CN2737799Y - Millipore filter culturing device for microbic near natural pure culture - Google Patents

Abstract

The utility model publishes a millipore filter culturing device for microbic near natural pure culture which takes a near-nature culture method as a principle. The millipore filter culturing device for microbic near natural pure culture is a perforated culture vessel, one to several layers of microporous filtering films are arranged in the vessel, and the millipore filter culturing device for microbic near natural pure culture can be used for microbic pure culture by adding the asepsis culture medium after sterilization; when culturing, the preforated vessel is arranged in the environment needed by the cultured microbe; thus the microbe in the membrane can exchange and communicate with the outside, but the microbe in the membrane is hard to escape or be contaminated, so the cultured microbe obtains a culture condition which is near nature. The millipore filter culturing device for microbic near natural pure culture can enhance the cultivable performance of the microbe in the natural environment and even makes possible to separate and purify the non-cultured microbe.

Description

The millipore filtration culture apparatus of the nearly natural pure culture of a kind of microorganism

Technical field

The utility model belongs to the microorganism culturing technical field, is specifically related to the millipore filtration culture apparatus of the nearly natural pure culture of a kind of microorganism.

Background technology

The occurring in nature microbial diversity more and more receives various countries scientist's concern, and scientist infers that according to the existing scientific evidence microbe species number should be 10 ⁵-10 ⁶But regrettably, prokaryotic organism below 5,000 kinds are only arranged by the document description mistake, popular Approved List of Bacterial Names has put down in writing 3,500 kinds, has write down 3,100 kinds in authority's the BergeyShi handbook.Bacterium may only account for environmental bacteria multifarious 1% or still less known to this just meaned.Obviously, 99% prokaryotic micro-organisms of not described has more heredity, metabolism and functional diversity certainly, is a kind of invisible national resources.

The research of microorganism is subjected to the restriction of cultural method and culture apparatus for a long time.German doctor Robert section is conspicuous, and (Robert Koch 1843-1910) once came culturing bacterium with the potato of scalding; 1887, Richard Petri delivered one piece of short essay, and the Koch plate technique has been done another improvement, had designed our today of employed culture dish (Petri dish).Ever since, pure culture technigne (pure culture technique) just by frequently with in Microbiological Lab, be used to routinely to separate, purifying, viable count and culturing micro-organisms, become the foundation stone of research microorganism, to such an extent as to that this method seems is also natural always.Over more than hundred years, microbiology has been obtained significant progress at this, and new microorganism constantly is separated, but this far is seldom a part of natural resources.Report such as Amann RI (1995), use standard microorganism to learn the mensuration of culture technique to different habitat microorganism cultivation property (culturability), the cultivation property of microorganism is about 0.001～0.1% in the seawater, fresh water is about 0.25%, soil is about 0.3%, and active sludge is about 1～15%.Some microorganism is confirmed it is active by many laboratories, but can not cultivate, and becomes a kind of active but can not cultivate that (viable but nonculturable, VBNC) state have at least 30 kinds of bacteriums of 16 genus this phenomenon that has been in the news to take place.Though cultivation, enrichment culture (enrichment culture) etc. provide appropriate nutrition and physiology combination for culturing micro-organisms naturally, yet last separation, purifying still be unable to do without traditional pure culture technigne.Therefore, the limitation of the pure culture technigne of microorganism has become a bottleneck of the further researchdevelopment of microbiology undoubtedly, still needs further to seek new cultivation means and method.

In recent years, along with molecular biological develop rapidly, utilize the non-culture method (culture-independent method) of molecular engineering research natural habitat microorganism to be widely used more and more, some even the technology of using some to study in position, these have crossed over necessary this bottleneck of pure culture of containment microbe research to a great extent, constantly disclose the new 16S rRNA sequence type of nature, make us can be familiar with increasing microorganism.Now, there have 40 bacterium monoids to become approximately to be known, and wherein 1/3 monoid is formed by not cultivating (nonculturable) bacterium fully, and they only go on record in 16S rRNA sequence mode.In many environment, these new bacteria monoids and pedigree are in the ascendance.But these microorganism sequence that obtain only are to obtain by inferring to the knowledge of aspects such as its form, physiological metabolism, heredity and ecological functions; In addition, those are only cultivated out the chemical substance that can produce in microorganism, nowadays also can not be studied and obtain.Therefore, though non-culture method has had significant progress, in order to understand the functional role of microorganism in physical environment fully, existing enrichment with separate strategy and just need to improve, still need to develop new cultural method.But strengthen up to now, microorganism cultivation property, more not culturing micro-organisms (nonculturable microorganism) be transformed into that can to cultivate still be microbiologist's a dream.

Why is numerous microorganisms difficult to be cultured on the artificial medium with the pure culture method? this is only specified microorganisms to be separated because the pure culture method itself is meant from occurring in nature, on artificial medium, breed, this means by culturing micro-organisms be in a kind of with under the isolated simple condition of other microorganism of nature and most of related substance.Yet natural bacterial flora has the height diversity, and it is complicated, much abundant that the natural habitat of the original existence of microorganism is wanted, and therefore is difficult to overlap culture apparatus and method with one and allows most of bacterial growths.In addition, also have a series of factor to hinder recovery and the cultivation of microorganism on artificial medium:

(1) recovery of many microorganisms need provide the original ecotope in the natural sample, these microhabitats (niche, or be called ecological niche) be difficult under artificial condition, restore, atomic little variation all might make its forfeiture vitality (viability).

(2) trophic level that extremely changes in the pure culture even may become main retardation of growth, especially the microorganism in oligotrophic water's body and those surviving policy (K-selection) are suitable for the kind of low nutrient contg and very low growth rate, provide the abundant substratum of nutritive ingredient to become on the contrary and hinder microorganism restorative important factor, even substrate quickens dead (substrate-accelerated death).

(3) to potential important ATP (its concentration in seawater fresh water can reach 1nM, is derived from plant plankton and other planktonic organism probably) and other active substance of microbial culture, the pure culture stage is difficult to be provided, and is difficult to provide with active condition.

(4) numerous species interdependence in the natural ecological environment of microorganism, get in touch countless ties between the microorganism in same habitat, communication is widely arranged between cell and the cell and get in touch, at the related cAMP of microbial gene expression under most of starvations, the N-acetylhomoserine lactone (N-acyl homoserine lactones) between most of Gram-negative bacteria all might be the signaling molecule of linking up between the cell, and these are disclosed by microbiological continuous development just gradually and prove.

(5) metabolism (cometabolism) mode is to be likely one of important model that microorganism survives at occurring in nature altogether, and commensalism, alternate, symbiosis are the different levels of common cooperation life style in the microorganism natural, ecological relation.

Obviously, traditional microorganism pure culture mode has completely cut off by culturing micro-organisms fully with extraneous interchange and communication, make it be difficult to obtain the active substance of specific habitat and particular organisms, therefore, will atrophy when most of microorganism of occurring in nature is pulled away from its primitive environment, wither, be difficult to be cultivated maybe cultivation property very low (lowculturability) by existing pure culture method.

For a long time, the microbiologist is in order further to strengthen microorganism cultivation property, to improve microorganism restorative situation from environmental samples, mainly study from two different angles: the one, test how to make microbial growth under field conditions (factors) as far as possible, be called the nature culture method; The 2nd, explore in common pure culture, adding which type of New Terms just can be widely near natural condition, both near natural culture method.The soil microorganisms scholar ties up promise Gladders base (Winogradsky) and takes much count of cultivating naturally of microorganism, sets up Winogradsky Column and is used for enrichment sulfuration bacterium and nitrifier, and become the pioneer that microbial enrichment is cultivated; Rossi-Cholodny buries sheet method (Rossi-Cholodny buried slide technique) and remains the best example that nature is cultivated, the report of Henrichi (1932) is thought, slide glass keeps certain hour in fresh water after, can be observed diversified unknown microorganism at microscopically; It also is a kind of multiplication culture under field conditions (factors) that the kapillary that Aristovskaya and Parinkina USSR (Union of Soviet Socialist Republics) soil microorganisms scholars such as (1962) proposes is surveyed the number method.Research to nearly natural pure culture, then mostly is as far as possible near the nutritional condition in nature active microorganism aspect the composition of proliferated culture medium, as using the soil extract as the basal component of cultivating soil microorganisms, river or seawater are cultivated microorganism in the water body separately as the interpolation of necessity, water adds substratum and is used for culturing plants pathogenic bacteria etc., but is not still obtaining revolutionary progress aspect this.The example that this thought is the most successful should be (2002) reports on the Science magazine such as Kaeberlein T, they have used millipore filtration, the device that the centre adds partition carried out cultivation to the sandy beach microorganism, simulation habitat, sandy beach, they have obtained success as a result, prove that this method can strengthen the cultivation property of part microorganism effectively, and the not culturing micro-organisms (nonculturable microorganism) of two bacterial strains before being proved to be arranged, but unfortunately they not form a cover complete, the nearly natural pure culture method and apparatus of microorganism that can be general.

Microbiology has used millipore filtration to come filter liquide or collects microorganism since the midium or long term, product has various various millipore filters; Also will collect film behind the microorganism in the filter membrane culture method (filter method) directly puts into substratum and cultivates, some companies even with its further commercialization, be designed to substratum and constitute one with millipore filtration, as check the portable millipore filtration enumeration plate of usefulness, also having laboratory millipore filtration product NPS (the Nutrient Pad Set) external member (filter membrane+substratum+culture dish) of German Sai Duolisi (Sartorius) company etc., all is the substratum external member that microorganism detection is used; Dialysis cultivation system in the microbiology is the bactogen for microorganism cells and nutrition and product are separated, and perhaps microorganism is carried out a kind of device of enrichment culture; In the patent and product of China and other national many zooblasts and tissue culture, a lot of millipore filtrations that all use are as the support of cultivating zooblast and tissue, as the Boyden chamber system in the functional analysis of cytochemistry chemotactic, the hyaline cell of Opticell is cultivated card etc.; In Chinese patent, application number is that 87106707 " equipment of microorganism culturing and detection and method " narrated optionally cultivation activity bacterium, is suitable for the recovery of part bacterium; Application number is to have used microporous membrane as the membranous method and apparatus that carries out gaseous interchange in 02120114.5 " liquid cultivating method of microorganism and cell and culture apparatus thereof ".These all devices or be can not make by culturing micro-organisms with extraneous cross streams, or be not suitable for microorganism pure culture thereon and form bacterium colony, or do not form the complete general experimental installation that carries out the microorganism pure culture.

Summary of the invention

The purpose of this utility model is the deficiency at existing culture apparatus existence, on the unfavorable factor basis of recovery of fully research obstruction nature microorganism and pure culture, a kind of nearly natural culture method thought inherited and develop has been proposed, for microorganism provides the microorganism of not cutting off active substance and communication source the pure millipore filtration culture apparatus of cultivating naturally, this device can guarantee can be exchanged and communication with external environment by cultured microorganism, but microorganism itself can not escape and be contaminated, reaches the cultivation property that strengthens the part microorganism, even will the part not culturing micro-organisms change the purpose that can cultivate (culturable) into.

The applicant is through finding in the years of researches experiment, when culturing micro-organisms, if when transplanting them, create one by the needed outside atmosphere of pure culture microorganism, provide compound in its primitive environment to form and the microorganism of community life, allowing it experience their all environment and neighbours does not change or changes and be enough to stand, these microorganisms just can not know that they have been moved so, will have many microorganisms to be cultivated out again.Sum up after deliberation, the applicant has invented this culture apparatus:

This device is the container underpunch in traditional culturing micro-organisms, allow it communicate with the external world, the inside of these porose containers is lined with one deck to several layers of millipore filtration, the function of millipore filtration is to make by culturing micro-organisms and outside atmosphere to separate, permission by culturing micro-organisms with extraneous cell exchange, communication, absorb the biologically active substance in the external environment, but do not allow microorganism escape and contaminated in the film; And porose container is to have supported and protection for millipore filtration provides, and can closely cultivate naturally not changing to finish under other condition of existing Microbiological Lab simultaneously, strengthens vitality of part microorganism and cultivation property widely.

The pore size of millipore filtration be according to do not allow microorganism cells itself by but permission film both sides cell communication determine with the requirement that exchanges active substance, select the millipore filtration of or low toxicity nontoxic to microorganism.Millipore filtration can the loosely liner in porose container, also further industrialization is integrated the two, can be coated with the whole internal surfaces of pore volume device, the also part of a porose inner surface of container, millipore filtration can be open or complete closure.

Substratum in the millipore filtration can be solid, liquid or semisolid medium.

The use-pattern of this device: after at first this culture apparatus sterilization being added aseptic culture medium, earlier culture apparatus is put into by the required physical environment of culturing micro-organisms, allowed in the culture apparatus substratum interpenetrate and be consistent with active substance in the external environment; Behind the balance certain hour, microbe inoculation under aseptic condition, continue to put back in the primitive environment and to cultivate until obtaining desired microorganisms, both allowed microorganism and exchange active substance but not allow the microorganism cells escape and pass through with extraneous communication by culture apparatus.

It should be noted in the use: (a) when carrying out the microbial solid cultivation, curing agent component content should be higher slightly than amount in the common medium preparation in the substratum, prevents to destroy the solid medium form owing to penetrating fluid in the external environment excessively enters; (b) cultivate preceding external environment liquid level and can be higher than inner substratum plane, pressure reduction promotes internal and external equilibrium; (c) during solid culture, the liquid level in the external environment should not be higher than the inside substratum height usually, and media surface does not have obvious water stain formation, the formation of bacterium colony when helping solid culture; Then need regulate inside and outside liquid level and osmotic pressure during liquid culture; (d) in the culturing process, the external environment material can not enter to remove the alternate manner that sees through millipore filtration; (e) its outside atmosphere is meant by the required any environment of culturing micro-organisms, can be physical environment, the also artificial environment of creating comprises various water bodys and the environment that contains liquid, as soil, sandy beach or marsh, even can be live plant or animal tissues and individuality.

Sharp device of the present utility model, on the basis of other condition that does not change existing Microbiological Lab, for recovery and the growth of many microorganisms provides required environment, the cultivation property of enhancing activity bacterium greatly not only, those are the microorganisms under the poor nutritional status especially, even the part in the not culturing micro-organisms that present stage microbiology field might be thought cultivates out, and making us seek more Microbial resources becomes possibility.

Description of drawings

Figure 1A is a kind of culture apparatus of the present utility model---porose culture dish stereographic map;

Figure 1B is the sectional view of porose culture dish;

Fig. 2 A is a kind of culture apparatus of the present utility model---porose triangular flask stereographic map;

Fig. 2 B is the sectional view of porose triangular flask;

Fig. 3 A is a kind of culture apparatus of the present utility model---porose test tube stereographic map;

Fig. 3 B is the sectional view of porose test tube.

Embodiment

Next the utility model is described with reference to the accompanying drawings:

This device has various ways, below exemplifies several:

Referring to Figure 1A and Figure 1B, this is lined with the porose culture dish of millipore filtration in being.1 is the ware body, and several holes 5 are arranged at its bottom; 2 are the ware lid; 3 is millipore filtration, is covered in the culture dish internal surface; 4 is solid medium.Microorganism culturing is when separating, and with 1,2,3 package sterilization, adds solid medium 4, and is stand-by after the condensation.Before the cultivation, at first this device is put into by the required environment of culturing micro-organisms, allows the external environment liquid level be higher than the surface of solid medium 4, and pressure reduction promotes internal and external equilibrium; Microbe inoculation continues to put back in the primitive environment and cultivates, and the external environment liquid level of this moment should be controlled to be the surface that is not higher than solid medium 4, and is obviously water stain to guarantee solid medium 4 surface nothings, helps microbe colony and forms.

Referring to Fig. 2 A and Fig. 2 B, this is lined with the porose triangular flask of millipore filtration in being.6 is triangular flask, and hole 5 is arranged at the bottom that it is characterized by bottom and bottle wall; Liner millipore filtration 3; Add tampon 8, the sterilization back adds aseptic liquid nutrient medium 7, can carry out the liquid culture of microorganism.Before cultivating this device is put into the external environment balance, continue to put into external environment after the inoculation and cultivate, should note regulating inside and outside liquid level this moment to keep the balance of osmotic pressure.This device can be used for the liquid culture of microorganism cells.

Referring to Fig. 3 A or Fig. 3 B, this is lined with the porose test tube of millipore filtration in being.9 is porose test tube, it is characterized by test tube bottom, hole 5 is arranged at the bottom; In be lined with millipore filtration 3; Add tampon 8, the sterilization back adds sterile solid substratum 4, makes the inclined-plane, can be used for microorganism strains purifying, switching and bacterial strain preservation etc. after the condensation.Porose test tube should be put into external environment before cultivating, allow the external environment liquid level be higher than solid medium 4 planes, pressure reduction promotes internal and external equilibrium; The liquid level of external environment is not higher than the inclined-plane foot during cultivation, and is obviously water stain to guarantee solid medium 4 surface nothings, helps microorganism and forms lawn.

More than the selection of millipore filtration requires in the device: 1. pore size should allow film both sides microbiological active material to exchange and communication, but microorganism cells can not pass through, and general using always is 0.2 μ m, 0.25 μ m, 0.45 μ m etc. can select littler, wide aperture filter membrane more for use when special; 2. film to microorganism nontoxic or low toxicity; 3. film is a wetting ability, as polycarbonate membrane, cellulose acetate film or the like; 4. still keep its primary characteristic after can bearing 121 ℃ of high-temperature sterilizations.

The use of millipore filtration: earlier it is wetting with a small amount of clear water or sterilized water, make it soft more, thereby can closelyr press close to (particularly porose test tube, tube wall is darker, is difficult for apt) mutually with porose inner surface of container.

More than device is for adopting under the laboratory scale.Porose container and millipore filtration can further be integrated, and can enlarge to carry out industrialization and mass production, other appellation can be arranged, but method feature are constant.

Use: microorganism culturing will be lined with porose culture dish (as Figure 1A) packaging sterilizing of millipore filtration when separating, and adds aseptic culture medium, and is stand-by after the condensation.Before the cultivation, at first this porose culture dish is put into by the required environment of culturing micro-organisms, allows the external environment liquid level be higher than the surface of substratum, and pressure reduction promotes internal and external equilibrium, allow in the culture dish substratum with active substance in the external environment interpenetrate, balance, be consistent.Behind the balance certain hour, at aseptic condition underscore or alternate manner microbe inoculation, continue to put back in the primitive environment and cultivate, the external environment liquid level of this moment should be controlled to be the surface that is not higher than substratum, to guarantee that media surface do not have obviously water stainly, help microbe colony and form; If but when using pour plate technology (pour plate technique), then pour this device at agar with by culturing micro-organisms after, the condensation of wait agar is directly put into by the required environment of culturing micro-organisms and is cultivated.The device of porose test tube and millipore filtration is then used in the microorganism strains preservation, and the sterilization back adds aseptic culture medium, condensation, and bevel is stand-by; During use, this device is put into the outside atmosphere balance, the external environment liquid level can be higher than the substratum internal surface, but media surface does not have obviously water stainly should guarantee to inoculate the time, helps the formation of lawn; During cultivation, the external environment liquid level is not higher than the lower end, inclined-plane usually.When using this method microbial liquid to cultivate, adopt the porose triangular flask of liner millipore filtration, add liquid nutrient medium, put into the external environment balance that microorganism needs; Behind the balance certain hour, inoculation continues to put into external environment and cultivates; During cultivation, should note regulating the balance of inside and outside liquid level and osmotic pressure.

The utility model is to propose after the drawback of the traditional pure culture method of further investigation and produce, therefore, the work that in traditional pure culture device, can carry out, utilize this device to carry out, can be used for microorganism pure culture, strains separation, viable count etc. equally as porose culture dish, porose triangular flask can be used for the production of strain liquid cultivation, meta-bolites etc., and porose test tube can be used for purebred separation, go down to posterity cultivation and bacterial strain preservation etc.But, this device for provided by culturing micro-organisms one with external environment link up may, need just can put it into information and the product that obtains in the primitive environment in the external environment as long as have, this be traditional pure culture device can not provide.Cultivate in the rough determination of comparison at this device of use and conventional apparatus, microorganism cultivation property in spring, thermal water, river, oligotrophic lake, rich lake, the active sludge is measured, under identical cultivation situation (as medium component, pH value, temperature), the colony number that occurs in the nearly natural millipore filtration culture method plate is many than in the culture-based method plate obviously, and statistics shows and manifests work or utmost point significant difference.

Claims (6)

1, the millipore filtration culture apparatus of the nearly natural pure culture of a kind of microorganism is characterized in that: this device is a kind of bottom foraminous structure of container, and container inside lining has one deck or several layers of millipore filtration, is added with substratum in the filter membrane.

2, millipore filtration culture apparatus according to claim 1 is characterized in that: in the porose container of millipore filtration loosely liner.

3, millipore filtration culture apparatus according to claim 1 and 2 is characterized in that: millipore filtration and porose container are integrated.

4, millipore filtration culture apparatus according to claim 1 and 2 is characterized in that: millipore filtration is coated with the part of whole internal surfaces of pore volume device or internal surface.

5, millipore filtration culture apparatus according to claim 1 and 2 is characterized in that: millipore filtration is open or complete closure form.

6, millipore filtration culture apparatus according to claim 1 and 2 is characterized in that: the substratum in the millipore filtration is solid, liquid or semisolid medium.

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