DE102007010866A1 - Device for immersing microcultivation and investigation of growth, morphology, and metabolism of microorganisms or cells, comprises tension-free unidimensional element and nutrient for growth of microorganisms or cells - Google Patents

Abstract

The device comprises a tension-free unidimensional element, and a nutrient for the growth of the microorganisms or cells. A hydrocolloid matrix is also provided, where the matrix of the element is held by the element, in which the matrix partly surrounds the element or the element covers the matrix or the matrix of the elements. The ratio of the length to middle diameter of the cultivation device (1) is greater than 500. Independent claims are included for: (1) a method for the microcultivation of microorganisms or cells, which involves applying cells of the microorganisms on the matrix in an inoculation area carried to the surface or into the interior of the matrix with an inoculation liquid; and (2) a method for manufacturing a device, which involves manufacturing an element by press over the outside cavity of a circular nozzle in the form a hose.

Description

contraption and methods for conducting emersed microcultures in microorganisms and cells

The The invention relates to an apparatus and a method for carrying out the same from emersed microculturing of microorganisms and cells, in particular for the study of growth, morphology and metabolism in an addressable and storable form.

microorganisms and cells have been used for decades on / in various media cultivated and examined.

The investigations to date are based on surface cultures in Petri dishes (emers cultures) which contain a layer of a hydrocolloid growth matrix (for example in the form of a nutrient agar) on whose surface the colonies grow or in liquid culture (submerged culture) ( Schlegel HG: General Microbiology, 6th edition, with the collaboration of K. Schmidt. Stuttgart, New York: Thieme, 1985 ).

there is the plane bottom of Emerskulturen the often drum-shaped Petri dish of a layer of nutrient agar as hydrocolloid Growth matrix covered. This contains the growth substrates and immediately supplies the moisture necessary for growth.

The Wall of the shell is part of the sterilizable space that passes through an overlapping, but not airtight resting Lid is closed. Through the closure with the lid forms a humid climate and the access of contaminants is prevented. The bottom of the Petri dish closes together with the overlying Growth matrix down the sterile room.

The Submerged cultures are used as standing or shaking cultures fermented.

Assuming that the diversity of human metabolic functions of microorganisms is far from exhausted, screening for new microorganisms will continue to be of great importance in the future ( RH Baltz, Marcel Faber Roundtable: Is our antibiotic pipeline unproductive because of starvation, constipation or lack of inspiration? J. Ind. Microbiol. Biotechnol. 33 (2006), 507-513 ; J. Berdy, Bioactive Microbial Metabolites. J. Antibiot. 58 (2005), 1-26 ; F. v. Nussbaum et al .: Antibacterial Natural Products in Medicinal Chemistry - Exodus or Renaissance. Angew. Chem. 118 (2006), 5194-254 ).

Screening for microorganisms with new metabolic activities requires the study of a large number of microorganisms. From the colony counts of plating on agar plates, average viable counts of about 10 ⁶ to 10 ⁸ microorganisms per gram of soil have been determined. In order to prevent overgrowth of microorganisms, a low cell density per unit area (ideally less than 100 germs on a 78 cm ² area Petri dish) must be achieved. Ie. In order to enable all microorganisms from 1 g of soil to grow as a pure culture and thus also to ensure the isolation of rarely occurring species, one million Petri dishes would be necessary. For example, to find the producer of a new antibiotic, an estimated 10 ⁷ microorganisms must be cultured and tested ( RH Baltz, Marcel Faber Roundtable: Is our antibiotic pipeline unproductive because of starvation, constipation or lack of inspiration? J. Ind. Microbiol. Biotechnol. 33 (2006), 507-513 ).

at The traditional isolation methods are very small quantities a suspension of a soil sample in dilutions on agar plates applied to obtain clones or pure cultures, respectively to go back to an individual. This only results a limited number of colonies, their number at not for the isolation of large numbers sufficient microorganisms.

A Possibility to circumvent these restrictions is the production of very small colonies, so-called microcultures, their diameter in the range of 10 .mu.m to 100 .mu.m should lie.

The isolation of microorganisms to obtain pure clones is suggested by several authors (Brehm-Stecher BF, Johnson EA, Single-cell Microbiology: Tools Technologies, and Applications. Microbiology and Molecular Biology Reviews, 2004, 68, 538-559 and Chan ECS, Pelczar MJ, War NR, Use of Pure Cultures - A prerequisite for the study of microorganisms. In: Laboratory Exercise in Microbiology. Chan ECS, Pelczar MJ, War N (eds). McCraw-Rill, New York, (1993) 123-152 and Huber R. The laser pipette as a basis for individual cultivation. Biospektrum 4, (1999), 289-291 ).

For the separation of microorganisms and microfluidic systems are used in which continuously injected sequences of small aqueous microculture droplets in a stream of water-immiscible release agent. They are transported with the separating agent stream into a capillary and incubated. After incubation, growth is preferably detected by optical methods. For example, fluorescence is used for detection zenzspektrometer suggested ( Martin K, Henkel T, Baier V, Grodrian A, Schön, T, Roth M, Köhler JM and Metze J. Generation of larger numbers of separated microbial populations by cultivation into segmented-flow microdevices. Lab on a chip 3 (2003) 202-207 ).

Another method of producing microcultures of isolated microorganisms consists in the production of small viscous drops, so-called gel microdroplets (GMD) from a sample (Weaver JC, Process for Measuring Microbial Active Material, US Pat. U.S. Patent 4,401,755 , 1981; Williams GB, Demain AL, Rapid microbial detection and enumeration using gel microdroplets and colorimetric or fluorescence indicator systems. J. Clinical. Microbiol. 1990, 28, 1002-1008 ; Manome A, Zhang H, Tani Y, Katsuragi T, Kurane R, Tsuchida T, Application of gel microdroplet and flow cytometry techniques to selective enrichment of non-growing cells. FEMS Microbiol. Lett. 2001, 197, 29-33 ). The GMDs are incubated together in a broth, and each bead is detected for growth.

With microstructured wafers or nanoplates, for example, 6000 chambers ( Mayer G, Wohlfart K, Schober A, and Köhler JM (1999) Nanotiterplates for synthesis and screening. In: Micorosystem technology: a powerful tool for biomolecular studies. Eds. Köhler JM, Mejaveia T, Saluz HP. P. 75-128, Birkhäuser Basel ) are cultivated on a plate.

With piezoelectric atomizers operating on the inkjet principle and producing up to 10,000 micro-drops per second with a volume of between 5 picoliters and a few nanoliters, the plates can be inoculated, incubated, detected and stored with isolated microorganisms ( Schober A, Günther R, Schwinhorst A, Döring M and Lindemann BF. Accurate high-speed liqiud handling of very small biological samples. BioTechniques (1993) 15, 324-329 ). An unsolved problem with this technique is the rapid drying out of the microcultures.

Textile fabrics of various shapes are used for the growth of microorganisms in the form of a nutrient-containing matrix (Tatsurn T, Shiro I, Katsutoshi A, Carrier for cultivation of cell and microorganisms. WO9821351 and JP 2154685 ). It is also known that unwanted microorganism growth can take place on textile surfaces. Arrangements consisting of hollow fibers are used to supply cultures with nutrients or as cultivation containers (see JP 63309177 and JP63071186 ).

The DE 100 53 394 A1 uses a variety of short fiber elements arranged to bind to them substances and perform analytical reactions to them after introduction of the elements in an analyzer. The longitudinal movement type of filaments associated with the liquid agent treatment is typical of many processes in the manufacture of textile filaments and textiles (see EP 1 369 521 A1 ).

Longitudinally moving, so-called "one-dimensional" solid phase reactors, e.g. In the form of a thread, are off DE 10 2004 008 319 A1 known. The term "one-dimensional" here describes elements whose length is very large compared to their average diameter and are therefore commonly referred to as thread, fiber, filament or wire. With them, substances, including microbial contaminants, bioaffected at the reaction site to the thread and determined qualitatively or quantitatively after passing through other reaction sites.

Of the Disadvantage of this technical solution is that an implementation from microbial growth and product formation processes longer periods on this one-dimensional Elements (threads) is not possible and none compact stacking and storage of the covered threads can be done.

Moving threads were also used to perform a sequence of different chemical reactions in micro-preparative work under the microscope. ( Mészáros L, Mészáros I .; Continuous thread reactor for micro-preparative purposes. Fats, soaps, paints (2006) 70, 940-941 ).

The WO 03/025113 discloses an arrangement and method for parallel cultivation of microorganisms wherein a coarse solids-liberated suspension or culture is added to a homogenous or heterogeneous microorganism population, nutrient substrates and / or effectors and / or microbial metabolites, then a volume of the microbial suspension containing N microorganisms is divided with a portioner in n ₁ partial volumes, wherein the number n ₁ between N and 100 N, preferably between N and 10 N is selected, then the Teilvolmina optionally with supply of nutrient substrates and / or effectors for inoculation of n ₂ separate Microcultures in microareas or microcavities, where n _{2 is} greater than or equal to n ₁ are used, then the microcultures incubated and optionally supplied during growth further nutrient substrates and / or effectors and / or metabolites and physiological parameters and the growth of the individual n microcultures with suitable measuring ver be detected driving.

According to this Revelation is miniaturized to singulate the microorganisms Fluid switch used in conjunction with a microinjection unit wherein the microorganisms in liquid segments in one closed microcapillary system are cultivated. (Serial River).

Of the serial flow of liquid segments is through periodic Change the volume flow rate by gas bubbles or by produces water immiscible separating liquids, where the liquid segments with a probability <5% more than one Cell per segment included.

adversely on this technical solution for the cultivation of microorganisms or cells in microcapillaries, however, is that consuming individual fluid segments must be generated and the wall of the capillaries or the liquid segments have special surface properties must be in order for the microorganisms or cells in the microcapillaries are not mixed or that not the microcapillaries by adhering to the wall, colony-forming microorganisms or tent associations be clogged.

The The object of the present invention is to overcome the disadvantages of the prior art to avoid the technique and a device and a procedure for the investigation of growth, morphology and metabolism of various kinds emers growing microorganisms or cells in addressable and storable To allow form in a simple way without it too a mixture of different cells and microorganisms comes.

The Object of the present invention is also that a huge variety of micro microorganisms can be produced in a form in which as small as possible dimensioned device, the microcultures are incubated, detected, addressed and also for subsequent withdrawals of inoculation material. To achieve this, the microcultures must be very small and dimensioned they are not supposed to be excessively large. cultivation areas to need in a compact two- or three-dimensional Arrangement can be stored.

to Solution of the problem is an inventive one-dimensional or filamentous culture device (KV) proposed that at least one tensile element and a growth-promoting, nutrient-containing matrix consists.

On or in the cultivation device according to the invention are linearly arranged microculture cultured, so that the microorganisms or cells are addressable cultivable and storable are.

According to the invention the micro-organisms to be applied to the KV in an inoculation room on the surface or in the interior of the KV with a Transfer vaccine fluid, the KV into an incubation room and after a culture time the KV with the grown microcultures transported to a detection room where preferred over optical detection devices or, for example, using bioaffinity detection methods, the microcolony growth or the linear Microculture sequence and the metabolism of microcultures in one the individual microcultures are detected in an addressable manner. Subsequently, the covered KV in a storage room transported, in the temperature suitable for storage, preferred between 0 ° C and 12 ° C and where accessibility insist on the individual microcultures.

The preferably present in the used inoculants sporadically present Microorganisms are thereby on the surface or in introduced the interior of a one-dimensional culture device. The Inoculated KV are then moved by moving in the longitudinal direction into an incubation room that is favorable for growth Conditions, transported and there in one possible arranged compact form. The humidity in the incubation room must be high, preferably between 90% and 100% relative humidity, to prevent drying of the hydrocolloid cultivation matrix.

Possibly done by spraying with finely dispersed water mist one additional humidification.

The Temperature in the incubation room is usually between 10 ° C and 80 ° C, preferably at 18 ° C to 40 ° C.

After the microorganisms or microcultures have grown, they are transported with the one-dimensional cultivation device or a plurality of them, from the incubation room into a room where the analysis of the metabolic products as well as the control of the growth takes place (detection space) and at the same time a possibility for addressing consists. Subsequently, in a manner similar to that in the incubation space, the transport takes place into a storage space, which as a rule is cooled in order to prevent further growth and metabolic activity. Again, they are stored, preferably in a compact form. The transport can be by longitudinal movement For example, in combination with the transport of the stacked surfaces as a whole done. The longitudinal movement is usually done by tensile forces or their vector.

In another version is inoculation, incubation and detection of KV continuously. In one embodiment the inoculation of the quasi-cultivated area is done by spraying with microdrops, in another by contact with a seeding liquid.

Prefers isolated microorganisms are used. The isolation This is due to a correspondingly high dilution of Inoculation fluids used as an inoculation culture be achieved.

A another application of the invention relates to the detection of substrates, Active ingredients, factors and hormones. The microcultures become this Substances exposed and their presence and concentration over Changes in growth behavior are determined.

The Essence of the inventive solution is "one-dimensional" or thread-like cultivation devices, in addition to the necessary resistance to tensile forces at the same time offer the possibility of succession in them or on them single or multiple microorganisms apply and their Promote growth.

According to the invention Microorganisms as Microcultures on a Cultivation Device (KV) cultivated, consisting of a tensile stable one-dimensional element and containing nutrients for growth Matrix exists and their relationship length of KV to mean KV diameter greater than 500 is. The longitudinal extent of the microcolonies on the thread is without limiting the invention, preferably less than 1000 microns and the distance between the outer edges adjacent growth areas should not be less than 1000 μm.

In In special cases, the tension-stable element can simultaneously have the function of the matrix containing nutrients.

The Nutrient-containing matrix consists of the as a growth support gel-forming and hydrophilic substances that have been used for a long time such as agar, pectin, chitosan, carrageenan, hyaluronic acid, Alginic acid, xanthan, cellulose, bacterial cellulose or their derivatives and salts or polyelectrolyte complexes or others hydrocolloid gelling agents or from water-storing silica gel, Alumina or silicon polymers.

The gel-forming substances are preferably immobilized, Hardening, crosslinking or mechanical forces or adhesion with structural irregularities with the tensile stable Anchored element.

The For example, texture bumps can be surface topographies in the form of holes, elevations, unidirectional furrows or superimposed furrows and / or convex or concave profiles or the shape of peaks, vaults or blocks exhibit.

The Nutrient-containing matrix may be inside the KV or on the surface as a continuously formed coat or discontinuous. In the discontinuous Arrangement, for example, gel droplets equidistant or also irregular on the surface the KV be distributed.

The KV may be formed monofilament, but are preferred polyfile one-dimensional structures used as yarns.

The KV can be made of staple fibers or of continuous filaments or mixtures Both types exist.

Several The fibers or filaments can be in the form of a knitted fabric or knitted fabric. Preference is given to a knitted fabric in the form of a Hose, wherein the knitted hose jacket regular openings or meshes used.

According to the invention also cultivation devices in which the tensile element by Pressing through an annular nozzle in the form of a Hose is made simultaneously over a Nozzle with a matrix containing nutrients, which contains living microorganisms, is filled.

The Threads can have different profiles like triangular, U-shaped or star-shaped. Also one Tape shape as in the film threads is then according to the invention, if they have a width / height ratio of 10 is not exceeded.

According to the invention For example, a foil thread on which in equidistant Distance from one or more gel droplets formed zones, which are separated by dangle-free zones, wherein the gel drops have the function of a matrix containing nutrients.

Preference is given to surface-structured CV with grooves which run in the longitudinal direction or have surfaces roughened by fibrillation or network-like fibrils. At In such surfaces, the nutrient-containing matrix is better protected against shearing, or its binding to the tensile member is firmer. Microporosity of KV or a capillarization of KV, as it is produced for example by absorbent fibers, is also beneficial because in the microstructures water and nutrients can be stored.

The CT can continue to have a bilateral structure such as side-by-side, Core / sheath or matrix / fibrils have. Threads with more hydrophilic properties are therefore also cheaper as threads with very hydrophobic properties. The diameters of such suitable and known technical filaments or threads is usually between 4 to 60 microns.

It Both staple fibers and endless naturally occurring ones are used Filaments of vegetable, animal, mineral, inorganic Origin or fibers consisting of natural polymers made of keratin, such as wool or silk, of polyamino acids, from polysaccharides, such as carrageenan, chitosan / chitin, alginates, Cellulose, viscose or cotton or their derivatives, salts or Polyelectrolyte complexes, from bioplastics such as polylactate or Polyalkanoates, of synthetic polymers such as polyethylene, polypropylene, Polyacrylamide, polystyrene, polyester, polyvinyl alcohol or polyamide or made of fibers of metal or glass.

The Fibers can be used as a microfiber fiber blend or as Composite available.

Advantageous on the proposed device and the proposed method is that a very large number of microcultures, preferred in the form of colonies grown on solid surfaces (emers growth) can be generated.

These Growth form, which also in the conventional agar plate cultivation occurs, is particularly favorable for the recognition or the detection of microbial products compared to those grown in liquid cultures forms.

Of the favorable for the production of secondary metabolites Differentiation state of the mature colonies as well as the general high cell density leads to the formation of higher ones and thereby better detectable concentrations.

Special applications of the invention further provide a number of advantages:
With conventional growth of microbial colonies on conventional agar plate cultures, the nutrient providing area increases with the square of the distance. This means that the colonies can become relatively large due to the large nutrient depot, because nutrients will diffuse for a very long time.

Advantageous on the one-dimensional shape according to the invention the KV is therefore the nutrient-providing area in the direction of the longitudinal extent of the KV only linear with the Distance grows. Due to the so limited diffusion of nutrients is slowed down the growth in the longitudinal direction, so that the culture during the cultivation time only the extent the KV and a relatively short section of the KV can overgrow.

The ultimately reached the size of microcultures is closely related to the cultivation parameters as the cultivation time, for different microorganisms specific growth rate, lag phases and especially with the competition with neighboring colonies for the nutrients in the overlapping areas as well with structural elements of KV acting as barriers.

These Barrier elements are inventively the textile thread structures or by introduced barriers formed and are related to the type of thread used KV.

Prefers in the spaces between the individual threads from which the filaments z. B. are composed as a knitted fabric, the nutrient-containing matrix can be applied. The resulting growth zones are then partially by the Filaments separated. But even monofile KV are inventive.

In a specific embodiment of the invention is the linear Microculture sequence of microcultures present on the KV Replicate duplicated by a second or even several KV in close contact with each other as loose multiple threads arranged or as interconnected multiple threads over Breakage zones are separated. They then ask for separation an identical replica of the microculture sequence, which is responsible for more Investigations can be used.

at Application of conventional methods for replication, for example when testing for antimicrobial activities Of the microcultures these are inevitably with the test germs contaminated or lost. By the possibility the identical replication according to the invention can use the replica on the unadulterated sequence of Microcultures on the KV can also be resorted to later.

The detection of the microcultures and / or the substances formed by them is carried out with known, preferably optical methods. So there is the possibility, according to the disclosure of DE 10 2004 008 319 A1 use specific binding to bioaffine substances, such as fluorescent antibodies, and perform the reactions directly on the longitudinally moving, covered with microcultures thread.

The Testing for antimicrobial activity can be up conventional way by placing the KV on one with a Test germ-covered agricultural surface, by means of a second, brought into close contact with the CT and inoculated with the test strain KV or by coating the KV with a test germ and nutrients containing gel-containing layer. In the places where the test germ does not grow, arises as in the classic Inhibition test for the detection of antibiotics not one with the microbial test germ overgrown Hemmhof.

In another embodiment becomes the quasi-cultural area covered with a gel-containing layer containing the test germ and then registering the zones of inhibition. The Hemmhofsignal can by using a marked z. B. fluorescent test bacteria or by using redox indicators such as tetrazolium compounds be strengthened.

Farther the use of the KV according to the invention is advantageous in the study of the morphological properties of the cultures, because the KV in a simple way under the lens of a microscope or other optical detectors along and thereby can be examined.

A Another solution of the inventive task, the KV in one to arrange as compact as possible or miniaturizable form, consists of a multitude of parallel one-dimensional KV's, including the loose and coupled multiple threads in the form of a generally rectangular area as so-called To arrange quasi-cultivated areas. This arrangement is in Comparison to a conventional agar culture surface achieved that much more microcultures per area can grow.

The Quasi-cultural area can also be like an agar plate culture as a whole inoculated with seed material, incubated and detected become. The KV become for example by Aufspannen in a construction the Aufspannrahmens fixed as a surface.

The Fixing the KV in the Aufspannrahmen done for example by Clamping parallel KV in the upper and lower frame parts with a clamping device. The clamping frames be successively by introducing the upper and lower frame boundaries into the quasi-cultivated areas emerging from the inoculation zone pushed in and by their appropriate raising or lowering the Quasi-cultural area raised or lowered and to the Quasi-cultural area after clamping with the Aufspannrahmen formed.

The Winding on frame leads to paired quasi-cultural areas. Due to the strength of the frame here is the distance between determines the paired quasi-cultural areas. The connecting threads between the individual surfaces can be severed by means of a cutting device and so the separate Aufspannrahmen with the spanned in them quasi-cultural areas separately to be edited.

Of the Transport of the KV from the inoculation space is preferably carried out by the tensioning of the KV in the Aufspannrahmen, in one embodiment doing a rotating movement, in which the traction vector, which arises during the rotation of the frame, the KV through the inoculation space and optionally through the coating room.

inventive but is also the winding of the KV on a spool or reel. There are as few points of contact as possible generated between the coil and the KV, spacers used or an inert layer z. B. in the form of a sheet placed between the quasi-cultural areas to a partial Destruction of the mechanically unstable nutrients containing matrix to avoid.

at Investigations on the influence of substrates, active substances, buffer substances or factors will contain these either in the nutrients Matrix deposited or during cultivation by dripping, Spraying or by direct addition to the KV or quasi-cultivated areas added.

One Another advantage is a substantial increase in the microcolon density per unit volume by a three-dimensional storage of KV. According to the Invention, a device is proposed in which by means of movable arrangements the quasi-cultural areas in parallel stacked to each other so that a three-dimensional Arrangement of the KV in the form of a stack results. The stack represents a compact form, with a very high microculture density can be realized.

The Three-dimensional arrangement can also be by a direct, without prior Formation as a quasi-cultural area was done in parallel or by a three-dimensional arrangement shifted by 90 ° the KV done.

The Microcultures become in the three-dimensional arrangement form, for example incubated and stored. The advantage is that at a sufficiently large Distance of KV or Quasikulturflächen each other, the Supply of oxygen as well as the maintenance of temperature and humidity also guaranteed inside the stack can be.

In a larger sized version of Invention inoculation as well as the growth and product formation studies within a period constantly and continuously.

The Inoculated KV is placed on a deceleration drum or deceleration reel coiled with a plurality of delay windings and the other end unwound at the same time, then a detector room supplied, detected and optionally subsequently the double thread separated into two monofilaments and different used. It can also have two or more threads over Predetermined breaking points are interconnected by mechanical Forces or chemical treatments are separated become.

potential Areas of application of the invention are the high-throughput screening for new microorganisms and studies on the influence of Substrates, agents or factors caused by microorganisms be formed or the analytical proof of physiological active substances with microorganisms as test germ.

The Invention will be described below with reference to the drawings and the embodiment explained in more detail. Show it:

1 a schematic representation of a first embodiment of the one-dimensional cultivation device according to the invention in a side view (time sequence of production and use),

2 a schematic representation of a second embodiment of the device according to the invention in a side view,

3 a schematic representation of a third embodiment of the device according to the invention in a side view;

4 a schematic representation of the flow of a variant of the method according to the invention using an embodiment of the device according to the invention in a side view;

5 1 is a schematic representation of an embodiment of the device according to the invention with a stack composed of several quasi-culture surfaces, which is located in a detection space, in a side view;

6 a schematic representation of the inoculation of one of a coil 9 unwound multipath in the form of a double thread in a side view and

7 a schematic representation of a covered with microcultures, designed as a double thread loose multiple thread in a cross section.

The 1 shows a side view schematically the timing of the production and use of a special type of the one-dimensional cultivation device according to the invention 1 (KV). Right in the 1 is the production of a one-dimensional culture device 1 (KV) after a continuous casting process by means of a spinneret 2 in their outer cavity 3 the material for the tubular tensile-stable jacket 7 the KV 1 and in an internal nozzle, the matrix material 4 , from the nutrient-containing matrix 8th formed, together with the isolated microorganisms used for inoculation 6 contains, shown.

When spinning the thread simultaneously becomes the interior with the material of the hydrocolloid matrix 7 which also contains microorganisms as a seed material.

After curing the tensile stable matrix, preferably by treatment in a precipitation bath 5 or other chemical or physical processes, the section of the functional KV shown on the left is formed 1 , Inside, it contains the most liquid or semi-solid cultivation matrix 8th with the grown colonies 6 , The cultivation matrix is surrounded by the usually oxygen-permeable jacket, which in this example is at the same time the tensile element 7 represents.

Selected Microcultures can be used in this type of execution by cutting out the selected microculture containing Section and subsequently for inoculation of subsequent cultures are used.

The 2 shows in side view another special type of one-dimensional KV 1 consisting of an internal tensile stable element 7 and a matrix containing nutrients as a coating 8th exists and with microbial colonies 6 is overgrown.

The 3 shows in side view one with colonies 6 overgrown other special type of one-dimensional cultivation facility KV 1 in which the nutrient-containing matrix 8th from an external tensile stable element 7 is enclosed in the form of a textile braid.

The 4 schematically shows the sequence of a variant of the method according to the invention using an embodiment of the device according to the invention in side view, in the zugstabile of a coil elements 7 be unwound in the form of a thread and the coating in the impregnation room 10 with the matrix material 4 he follows.

By means of pressure or deflection rollers 11 In this case, the thread is in the liquefied matrix material 4 pressed.

The one-dimensional culture device KV formed after solidification of the matrix material 1 is then in a vaccination room 12 through the roles 11 pressed into the inoculum. The KV 1 is then by tensile forces 14 in an incubation room 13 transports them in the form of a stack for the duration of the cultivation period 15 remains.

The 5 schematically shows one of several quasi-cultivated areas 17 composite pile 15 who is in a detection room 16 located.

Each quasi-cultural area consists of a frame 18 on which the KV 1 are clamped parallel to each other.

The detection of the KV 1 grown cultures or colonies 6 takes place, for example, contactless via a known per se optical detector system consisting of a light source 19 that with a ray of light 20 the KV 1 or the microcultures on them and scanned with a fluorescence receiver 21 with a connected computer 22 evaluates the fluorescence signals.

The 6 shows in side view the inoculation of one of a coil 9 unwound multiple thread in the form of a double thread 23 or a double KV 1 in an inoculation room 12 inoculated, on a deceleration drum 24 wound with a plurality of windings and the other end unwound at the same time, a detector space 16 fed, detected and then the multiple thread 23 is separated into two individual threads. The in the picture below again in the supervision 25 shown delay drum shows the winding pattern of the double thread.

The 7 shows in cross-section one with microcultures 6 overgrown, designed as a double thread loose multiple thread 26 in which the growth matrices of the two threads are in close contact and further coupled multiple thread 27 in which the tensile elements of two parallel threads with each other by a predetermined breaking point 28 are connected. The KV consist here of a tensile stable element 7 and a nutrient-containing matrix 8th ,

In an embodiment not represented by illustrations the device according to the invention become a, two or a plurality of moving in the longitudinal direction Cultivators (KV) continuously inoculated, cultured and detected. By separating yourself in one execution of the invention in close contact during incubation one-dimensional cultivation devices (multiple threads) it is possible to replicate the microculture sequence identically. In another embodiment, one arranged on a frame Plurality of one-dimensional cultivators arranged in parallel, which form a quasi-cultural area, in a compact, two- or after stacking also three-dimensional arrangement incubated, then detected area by area or in thread form and then stored in compact form ready for access.

One preferred field of application of the invention is the cultivation large numbers of microorganisms, preferably in the form of Cloning, characterizing and typing their growth and metabolic properties as well as the development of morphological features related to chemical Influencing factors and cultivation parameters. Furthermore it is applicable for high throughput screening for new Microorganisms with special metabolic benefits and product formations.

All in the description and the claims which follow Features can be used individually or in any combination with each other be essential to the invention.

1

Culture device (KV)

2

spinneret

3

outer cavity

4

matrix material

5

precipitation

6

cultures colonies

7

zugstabiles element

8th

hydrocolloid Nutrient-containing matrix

9

Kitchen sink

10

impregnation chamber

11

Deflection or pressure rollers

12

Beimpfungsraum

13

incubation

14

tensile forces or direction of movement

15

stack

16

detection space

17

Quasi-growing areas

18

mounting frame

19

light source

20

beam of light

21

fluorescence receiver

22

computer

23

double thread

24

Deceleration drum, sideview

25

Deceleration drum, At sight

26

loose Multiple yarn

27

coupled Multiple yarn

28

Breaking zone

29

Storage space

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• US 4401755 [0014]
• WO 9821351 [0017]
• - JP 2154685 [0017]
• JP 63309177 [0017]
• JP 63071186 [0017]
• - DE 10053394 A1 [0018]
• EP 1369521 A1 [0018]
• - DE 102004008319 A1 [0019, 0065]
• WO 03/025113 [0022]

Cited non-patent literature

• - Schlegel HG: General Microbiology, 6th edition, with the collaboration of K. Schmidt. Stuttgart, New York: Thieme, 1985 [0004]
• - RH Baltz, Marcel Faber Roundtable: Is our antibiotic pipeline unproductive because of starvation, constipation or lack of inspiration? J. Ind. Microbiol. Biotechnol. 33 (2006), 507-513 [0008]
• - J. Berdy, Bioactive Microbial Metabolites. J. Antibiot. 58 (2005), 1-26 [0008]
• - Nussbaum et al .: Antibacterial natural products in medicinal chemistry - Exodus or Renaissance. Angew. Chem. 118 (2006), 5194-254 [0008]
• - RH Baltz, Marcel Faber Roundtable: Is our antibiotic pipeline unproductive because of starvation, constipation or lack of inspiration? J. Ind. Microbiol. Biotechnol. 33 (2006), 507-513 [0009]
• - Microbiology and Molecular Biology Reviews, 2004, 68, 538-559 [0012]
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Claims (35)

Device for the microbial fermentation of microorganisms or cells comprising a tensile stable one-dimensional element ( 7 ) and a hydrocolloid matrix containing nutrients for the growth of microorganisms or cells ( 8th ), where the matrix ( 8th ) of the element ( 7 ), in which the matrix ( 8th ) the element ( 7 ) at least partially surrounds or the element ( 7 ) the matrix ( 8th ) or the matrix ( 8th ) of the element ( 7 ), and the ratio of the length to the mean diameter of the culture device (KV, 1 ) is greater than 500. Device according to claim 1, characterized in that the matrix ( 8th ) in the form of discontinuously distributed zones or spots or droplets or aggregates, possibly in a seeded form, with regular or irregular distribution on the CV ( 1 ) is available. Devices according to claim 1 or 2, characterized in that the nutrients containing Matrix water-binding polysaccharide compounds such as agar, Pectin, chitosan, alginic acid, hyaluronic acid, xanthan gum, Carrageenan, cellulose, bacterial cellulose or derivatives thereof or Salts or polyelectrolyte complexes or inorganic materials such as silica gel or clays or silicon polymers. Devices according to claim 1 or 3, characterized in that the element ( 7 ) and the matrix ( 8th ) from an incubation room ( 13 ) are surrounded. Devices according to claim 4, characterized in that the incubation room ( 13 ) with a detection space ( 16 ) connected is. Devices according to claim 5, characterized in that the detection space ( 16 ) with a storage room ( 29 ), wherein the detection space ( 16 ) between the incubation room ( 13 ) and the storage room ( 29 ) is located. Device according to one or more of the preceding claims, characterized in that in the incubation room ( 13 ) a high humidity is adjustable. Device according to one or more of the preceding claims, characterized in that in the incubation room ( 13 ) a temperature of 10 ° C to 80 ° C is adjustable. Device according to one or more of the preceding claims, characterized in that two or more KV ( 1 ) by a longitudinal predetermined breaking zone, which can be separated by mechanical or chemical / biochemical action, as coupled multiple threads ( 27 ) are interconnected. Device according to one or more of the preceding claims, characterized in that the element ( 7 ) from biopolymers in the form of keratin, wool or silk, from polyamino acids, from polysaccharides, in the form of carrageenan, chitosan / chitin, alginates, cellulose, from viscose or from cotton or derivatives thereof, from salts or from polyelectrolyte complexes, from bioplastics in the form polylactate or polyalkanoates, of synthetic polymers in the form of polyethylene, polypropylene, polyacrylamide, polystyrene, polyester, polyvinyl alcohol or polyamide, or of metal or glass or carbon fibers. Device according to one or more of the preceding claims, characterized in that the element ( 7 ) consists of textile fibers or filaments in the form of knitting, knitted, knitted or braided or tubular fabric. Device according to one or more of the preceding claims, characterized in that the element ( 7 ) consists of textile yarns or sewing silk. Device according to one or more of the preceding claims, characterized in that the element ( 7 ) consists of biocomposites in the form of polyalkanoates, polylactic acid, semisynthetic or synthetic plastics in the form of polyamide, polyethylene, polypropylene, polystyrene or polyvinyl chloride. Device according to one or more of the preceding claims, characterized in that the KV ( 1 ) has a flat oval to circular profile with a height / width ratio of 0.1 to 1.0. Device according to one or more of the preceding claims, characterized in that the KV ( 1 ) in the longitudinal direction extending longitudinal grooves or a star-shaped profile. Devices according to one or more of the preceding claims, characterized in that the element ( 7 ) or the element ( 7 ) with matrix ( 8th ) on a spool ( 9 ) can be wound up. A method of microculturing microorganisms or cells comprising a device according to one or more of the preceding claims claims is used. A method according to claim 17, characterized in that on the matrix ( 8th ) to be applied to cells of the microorganisms in an inoculation space ( 12 ) on the surface or in the interior of the matrix ( 8th ) with a seeding fluid, the matrix ( 8th ) into the incubation room ( 13 ) and after a cultivation time the microcultures to the detection space ( 16 ), in which by means of detection devices ( 19 . 20 . 21 . 22 ) the microcolonia growth and the linear microculture sequence are analyzed and metabolites of the microcultures are detected in a manner which can be addressed by the individual microcultures and the matrix ( 8th ) then into a storage room ( 29 ), in which suitable temperatures for the survival of the microorganisms to be stored are present, so that the individual microcultures can be specifically accessed. Process according to claim 18, characterized in that in the incubation room ( 13 ), a high humidity, between 90% and 100% relative humidity, is present, or by spraying with finely dispersed water mist additional humidification of nutrients containing matrix ( 8th ) and the temperature is between 10 ° C and 80 ° C. Method according to one or more of the preceding claims, characterized that the microcultures substrates, agents, factors, among others by spraying, fed and their presence and concentrations analytically about changes in the growth behavior of the microcultures. Method according to one or more of the preceding claims, characterized in that in the course of a replication one or more longitudinally closely contacting KV ( 1 ) as loose multiple threads ( 26 ) are incubated together and that later one or more of them are separated and stored in a storage room ( 29 ) are kept as identical replicates of Mikrokoloniesequenz or used. Method according to one or more of the preceding claims, characterized in that the KV ( 1 ) continuously before passing through the inoculation space in an impregnation space ( 10 ) with the nutrient-containing matrix ( 8th ) is prepared. Method according to one or more of the preceding claims, characterized in that the inoculation of the KV ( 1 ), including touching KV ( 1 ) for replicating, contact with a microorganism-containing seeding liquid, or by applying the seeding liquid with a pipettor. Method according to one or more of the preceding claims, characterized in that upon contact of the surface of the KV ( 1 ) and the inoculation fluid per mm of KV ( 1 ) is transferred on average no more than one living microorganism cell or that in the per mm of liquid transferred amount of the inoculation liquid is on average not more than one cell. Method according to one or more of the preceding claims, characterized in that the transport of the KV ( 1 ) when inoculated in the longitudinal direction ( 14 ) he follows. Method for producing a device according to one or more of Claims 1 to 15, in which the element ( 7 ) is made by pressing over the outer cavity of an annular nozzle in the form of a tube and this at the same time via a second nozzle disposed within the annular nozzle with a matrix containing nutrients and microorganisms for inoculation ( 8th ) is filled. Method for producing a device according to one or more of claims 1 to 15 according to claim 22, characterized in that the matrix ( 8th ) sheathed element ( 7 ) is cured chemically or physically. Method for producing a device according to one or more of Claims 1 to 15 according to Claim 22, characterized in that the method steps of inoculation, incubation and detection are carried out continuously on longitudinally moved KV ( 1 ) respectively. Use of a device according to one or more of claims 1 to 16, characterized in that a plurality of KV ( 1 ) arranged in parallel, in a Aufspannrahmen to a quasi-cultural area ( 17 ), in which the KV ( 1 ) on the Aufspannrahmen ( 18 ) are fixed and transported together with this, incubated, detected and / or stored. Use according to one or more of the preceding claims, characterized in that the transport of the KV ( 1 ) when inoculated in the longitudinal direction ( 14 ) he follows. Use according to one or more of the preceding claims, characterized in that the tension-stable element of the KV ( 1 ) or the complete KV ( 1 ) on a spool ( 9 ), from which they are subject to tensile forces ( 14 ) unpacked becomes. Use according to one or more of the preceding claims, characterized in that a plurality of KVs arranged in parallel ( 1 ), optionally also coupled multiple threads ( 27 ) or loose multiple threads ( 26 ), in a framework to a quasi-cultural area ( 17 ) clamped on the Aufspannrahmen ( 18 ) and with the Aufspannrahmen ( 18 ) are transported, incubated, detected and / or stored together. Use according to one or more of the preceding claims, characterized in that the KV ( 1 ) in the incubation room ( 13 ) and in the storage room ( 29 ) on coils ( 9 ) or reels or by means of the Aufspannrahmens ( 18 ) in tensioned form horizontally or vertically as a stack ( 15 ) to be ordered. Use according to one or more of the preceding claims, characterized in that during the incubation and / or storage a direct contact of adjacent KV ( 1 ) and adjacent quasi-cultural areas ( 17 ) is prevented by gaps or a non-contact arrangement is optionally ensured by spacers. Use according to one or more of the preceding claims, characterized in that the process steps inoculation, incubation and detection are carried out continuously on longitudinally moved KV ( 1 ) he follows.