after oxygen method screens, in settling, the flora number of strictly anaerobic bacterium and strict aerobic bacteria is almost nil, in resulting amphimicrobian flora, does not have strictly anaerobic bacterium and strict aerobic bacteria to mix.And utilize intermittently gradient to expose to the sun after oxygen method screens, the flora number of facultative anaerobe (Log (MPN)/g) is 4.7, illustrate and utilize intermittently the gradient oxygen method of exposing to the sun can filter out comprehensively and effectively the amphimicrobian flora in settling, avoided not growing of some facultative anaerobe that traditional method causes because of the unexpected oxygen that exposes to the sun for a long time.
From the present embodiment, intermittently expose to the sun the present invention's utilization used to filter out comprehensively and effectively amphimicrobian flora be not the facultative anaerobe in the environmental sample of dominant microflora for the facultative anaerobe screening novel method of oxygen method improvement by gradient, and in the amphimicrobian flora filtering out, not strict aerobic or strictly anaerobic bacterium does not mix.
Case study on implementation 3:
In the present embodiment, utilize the expose to the sun amphimicrobian bacterial screening method of oxygen new technology improvement of gradient intermittently, from lakeside, Taihu Lake Wetland Sediments (with embodiment 1), filter out amphimicrobian flora, comprise the steps:
Step (1) is identical with embodiment 1 to step (3).
Step (4) is also basic identical, and different is that the first aerobic incubation time is different, and in the implementation case, the first aerobic incubation time is 4 hours.
Step (5) is basic identical with the step (5) in embodiment 1, and different is the asynchronism(-nization) that aerobic is cultivated, and the time that aerobic is cultivated is in the present embodiment respectively 4 hours, 8 hours, 12 hours, 24 hours, 48 hours and 72 hours.
Step (6) is identical with the step (6) in embodiment 1.
Below in conjunction with Fig. 3, the selection result is carried out to analytic explanation:
As can be seen from Figure 3, the flora number of strict aerobic bacteria, strictly anaerobic bacterium and facultative anaerobe in initial settling (Log (MPN)/g) is respectively 4.1,4.2 and 4.3, and utilize intermittently gradient to expose to the sun after oxygen method screens, in settling, the flora number of strictly anaerobic bacterium and strict aerobic bacteria is almost nil, in resulting amphimicrobian flora, does not have strictly anaerobic bacterium and strict aerobic bacteria to mix.And utilize intermittently gradient to expose to the sun after oxygen method screens, the flora number of facultative anaerobe (Log (MPN)/g) is 5.7, illustrate and utilize intermittently the gradient oxygen method of exposing to the sun can filter out comprehensively and effectively the amphimicrobian flora in settling, avoided not growing of some facultative anaerobe that traditional method causes because of the unexpected oxygen that exposes to the sun for a long time.
From the present embodiment, intermittently expose to the sun the present invention's utilization used to filter out comprehensively and effectively amphimicrobian flora be not the facultative anaerobe in the environmental sample of dominant microflora for the facultative anaerobe screening novel method of oxygen method improvement by gradient, and in the amphimicrobian flora filtering out, not strict aerobic or strictly anaerobic bacterium does not mix.
Case study on implementation 4:
In the present embodiment, utilize the expose to the sun amphimicrobian bacterial screening method of oxygen new technology improvement of gradient intermittently, from lakeside, Taihu Lake Wetland Sediments (with embodiment 1), filter out amphimicrobian flora, comprise the steps:
Step (1) is identical with embodiment 1 to step (3).
Step (4) is also basic identical, and different is that the first aerobic incubation time is different, and in the implementation case, the first aerobic incubation time is 8 hours.
Step (5) is basic identical with the step (5) in embodiment 1, and different is the asynchronism(-nization) that aerobic is cultivated, and the time that aerobic is cultivated is in the present embodiment respectively 8 hours, 12 hours, 24 hours, 48 hours and 72 hours.
Step (6) is identical with the step (6) in embodiment 1.
Below in conjunction with Fig. 4, the selection result is carried out to analytic explanation:
As can be seen from Figure 4, the flora number of strict aerobic bacteria, strictly anaerobic bacterium and facultative anaerobe in initial settling (Log (MPN)/g) is respectively 3.8,3.9 and 4, and utilize intermittently gradient to expose to the sun after oxygen method screens, in settling, the flora number of strictly anaerobic bacterium and strict aerobic bacteria is almost nil, in resulting amphimicrobian flora, does not have strictly anaerobic bacterium and strict aerobic bacteria to mix.And utilize intermittently gradient to expose to the sun after oxygen method screens, the flora number of facultative anaerobe (Log (MPN)/g) is 6.2, illustrate and utilize intermittently the gradient oxygen method of exposing to the sun can filter out comprehensively and effectively the amphimicrobian flora in settling, avoided not growing of some facultative anaerobe that traditional method causes because of the unexpected oxygen that exposes to the sun for a long time.
From the present embodiment, intermittently expose to the sun the present invention's utilization used to filter out comprehensively and effectively amphimicrobian flora be not the facultative anaerobe in the environmental sample of dominant microflora for the facultative anaerobe screening novel method of oxygen method improvement by gradient, and in the amphimicrobian flora filtering out, not strict aerobic or strictly anaerobic bacterium does not mix.
Claims (6)
1. a method of facultative anaerobe in sample being screened, described method comprises step:
(1) under oxygen free condition, described sample anaerobic is cultivated to the first anaerobic time;
(2) then under aerobic conditions, described sample aerobic is cultivated to the first aerobic time;
(3) above step (1) and (2) are repeated at least 4-15 the cycle again, wherein in repeating: identical or different and aerobic incubation time of time that anaerobic incubation time and anaerobic are last time cultivated is longer than last time aerobic incubation time at every turn,
Wherein anaerobic incubation time is 12-48 hour;
The first aerobic time was 0.5-12 hour, and the time that last aerobic is cultivated is 72 hours.
2. the method for claim 1, also comprises step:
(4) culture that step (1)-(3) is obtained to environmental sample carries out separation, obtains single strain.
3. claim 1 or 2 method, the aerobic incubation time of wherein said follow-up aerobic in cultivating is all 2 times of aerobic incubation times last time.
4. claim 1 or 2 method, repeating in step (3) is 8 cycles, wherein said anaerobic incubation time is all 24 hours, and aerobic cultivated for the first aerobic time and follow-up aerobic incubation time is respectively 0.5,1,2,4,8,12,24,48 and 72 hour.
5. claim 1 or 2 method, repeating in step (3) is 5 cycles, and wherein said anaerobic incubation time is all 24 hours, and aerobic cultivated for the first aerobic time and follow-up aerobic incubation time is respectively 4,8,12,24,48 and 72 hours.
6. claim 1 or 2 method, repeating in step (3) is 4 cycles, and wherein said anaerobic incubation time is all 24 hours, and aerobic cultivated for the first aerobic time and follow-up aerobic incubation time is respectively 8,12,24,48 and 72 hours.
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