DE20306346U1 - System culturing small aquatic organisms in particle-free suspension, comprises upper and lower chambers with porous partition between suspension and nutrient - Google Patents

Abstract

The lower of two chambers (1, 2) is filled with solid nutrient (6). The upper is a culture chamber (1), a reservoir for a particle-free suspension of the organism. The chambers are separated by an open-pore physical barrier (3). Upper chamber sealing against the environment, maintains sterility whilst permitting oxygen exchange. The system is either single-part, forming a single cultivation unit, or a two-part culturing system, in which both chambers are completely separated. The unit is designed as a cuvette for direct photometric determination of organism density in the culture chamber. The oxygen-permeable seal is a penetrable septum. The implementation format is a 96-cell microtitration plate. It is alternatively a standard glass container with screw closure and inner septum.

Description

The invention relates to cultivation systems for the extraction of particle-free long-term cultures of water-living micro- and small organisms in which the organisms in a two-chambered Cultivation system can be kept sterile closed.

Protozoa as small aquatic organisms are unicellular eukaryotic organisms. There are a number in the laboratory by representatives of this group of organisms for several decades in the center of biochemical and molecular genetic interest (Gall J G, 1986, The Molecular Biology of Ciliated Protozoa, Academic Press, London; Lwoff A, 1951, Biochemistry and Physiology of Protozoa, Academic Press, London; Levandowsky M, Hutner S H, 1981, Biochemistry of Protozoa, vol. I-V. Academic Press, London.). Most recently In the past, some protozoan species have also been increasing Importance in the field of toxicology, environmental pollutant research and biotechnology (Gilron G L, Lynn D H, 1997, Ciliated Protozoa as Test Organisms in Toxicity Assessment. In: Microscale Testing in Aquatic Toxicology, Wells P G, Lee K, Blaise C (eds), CRC Press, Boca Raton; Tiedke A, 2001, Biotechnology with Protozoa. In: Biotechnology, Brehm H-J, Wiley-VCH, Weinheim).

An important requirement for biochemical, molecular genetic and toxicological studies is the possibility for the cultivation of the biological raw material in pure culture. Foreign organisms would through their own biology or through the additional presence of metabolic processes and biomolecules contaminate the sample and thus interfere with the analysis of the actual object to be examined or even prevent.

As a rule, they have for laboratory tests protozoan species used no cryptobiotic life stages, so they are not poor in metabolism Permanent forms (cysts) like many of their relatives form but keep theirs even under the conditions of long tent cultivation active, comparatively metabolism-intensive lifestyle, which on permanent Presence of food or nutrients is instructed. The pure cultivation of these laboratory-typical species is therefore usually done in full media based on proteose peptone / yeast extract (PPY) cultivated (Cell Biology, 1994, A Laboratory Handbook, Celis J (ed), Academic Press, London); synthetic media are less common with chemically defined composition use (Szablewski et al., 1991, Tetrahymena thermophila; growth in the synthetic nutrient medium in the presence and absence of glucose, J. Protozool, 38, 62-65). In addition to cultivation in dissolved nutrients also become watery Media related as a nutrient substrate solid components of plants (mostly cereals such as rice or cereals) or of animal origin (meat) (e.g. Father Dobberstein B, Hilfrich H-G, 1982, experiments with unicellular organisms, Kosmos, Stuttgart). The latter form, in particular, with a solid nutrient substrate is often used Strain cultivation of the organisms chosen.

The stem cultivation forms the The basis of the cultivation of biological material. From regular cultures aquatic small and very small organisms, e.g. the actual protozoan test cultures were drawn, their culture medium adapted to the respective biological / biochemical question is. Strain cultivation primarily serves the purpose of organic To keep raw material long-term and at the same time genetic Displacement ("genetic shift ") which is why the organisms here under conditions very low or stationary Growth, i.e. largely avoiding the possibility of spreading by mutants. The organisms are kept thereby in sterile closed vessels, whereby substrate and cells be united in one cultivation room.

Forms with age in stem cultures by dying organisms cell debris [fragments dead (body) cells, Cell fragments]. Likewise, this initially decomposes over time still consistent, from solid constituents of plant (cereals) or animal (e.g. meat) origin nutrient substrate. With conventional Process of cultivation comes in a single cultivation room it - especially when taking cells - easily to mix these particular Degradation products with the cell suspension. An accurate one necessary for many purposes It is used to determine the cell densities of such aged cultures considerably more difficult. common photometric methods are the result of mixing downturn disturbed, and the usual ones too electronic cell counting are due to the particular Impurities cannot be used directly. To the actual nevertheless Determining the cell density of these stem cultures must be based on time and laborious microscopic determination methods or the cells need to through cumbersome further work steps (e.g. centrifugation) separated from the culture medium become.

The object of the invention is Systems for stem cultivation of aquatic small and very small organisms to create, with which the disadvantages of root cultivation can be overcome and which are a regular cultivation of aquatic small and microorganisms in particular allow such that mixing dead cell components (cell debris) and particulate nutrient substrates can be avoided when removing live cells. Another The object of the invention is devices for such To develop stem cultivation.

To start from known options for the stem cultivation of aquatic small and very small organisms, especially of protozoa (unicellular organisms), free of detritus and nutrient particles for long periods To achieve organism suspensions / cell suspensions (detritus: mushy or crumbly remains crumbled tissue u. Cell parts), is proposed according to the invention, that the cells in one of two superimposed chambers existing, sterile sealed cultivation system in liquid held for at be solid nutrient substrate or particulate Nährmediumbestandteile only be added to the lower chamber (supply chamber), and the upper chamber as a reservoir (organism reservoir / cell reservoir) particle-free organism suspension / cell suspension (culture chamber) serves. Both chambers protrude one or more openings in connection with each other. The cells receive their nutrients preferably from solid, particulate nutrient substrates such as, for example Cereals or meat. According to their compared to the organism suspension / Cell suspension higher Density and its size that is many times larger than the cells they added to the lower part of the cultivation system. The opening or The pore size of the separating layer between the two chambers is so dimensioned that particles are unhindered by the size of the organism can happen the nutrient particles but held back become. At the same time, the opening or pore size selected be that the capillary forces acting in the separating layer are strong enough are, liquid to hold even after the cultivation unit is tilted. So that is guaranteed that cell debris that forms over time moves down into the Supply chamber can sink and accumulate there. Vice versa becomes the particular nutrient substrate due to the small pore size of the separating layer in the lower part retained.

The objects of the invention will be by devices according to the characteristics of protection claim 1 solved. Advantageous further developments are described in the subclaims.

The cultivation system according to the invention Obtaining particle-free long-term pure cultures of protozoa consists in that the organisms in one of two superimposed chambers existing cultivation system, the lower chamber with solid nutrient substrate filled is (supply chamber), and the upper chamber as a reservoir (organism reservoir / Cell reservoir) particle-free organism suspension / cell suspension serves (culture chamber) and supply and culture chamber by one open-pore physical barriers are separated, where is that

• - the Organisms are sterile and cultivated as a pure culture,
• - the Cultivation unit sterile, but sealed oxygen permeable is
• - particulate, solid nutrient of vegetable or animal origin the starting substrate of the Form culture and
• - the physical barrier between the two chambers one or more openings which is large enough are the active transition of cells to ensure as well as a passive sinking of dead cells or cell components to allow but are small enough by capillary culture fluid withhold and mixing even when the cultivation unit is tilted prevent or significantly reduce the two chamber contents.

The cultivation system can be used as one-piece or two-piece system designed in which the upper Chamber can be removed from the bottom, i.e. both chambers Completely are separable from each other. In the case of a split, the organisms directly to the chamber of culture without any additional aids remove from the supply chamber.

Another feature of the invention consists in the fact that an oxygen-permeable closure consists of a pierceable septum, through which cells from the outside by means of a syringe can be removed sterile, without opening the cultivation unit to have to. A septum closure has the possibility of repeated Withdrawal still another advantage without having used sterile room conditions or old liquid to be replaced or exchanged in the cultivation chamber.

Another preferred embodiment is that the cultivation unit is designed as a photometer cuvette is. With that leaves the density of the organisms is non-invasively directly in the cultivation system determine even before removal and a defined number of organisms remove.

In a preferred training are used as a cultivation system in standard 96-well microtiter plate format used. This facilitates the semi or fully automatic filling of the Cultivation system and enables the production of the stock cultures in large numbers.

As water-living small and very small organisms For the purposes of this invention, bacteria, algae, Mushrooms, protozoa and small metazoa (multicellular organisms) understood.

Among particle-free cultures in For the purposes of this invention, those are understood that are largely free of non-living particulate Ingredients such as cell debris or nutrient substrate particles.

It is a crucial and surprising property of the present invention that the compartmentalization of the cultivation unit achieves an organism suspension / cell suspension free of cell debris and nutrient substrate particles in the overhead culture chamber: Favored by gravity, cell material that dies in the upright, two-chambered cultivation system sinks so like particulate metabolites into the supply chamber below. Conversely, the particulate nutrient substrate is retained in the lower part due to the small pore size of the separation layer. If the cultivation unit is tilted, for example to remove cell material or during transport, the separating layer or the liquid retained there due to capillary forces prevents the two chambers from mixing. This separation eliminates the need for complex physical steps such as centrifugation or filtration to separate particles from the culture. The modular structure of the cultivation system according to the invention fulfills an essential requirement for the use of these organisms for a number of biochemical / molecular genetic and toxicological questions.

In addition to the main advantage of a of unwanted particulate Contamination free organism suspension / cell suspension in the upper chamber also causes the vertical arrangement of the chambers, that much of it of the organisms present in the culture device in the above lying culture chamber: First, protozoa show one general tendency towards aerotaxis, i.e. themselves along oxygen gradients to orientate yourself and in places higher Oxygen content, i.e. above to accumulate near the breech. And secondly the vertical arrangement uses what is often found in protozoa negative geotactic behavior of the cells leading to movement against gravity and thus to a relative enrichment of cells in the upper part of the liquid column.

The cultivation system according to the invention enables

• - cells several times to be taken sterile from the culture chamber and the removed Volume due to aqueous medium components to replace and thus a semi-continuous cultivation to reach the cells;
• - the Organism density / cell density without prior removal of organism suspension / Determine cell suspension directly in the culture chamber;
• - the Culture chamber with the particle-free organism suspension contained therein / Easily separate the cell suspension from the supply chamber.

The features of the invention go from the elements of the claims and from the description, both individual features and also represent several advantageous designs in the form of combinations, for the protection is requested with this writing. These characteristics exist from elements known per se, in their new combination to the advantageous according to the invention Cultivation systems and which in their entirety result in a synergistic effect, which is that for the first time cultivation systems for extraction particle-free long-term pure cultures of mobile microorganisms or small multicellular cells.

The new cultivation systems according to the invention are suitable for obtaining particle-free long-term pure cultures of Organisms and continue

• - as cuvettes for the direct photometric determination of the organism density / cell density in the culture chamber and as a photometer cuvette of the septum-closed cultivation unit
• - as Cultivation vessels in the 96s Microtiter plate format
• - as Devices for implementing the cultures according to the invention such as
• - to Determination of organism density / cell density without prior removal of organism suspension / cell suspension directly in the culture chamber.

The invention is based on exemplary embodiments are explained in more detail without being limited to these examples to be.

embodiments

According to the invention, the cultivation containers are Can be manufactured and used in several variants. embodiments are shown in the following drawings and are shown in following described in more detail.

Legend to the characters

Show it

1 is a schematic overview of a cultivation device according to the invention with removable screw cap

2 a closure with an inserted pierceable septum

3 an arrangement of cultivation units in 96 microtiter plate format

4 a cultivation unit as a photometer cuvette / schematic overview of a septum-closed cultivation unit designed as a cuvette

5 a cultivation unit consisting of a supply that can be separated from the culture chamber supply chamber / schematic representation of a cultivation unit with a separable culture chamber.

reference numeral

In a preferred embodiment of the cultivation unit according to 1 become both chambers 1 . 2 enclosed by a cylindrical vessel (preferably made of glass or plastic) and by an open-cell foam 3 separated from each other. Open-pore foam (e.g. polyurethane, polyester and polyether foams) is inexpensive and available in almost any pore size. It can be easily adapted to the respective dimensions of the cultivation unit and can be penetrated by the cells if the pore size is selected accordingly. If a layer thickness of the foam of several millimeters is selected and the pore size is a maximum of about two mm, it can also be achieved that even when the cultivation unit is tilted, no components from the supply chamber 2 to the chamber of culture 1 exceed and the organism suspension / cell suspension 5 contaminate because it is in the fine pores of the foam 4 one of the two chambers due to capillary forces 1 . 2 separating, stationary aqueous layer forms practically without flowing movements. At the top, the cultivation unit is sterile, but oxygen-permeable through a stopper or a cap 7 locked. After removing the clasp 7 can the organism suspension / cell suspension 5 can be removed without contamination from cell debris, detritus or nutrient substrate particles. If this is done under sterile conditions, cells can be removed repeatedly. If the removed volume is replaced sterile by aqueous medium components, a semi-continuous further cultivation of the stock culture can be achieved.

An advantageous modification according to 2 the cultivation unit sees oxygen-permeable pierceable septa 9 (e.g. made of silicone or silicone / Teflon) as a closure 7 in front. After piercing the septum 9 can - without opening the cultivation unit - using a sterile syringe 10 multiple organism suspension / cell suspension 5 can be removed and transferred without the need for further precautions for sterile working (e.g. sterile workbench, working on the flame). Likewise, the removed suspension volume or old culture fluid can be replaced with fresh medium components without opening the cultivation unit. Glass tubes such as those used in the field of chromatography have proven their worth here. They are available inexpensively in different versions and are characterized by a (removable) screw cap 8th with inserted silicone / teflon septum 9 out. The septum 9 is permeable to oxygen and can also be pierced several times with a cannula to remove or add liquid without damaging the sterile closure.

In order to achieve a semi or fully automated production of the stock cultures in large numbers, it has proven to be particularly advantageous to use glass or plastic tubes as the cultivation system, which are already available on the market in a 96-well microtiter plate format. 3 shows such an arrangement of tubes. The use of such standard formats offers not only automation, but also a wide range of very different culture vessels and also allows the acquisition and production costs to be kept very low.

Another preferred embodiment is according to 4 in that the cultivation unit is designed as a photometer cuvette. In this way, an accurate and non-invasive determination of the organism density / cell density is carried out in advance in the culture chamber 1 allows. For this purpose, the cultivation unit is introduced into a photometer in such a way that the light beam passes through the culture chamber 1 with the organism suspension / cell suspension contained therein and free from interfering particles 5 falls. Based on the optical density (or the extinction value) at a given light wavelength and a calibration curve, the exact organism density / cell density in the culture chamber can be determined 1 calculate and extract a defined number of organisms by taking a certain volume. In principle, any light wavelength can be used for the determination, however, the relationship between optical density and organism density / cell density (calibration curve) must be known. Used as a clasp 7 a pierceable septum 9 selected, cells can also be used under non-sterile room conditions, for example using a sterile syringe 10 Taken several times and sterile in precise numbers become.

As an alternative to the embodiment in which both the culture and supply chamber are firmly enclosed by a container, has according to 5 an inventive design of the cultivation system in which the culture chamber 1 with the organism suspension / cell suspension contained therein 5 from the supply chamber 2 can be removed. At the in 5 illustrated embodiment, two tubes are inserted into each other, the bottom of the culture chamber 1 the interface 3 to the supply chamber 2 forms and a hole 11 in the floor of the culture chamber 1 (upper tube) the connection to the supply chamber 2 manufactures.

The invention is not restricted to the illustrated embodiments. Rather, the culture chamber, the separating layer, the supply chamber and the closure devices can be modified depending on the application. For example, the volume of the supply chamber 2 be designed to be many times larger than the volume within the cultivation chamber 1 , Furthermore, in deviation from the three-dimensional structure of the open-pore separation layer, a two-dimensional network-like matrix can occur. Further modification options relate to the shape of the chambers and the cultivation unit and the selection of the closure device.

Claims (7)

Cultivation systems for the production of particle-free Long-term pure cultures of aquatic micro and small organisms, consisting of two on top of each other arranged chambers, the lower chamber as a supply chamber with solid nutrient substrate filled and the upper chamber represents a culture chamber that acts as a reservoir particle-free organism suspensions and through both chambers an open-pore physical barrier is separated. Cultivation systems according to claim 1 as devices for obtaining particle-free long-term pure cultures of aquatic species Micro and small organisms - bacteria, Algae, fungi, protozoa or small metazoa -, characterized, that they are two vertically on top of each other arranged chambers exist, of which the top - sterile, but permeable to oxygen outward locked up - cultural chamber represents the organism reservoir and the lower represents the supply chamber. Cultivation systems according to claim 1 and 2, characterized characterized them 3.1. as a one-piece cultivation system form a single cultivation unit and 3.2. as a two-part Cultivation system exist, with both chambers completely apart be separated. Cultivation systems according to claims 1 to 3, characterized characterized in that the cultivation unit as a cuvette for direct photometric determination of the organism density in the Kulturkammer is designed. Cultivation systems according to claim 1 to 4 thereby characterized in that the oxygen-permeable closure from a pierceable septum. Cultivation systems according to claim 1 to 5 thereby characterized that they from cultivation vessels in 96 microtiter plate format consist. Cultivation systems according to claim 1 to 5 thereby characterized that they consist of standard glass jars with screw caps and inset septum.