GB2269391A - Apparatus for cultivating microorganisms - Google Patents

Abstract

Apparatus for cultivating microorganisms comprising a baseplate including culture media retaining cavities, wherein each cavity is sealed by means of a flexible membrane permeable to oxygen and which prevents the escape of culture medium.

Description

APParatus for Cultivating Microorganisms The present invention relates to apparatus for cultivating microorganisms in microtitre plates, in which the microtitre plates are sealed by membranes which on the one hand prevent liquid from escaping and on the other hand are permeable to oxygen.

The use of microtitre plates for cultivating microorganisms when testing numerous cultures is known and a number of publications deal with their use in the qualitative and quantitative detection of microorganisms or substances produced by microorganisms.

FR-A-2556741 describes the use of microtitre plates for detecting the effect of certain substances on microorganisms or mixtures of microorganisms, in which the microorganisms are present in a lyophilic medium and the substance acting thereon is added to the culture medium. The microtitre plate is then incubated at an appropriate temperature. Microtitre plates for antibiotic sensitivity tests which are stored at low temperatures, e.g. -70 C, in order to maintain the stability of the antibiotics, are described by P.L.

Kuglin Bailey and M.B. McGuckin in Am. J. Med. Technol 45 (6): 517-522 (1979).

DE-OS-3336738 also describes microtitre plates for use in antibiotic sensitivity tests. The microtitre plates have exchangeable modules with cavities, the cavities being capable of absorbing specific volumes, e.g. of suspensions of bacteria, and having openings for the exchange of gases.

German Utility Model Application No. G 91 07 670.6 discloses modified microtitre plates of a known composition having a transparent polymer coating containing a plurality of epoxy groups on their surface, for fixing biologically significant materials containing amino or sulphohydride groups. US-A-4680269 describes microtitre plates the cavities of which are covered with a porous coating impregnated with an acidic component.

This coating is capable of neutralising gaseous basic products such as ammonia which may be formed during the growth of bacterial cultures, thereby avoiding errors in pH-sensitive biochemical tests.

When cultivating microorganisms, during the screening phase it is frequently necessary to test a very large number of cultures. Accordingly, every effort is made to achieve high cell densities or high biomass concentrations in the individual cultures, particularly when the formation of a desired product of cell metabolism, e.g. a fermentation product, is dependent on the biomass concentration in the culture. In aerobic processes, for example, the formation of biomass depends primarily on the quantity of oxygen introduced into the culture. In order to achieve high biomass concentrations, Erlenmeyer flasks with chicanes are generally used which are incubated on shaking apparatus.

Typically, the shaking flasks have a total volume of 100 ml and are filled with about 20 ml of culture medium. Compared with a miniaturised setup the production of a large number of shaking flask cultures is time consuming and expensive in terms of materials.

If microtitre plates according to the prior art are used in the aerobic processes described above, the biomass is first formed on the surface of the culture medium in the cavities and in this way further diffusion of oxygen into the interior of the culture medium is inhibited.

This diffusion barrier can be partly overcome by shaking, whilst, in the normal two-dimensional shaking technique, the permeation of gas is only slightly improved by the high surface tensions and by the concentration of the specifically lighter biomass on the surface of the culture medium.

There is still therefore a need to be able to improve the permeation of gas into the cavities of microtitre plates during cultivation of microorganisms, whilst at the same time miniaturising the setup of the cultures.

We have been able to achieve an improvement in this respect.

Thus, the present invention provides apparatus for cultivating microorganisms comprising a baseplate including culture media retaining cavities, wherein each cavity is sealed by means of a flexible membrane permeable to oxygen and which prevents the escape of culture medium.

The baseplate is preferably agitated at constant temperature for the purposes of intensive mixing and hence for achieving a satisfactory concentration of oxygen in the cavities. The flexible membrane preferably comprises a plastics material having a permeability for oxygen, expressed by the gas permeation coefficient P of more than 10-9 cm3

( 2 cm ), cmZ.s.Torr and more preferably comprises a polymer consisting of siloxane units (ie. an organopolysiloxane).

Preferred embodiments of the invention will now be described by way of example and with reference to the accompanying Figure 1 which shows a plan view of a microtitre plate (1) according to the invention.

The microtitre plate (1) consists of a variable number A of cavities (2). Usually, the cavities (2) and the baseplate (2a) consist of a transparent material, preferably plastics, and the cavities (2), which are preferably cylindrical, are open at one end. The number A of cavities (2) is generally between 12 and 96 per baseplate. After the cavities (2) have been filled with the liquid culture medium, e.g. microorganisms in an aqueous nutrient solution, the open ends of the cavities are covered with a flexible membrane (4) permeable to oxygen.The oxygen permeability of the membrane (4) may be expressed, for example, by the gas permeation coefficient P which is inserted in Fick's transport law: N = P.F.p.t , (I) d wherein N = the quantity of gas passing through the membrane (4) P = gas permeation coefficient F = surface area of passage ap = vapour pressure difference of the gas t = transit time and d = thickness of the membrane (4).

Preferably, the flexible membrane (4) comprises a plastics with a gas permeation coefficient P of more than 10-9 cm3

cm cm2. s . Torr whilst it is particularly preferred to use plastics which are corrosion-resistant in the presence of the culture medium (see for example Vieweg/Braun, Kunststoff-Handbuch, Volume 1, pages 936 ff, Carl Hanser, Munich, 1975).

Plastics which may be used for the membrane (4) include, for example, polystyrene, poly(meth)acrylates, polycarbonate, polyethylene having a density of less than 0.94 g/cm3, styrene-butadiene-rubber and especially organopolysiloxanes such as for example dimethylpolysiloxane.

The membrane (4) must be sufficiently flexible that any small perforations which may occur as nutrient is supplied into individual cavities (2) by means of syringes will close up immediately after the syringe has been withdrawn and consequently no culture medium can escape.

In order to fix the membrane (4), a fixing plate (3), conveniently made of the same material as the baseplate (2a) and about 25 to 50% larger than the membrane, is placed over the membrane (4). The fixing plate (3) is provided with bores (3a) which enable the individual cavities (2) to be supplied with nutrient liquid, cultures or other ingredients of the culture medium by means of a syringe. The fixing plate (3) is connected to the baseplate (2a) by screw connections (6), whilst the brackets (5) may be used for additionally fixing the membrane (4) and fixing plate (3) on the cavities (2).

The microtitre plate (1) thus assembled is preferably agitated in a medium at constant temperature in such a way that the liquid culture media are thoroughly mixed in the cavities (2). Preferably, air is used as the tempering medium and this air is heated, by means of a heating thermostat, to the required temperature, preferably between 30 and 60"C, and circulated in an incubator. The incubator preferably consists of a transparent housing which is preferably made of plastics and, more particularly, of a polymethylmethacrylate (Plexiglas@). The microtitre plates (1) are mounted on a retaining frame which is located on a motor-driven shaft. The intensive mixing of the liquid culture medium in the cavities (2) is carried out by a rotating movement of the microtitre plates (1) about the shaft.

EXAMPLE The microtitre plates (1) used consist of polystyrene (Costar@, made by Falcon) and have 12 to 96 cavities (2). The membrane (4) consists of a film of crosslinked polydimethylsiloxane and has a thickness of 1 mm.

The fixing plate (3) and baseplate (2a) are polymethylmethacrylate plates 5 mm thick, the dimensions (length, width) of which are approximately 25% larger than the microtitre plate (1), whilst the number and position of the bores (3a) correspond to those of the cavities (2) on the microtitre plate (1).

The shaft to which the microtitre plate (1) is fixed is driven by an electric motor. The incubator consists of transparent polymethylmethacrylate (PlexiglasQ), whilst the required temperature in the incubator, which is to be maintained at between 30 and 60"C, preferably between 35 and 40or, is adjusted by means of a heating thermostat with circulation of air.

With the apparatus according to the invention, consistently high oxygen concentrations are maintained in the liquid culture media contained in the cavities (2), by intensive mixing of the culture media and by the constant replacement of the used-up oxygen through the flexible membrane (4). In addition, the intensive mixing prevents the biomass from settling on the surface of the culture liquid, which is another cause of inhibiting oxygen diffusion and hence the biomass formation. In addition, in screening tests, the small volume of the cavities (2) in the microtitre plates (1) is advantageous, since the quantity of culture medium required can be reduced significantly. Furthermore, the production of a number of shaking flask cultures is extremely time consuming and expensive in terms of material, compared with the miniaturised setup according to the invention.

Claims (9)

1. Apparatus for cultivating microorganisms comprising a baseplate including culture media retaining cavities, wherein each cavity is sealed by means of a flexible membrane permeable to oxygen and which prevents the escape of culture medium.

2. Apparatus as claimed in claim 1 wherein said flexible membrane is held in position by means of a fixing plate provided with bores, the number and position of said bores corresponding to those of the cavities.

3. Apparatus as claimed in claim 1 or claim 2 wherein said flexible membrane comprises a plastics material having an oxygen permeability P of more than 10-9 cm3

cm cm2.s.Torr

4. Apparatus as claimed in claim 3 wherein said plastics material comprises a polystyrene, poly(meth)acrylate, polycarbonate, polyethylene, styrene-butadiene rubber or an organopolysiloxane.

5. Apparatus as claimed in claim 4 wherein said organopolysiloxane is dimethylpolysiloxane.

6. Apparatus for cultivating microorganisms substantially as herein described with reference to the Example and/or Figure 1.

7. A method for cultivating microorganisms comprising introducing said microorganisms into one or more culture media retaining cavities included in a baseplate, each cavity being sealed by means of a flexible member permeable to oxygen and which prevents the escape of culture medium.

8. A method as claimed in claim 7 wherein cultivation is assisted by agitating said baseplate.

9. A method as claimed in claim 7 substantially as hereinbefore described with reference to the Example and/or Figure 1.