WO2022067369A1 - Incubation chamber - Google Patents

Abstract

The invention relates to an incubation chamber, comprising a base plate (1), a stamp plate (2), and a lid (3), wherein: the base plate (1) has a bottom (1') and side walls (1''); the bottom (1') and the side walls (1'') form a chamber (5); the bottom (1') has at least one recess (6), which is preferably circular; the stamp plate (2) has at least one stamp (2'); the at least one stamp (2') can be pushed through the at least one recess (6) in the bottom; the height of the at least one stamp (2') is preferably greater than the height of the side walls (1''); the chamber (5) formed by the base plate can be closed without contamination using the lid (3) for air-permeable closure. The invention also relates to a method for incubating sample cultures using an incubation chamber of the invention, and for microscopy of an incubated sample culture.

Description

INCUBATION CHAMBER

The present invention relates to an incubation chamber comprising a base plate, a stamp plate and a cover. Furthermore, the invention relates to a method for incubating or microscopy a sample with an incubation chamber according to the invention.

BACKGROUND OF THE INVENTION AND PRIOR ART Incubation chambers are used to cultivate and propagate bacteria and other microorganisms such as filamentous fungi on suitable media. This allows controlled external conditions to be created and maintained for various development and growth processes. An incubation chamber allows the creation and maintenance of a microclimate with tightly controlled humidity and temperature conditions.

The usual pre-cultivation of microorganisms takes place on agar nutrient medium in standard Petri dishes made of disposable polystyrene (PS). To prepare samples of filamentous fungi for live cell microscopy, a section of the colony approx. 2 - 4 cm ² in size is usually cut out with a scalpel and inverted onto a coverslip. The advantages of this method are the very high resulting microscopic quality and the possibility of making long-term observations of up to 2 hours with the same sample due to the relatively large available medium volume of approx. 1.2 - 2.4 mL. After more than 2 hours, however, the sample begins to dry out quickly and is lost. The cut injury and the mechanical stress when transferring from the Petri dish to the coverslip generate significant cell stress, which becomes apparent under the microscope, for example, from the momentarily altered growth behavior of fungal filaments (hyphae). In addition, the natural organization of the colony network (mycelium) is destroyed and the reactions of an intact colony can no longer be examined. In addition, the sample cannot be reused, i.e. it cannot be microscopically examined, further incubated and microscopically examined again at a later point in time. Furthermore, the cultured colony from which the sample was taken also remains damaged.

Stress-free sample preparation is very often essential for live cell microscopy. The preservation of the natural, stress-free state, for example of a fungal colony, is generally important for all investigations, especially if a desired stress is to be explicitly induced and the cellular reaction is to be observed. One way of stress-free sample preparation is to pour small amounts (approx. 0.15 - 0.5 mL) of agar nutrient medium onto microscopy cover slips. However, the layers of culture medium that can be generated are too thin to survive long-term incubations and often result in an uneven or slightly convex surface. Due to the very small media volume, the colony size that can be cultivated is also severely limited. Due to the rapid drying out on the microscope, no long-term or repeated observations can be carried out. In addition, sterile handling is complicated. Other disadvantages of this method of freehand pouring are limited reproducibility due to non-standardization and the poor resulting optical quality of the microscopic images.

BRIEF DESCRIPTION OF THE INVENTION

The object of the present invention is therefore to provide injury-free sample preparation without mechanical stress for live-cell microscopy of microbial cultures, in particular of filamentous fungi.

This object is achieved by an incubation chamber comprising a base plate, a stamp plate and a cover, the base plate having a floor and side walls which together form a chamber. In addition, the base has at least one preferably circular recess and the stamp plate has at least one stamp. The at least one stamp can be pushed through the at least one recess in the floor and the height of the at least one stamp is preferably greater than the height of the side walls of the base plate. The chamber formed by the base plate can be closed with the cover.

The lowered edge of the lid of the incubation chamber is used for contamination-free incubation of a sample. The sample can be removed from the chamber using the stamp plate. The stamp plate thus enables the sample to be removed from the incubation chamber without causing injury, without subjecting the sample to mechanical stress.

Stamps and recesses can have different shapes, it always having to be the case that the at least one stamp passes through the at least one recess in the floor can be pushed. If there are several stamps and recesses, the stamps must be arranged on the stamp plate in such a way that all stamps can be pushed into a recess when the base plate is placed on the stamp plate.

Stamps and recesses can essentially have the same basic shape. In the case of circular recesses, the stamps can then have a cylindrical shape, with the diameter of the cylindrical stamps being slightly smaller than the diameter of the circular recesses, so that the stamps fit into the recesses. However, the recesses and stamps can also have any other shape and do not have to be the same in their basic shape. More precisely, the stamps can have a cylindrical shape, for example, and the recesses can be square or vice versa, with the stamps in turn fitting into the recesses so that they can be pushed through them.

In addition, the incubation chamber may include a microscopy coverslip, wherein the microscopy coverslip is substantially congruent with the inner surface of the bottom of the base plate. Therefore, the microscopy cover slip can be placed in the chamber formed by the base plate.

After the microscopy cover glass has been placed in the chamber, the at least one recess in the bottom of the base plate is thus covered. Furthermore, a defined layer of nutrient medium, preferably an agar nutrient medium, can then be applied to the cover glass in a standardized manner. Preferably the layer is between 3 and 8 mm thick. This allows a culture medium volume of up to 30 mL. Microcolonies of filamentous fungi with a diameter of preferably 1 to 2 cm can be cultivated on the layer of nutrient medium. Due to the large reservoir of culture medium, long-term observations of the sample colonies can be carried out macroscopically and microscopically for more than 96 hours. Furthermore, the sample dries out much more slowly compared to other sample preparation methods on the microscope. More specifically, it can be observed continuously for up to 12 hours without any significant shift in the focal plane. The stability of the sample and the resulting possible duration of long-term observation can be further improved by placing a humidity chamber on top. In addition, by being able to continue an incubation after examining the sample, even repeated long-term observations can be made. Since the at least one stamp protrudes over the lateral walls of the chamber, ie the stamp has a length which is greater than the height of the lateral walls of the chamber, the sample on the microscopy cover slip can be rotated slightly and then lifted. The cover glass with the sample culture on it can thus be removed in a simple manner and transported to a microscope. It is also possible to put the sample culture and cover glass back into the incubation chamber after an examination and continue incubating. The resetting in turn works via the stamp plate, on which at least one stamp the cover glass can be placed. By slowly lifting the base plate from the stamp plate, the cover glass with the culture on it is lowered into the chamber again without injuring it. At a later point in time, the sample can then be removed again in reverse order using the stamp plate and examined. Thus, the incubation chamber according to the invention allows an injury-free and thus cell-stress-free preparation and multiple macroscopic and microscopic observation of a sample.

In a further embodiment, the base plate, the stamp plate and the cover are made of plastic, preferably polylactic acid (polylactic acid, PLA), glycolized polyethylene terephthalate (PETG), acrylonitrile butadiene styrene (ABS) or acrylonitrile styrene acrylate (ASA). These plastics offer different advantages in terms of shape, surface and function options, as well as temperature and UV resistance and environmental friendliness. For example, the incubation chamber is manufactured using a 3D printing process. As a result, different dimensions and designs of the incubation chamber according to the invention and of the microscopy cover glass can also be very easily adapted as required and immediately implemented as a 3D print. Starting from the basic model, other functional changes and user-specific requests can also be easily implemented. For example, the incubation chamber can be adapted to the special needs of bacteria, yeast or plant seedlings.

Another method for producing the incubation chamber according to the invention is by means of 3D milling. Preferred materials for this are the plastics polypropylene (PP) and polyetheretherketone (PEEK) or also glass ceramics such as Macor™ or Shapal™. In addition, the production can also be carried out by 3D lithography, with polyphenylsulfone (PSU) or photopolymer synthetic resins being preferred as materials. Furthermore, the incubation chamber according to the invention is very easy to clean and to sterilize, for example, using ethanol and UV radiation.

Another advantage is that all parts of the incubation chamber can be reused many times. Therefore, there is much less plastic waste than with disposable plastic Petri dishes, which are used as standard for mushroom cultivation. The use of PLA is particularly environmentally friendly: PLA is a bioplastic that is obtained from renewable resources such as potato or corn starch and can be easily disposed of and is biodegradable. Of course, this does not apply to petroleum-based types of plastic such as PS or ABS/ASA.

In addition, the base plate, the stamp plate and the cover can also be made of autoclavable plastic or synthetic resin. The ability to be autoclaved allows the incubation chamber according to the invention to be used in scientific laboratories with a high biosafety standard and for clinical applications.

Further material alternatives for the incubation chamber according to the invention are plastics that can be milled, such as polypropylene (PP) or polyetheretherketone (PEEK), as well as photopolymeric resins, such as monomeric styrenes, acrylates or methyl acrylates, and ceramics. All three groups of materials enable the microfabrication of complicated shapes and functions due to their very high level of detail. Application examples include e.g. For example, at least one diffusion barrier can be applied to the cover glass. Diffusion barriers can be used to spatially control chemical communication between co-cultured samples. This means that two different sample cultures can be incubated with just one cover glass at the same time, which can only interact with each other in certain areas, to a limited extent or not at all.

In a preferred embodiment, the cover of the incubation chamber has support corners. The corners of the side walls of the base plate facing away from the ground can rest on these support corners. This makes the chamber permeable to air and allows aerobic incubation of the sample cultures. In a further embodiment variant, the cover can be designed to be essentially transparent. On the one hand, this makes it possible to view the growing cultures with the naked eye without having to lift the lid. On the other hand, there is a transparency that promotes the natural development and production quality of the sample cultures during incubation.

In addition, the base plate can also be designed in different colors. This allows defined contrasts for visual assessment of the growing cultures of differently colored microorganisms with the naked eye.

In one embodiment, the cover of the incubation chamber according to the invention can also include a light filter. These light filters could be designed in such a way that they only let through light with a certain wavelength. Thus, any test sample can be easily incubated using the same incubation chamber and different lids with just a single white light source, since the integrated light filters allow selective passage of light qualities and quantities. Light plays a crucial role in cell function, development and thus breeding and experimental study of microorganisms and plants. This would also eliminate the costly use of different monochromatic light sources or the complex construction of incubation chambers for individual light wavelengths. Since the production quality and quantity of the test samples depends very much on the given light quality and quantity, this simple incubation using a filter cover results in significant advantages. Light could thus be controlled more specifically as an experimental factor, and the cellular effects of different light conditions could be studied directly using live cell microscopy. Also for biotechnological process optimization, e.g. the light dependence in the production of antibiotics, standardized cultivation and examination methods in which the light factor can be specifically controlled are decisive.

Furthermore, the surface of the base plate can be made very smooth by selecting a suitable material and manufacturing process or by applying a non-stick coating. A smooth surface prevents menisci from forming on the side walls of the base plate after the culture medium has been poured into the Chamber inlaid coverslip. These menisci can impair or make impossible microscopy at the edge of the sample culture. By avoiding edge mensics, the incubation chamber according to the invention produces a nutrient medium block with a smooth and completely even surface, so that microscopy can be carried out over the entire surface.

A further aspect of the present invention relates to a method for incubating sample cultures with an incubation chamber according to the invention. The procedure includes the steps

• Insertion of a microscopy cover glass in the base plate,

• Pouring a nutrient medium onto the cover slip, preferably between 10 and 30 mL being poured in,

• Application of a sample culture to the solidified nutrient medium,

• Place the lid on the base plate and incubate the sample culture under the desired temperature, humidity and light conditions.

The method for generating a sample culture with the incubation chamber according to the invention enables a simple pouring of a block of nutrient medium with a smooth and level surface in a standardized chamber. The closed lid allows the samples to be incubated in the incubation chamber without contamination or drying out.

In addition, the invention also relates to a method for microscopy of a sample culture incubated in an incubation chamber according to the invention, the sample culture being applied to a microscopy cover glass which lies in the chamber formed by the base plate, and the sample culture together When the lid is open, the microscopy cover glass is pushed up using the stamp plate and can thus be removed.

With the incubation chamber according to the invention, the sample can be easily transported and just as easily removed by means of the stamp plate. The preparation of individual samples in individual chambers also ensures their safe and cross-contamination-free storage and transport, even in a container. In summary, the incubation chamber according to the invention allows a method which includes the contamination-free preparation, removal and renewed incubation of a sample culture before and after a microscopic examination.

A further variant provides for the application of an additional cover glass of the same size after removal of the sample culture together with the microscopy cover glass from the incubation chamber. Depending on the size and surface condition of the sample culture, approx. 1 - 2 mL of sterile liquid medium or physiological saline solution are preferably dropped onto the sample and then the second cover glass is placed. Using this glass-broth+culture-glass sandwich construction, the sample can then be placed inverted on a microscope stage, making it suitable for use on an inverted microscope. Thus, the method can be used equally for upright and inverted microscope configurations. After microscopy, after carefully removing the second cover glass, the sample can be put back into the chamber using the stamp plate and incubated further. The short-term mechanical stress on the fungal culture caused by the application and repeated removal of the second coverslip is usually well tolerated.

The sandwich method according to the invention therefore allows recordings with transmitted light, differential interference contrast or other contrasting optics, as well as fluorescence microscopy. Alternatively, the use of the second coverslip can be avoided altogether by using immersion objectives and an upright microscope. This approach also ensures optimal optical quality of the recordings, since there is no cover slip between the sample colony and the objective lens.

DETAILED DESCRIPTION OF THE INVENTION

In order to better illustrate the invention, the essential features are shown in the following figures on the basis of preferred embodiments of the incubation chamber according to the invention.

Show it:

1 and 2 show a plan view and a sectional view, respectively, of the base plate of the incubation chamber according to the invention.

3 and 4 are top and front views of the stamping plate of the incubation chamber according to the present invention.

5 and 6 show a bottom view and a sectional view of the incubation chamber cover according to the invention.

7 shows a sectional view of the incubation chamber with the lid closed and the stamp plate pushed in.

8 shows a sectional view of the incubation chamber with the lid closed, inserted microscopy cover slip with poured agar nutrient medium and without stamp plate.

9 shows a sectional view of the incubation chamber without a cover, with the stamp plate pushed in and the microscopy cover glass raised with agar nutrient medium poured on.

10 shows a contrast-inverted living cell fluorescence microscopy example of a fungal co-culture which was prepared and microscopically examined with the aid of the incubation chamber according to the invention.

The incubation chamber according to the invention comprises the base plate 1, the plunger plate 2 and the cover 3 as essential components, as shown in FIGS. 1, 3 and 5. As can be seen in FIGS. 1 and 2, the base plate 1 is composed of a floor 1' and side walls 1". The base 1' and the lateral walls 1'' form a chamber 5 (see FIG. 2), the base 1' preferably having circular recesses 6.

As can be seen in FIG. 4, the stamp plate 2 has at least one stamp 2'. In the embodiment variant shown in FIGS. 3 and 4, the stamp plate 2 has five stamps 2'. The stamps 2' are designed in such a way that they can be pushed through the recesses 6 in the bottom 1' of the base plate. If stamp 2 'and recesses 6 circular are formed, the diameter of the punches 2' can be slightly smaller than the diameter of the recesses 6. The stamps 2' can thus be pushed into the chamber 5 on the underside of the base plate 1. However, the stamps 2' and recesses 6 are not limited to the shapes shown in the figures and can have any basic shape. The cross-sectional area of the stamps 2' does not necessarily have to match the area of the recesses 6, as long as the stamps 2' can be pushed through the recesses 6. For example, the recesses 6 can be circular and the stamps 2' have a rectangular or square cross-sectional area.

The height of the at least one stamp 2' is preferably greater than the height of the side walls 1", so that the stamps 2' protrude slightly beyond the side walls 1" after they have been pushed completely through the recesses 6.

As shown in FIGS. 5 and 6, the lid 3 preferably has support corners 3'. The cover 3 allows the chamber 5 formed by the base plate to be closed without contamination but still permeable to air. This is an essential prerequisite for successful incubation of sample cultures. One way to design the cover 3 accordingly is by using the support corners 3'. In addition, as can be seen in FIG. 6, the cover 3 has a pulled-down edge 3″ which partially extends over the lateral walls 1″ of the base plate 1. The rim 3" enables a contamination-free incubation of the sample with the lid closed. In addition, the 3" edge prevents the lid 3 from slipping when the incubation chamber is transported.

7 shows an embodiment of the incubation chamber according to the invention, wherein the base plate 1, stamp plate 2 and cover 3 are assembled. This means that the stamps 2' of the stamp plate 2 have been pushed through the recesses 6 in the bottom of the base plate 1', so that the stamp plate 2 is located directly under the base plate 1. The cover 3 is designed in such a way that it allows the chamber 5 to be closed despite the plunger 2'.

If the stamp plate 2 is not connected to the incubation chamber, a microscopy cover glass 4 can be inserted into the chamber 5, as shown in FIG. The microscopy cover glass 4 is preferably essentially congruent with the inner surface of the bottom of the base plate 1'. This prevents the cover glass 4 from slipping and makes it easier to pour a nutrient medium onto the cover glass 4, since the edges of the cover glass 4 connect directly to the side walls of the 1" base plate. As can also be seen in FIG. 8, the inserted microscopy cover glass 4 closes the recesses 6 in the bottom of the base plate 1'. Using the cover glass 4 in the chamber 5, the nutrient medium can simply be poured in and form a flat surface. In addition, a large amount of nutrient medium can be poured.

In one embodiment, the cover 3 of the incubation chamber is transparent, so that the sample can be viewed through the cover 3 during the incubation shown in FIG. 8 .

In a further embodiment, the cover 3 can comprise light filters. The light filters only let through light with a certain wavelength. Therefore, test samples can be incubated with the incubation chamber according to the invention with a single white light source, since the covers 3 allow different light qualities and quantities. Expensive additional light sources are not necessary for this.

When the cover is open, the microscopy cover glass 4 can be lifted out of the chamber 5 with the aid of the stamping plate 2, as shown in FIG. After that, the cover glass 4 can be transported onto a microscope table, for example, for further investigations. To make it easier to remove the cover glass 4 from the stamps 2', the cover glass 4 can be rotated slightly beforehand so that its edges no longer run parallel to the edges of the side walls of the base plate 1". Conversely, the cover slip 4 can also simply be placed back into the base plate 1 after microscopy by first guiding the stamps 2' of the stamp plate 2 through the recesses 6 in the base of the base plate 1', so that the stamp plate 2 is connected to the base plate 1 . Then the cover glass 4 can again be placed on the stamp 2' and turned in such a way that the edges of the cover glass 4 again run parallel to the edges of the lateral walls of the base plate 1". The base plate 1 is then slowly lifted upwards so that the plungers 2' move out of the recesses 6 and the cover glass 4 is slowly guided back towards the bottom of the base plate 1'. After the cover glass 4 is again lying on the bottom 1', the lid 3 can be put on and the sample can be further incubated.

The incubation chamber according to the invention thus enables a very simple and error-free incubation of microbial cultures. In addition, the production of Incubation chamber very inexpensive and fast. The incubation chamber can be reused, which significantly reduces the environmental impact. In addition, the incubation chamber saves a considerable amount of time when examining the sample under the microscope due to the large reservoir of culture medium. This allows both long-term observations and repeated observations of the sample to be carried out. After microscopy, the sample on the coverslip can be placed back into the incubation chamber and incubated further. Due to the numerous possible embodiments of the incubation chamber, it is also possible to respond very specifically to the most varied of requirements in the incubation and in the microscopy of cultures in living cell microscopy. For example, the optical quality of the recordings ultimately achieved can therefore also be optimized with the aid of the incubation chamber according to the invention.

EXAMPLES

The incubation chamber according to the invention can be used, for example, to investigate fungal-fungal interactions, such as between the mycroparasite Trichoderma atroviride (Ta) and its prey fungus Botrytis cinerera (Bc). In the process, microcolonies of both fungi grow towards one another in the chamber 5 . In the contact zone, there is a mycroparasitic interaction, which can then be examined without stress or injury by lifting the sample co-culture out of the chamber 5 using the stamp plate 2, for example by means of fluorescence microscopy. In Fig. 10 is the interaction zone of the two fungi Ta and Bc, which are treated with a Plan Apo 20x 0.75 N.A. Objective imaged on a Nikon Eclipse Ti2-E brightfield inverted fluorescence microscope with subsequent deconvolution. On the right-hand side in FIG. 10, the area marked with a square on the left-hand side is shown enlarged.

Claims

EXPECTATIONS Claims 1. Incubation chamber comprising • a base plate (1), • a stamp plate (2) and • a cover (3), the base plate (1) having a bottom (1') and side walls (1"), the bottom (1') and the side walls (1") forming a chamber (5), the base (1') having at least one preferably circular recess (6), the stamp plate (2) having at least one stamp (2'), the at least one stamp (2') passing through the at least one recess (6) in the The height of the at least one plunger (2') is preferably greater than the height of the lateral walls (1"), the chamber (5) formed by the base plate being closable with the cover (3). 2. Incubation chamber according to Claim 1, characterized by a microscopy cover glass (4), the microscopy cover glass (4) being essentially congruent with the inner surface of the bottom of the base plate (1'), the microscopy cover glass (4) being in the chamber (5) formed by the base plate can be inserted. 3. Incubation chamber according to one of claims 1 or 2, characterized in that the base plate (1), the stamp plate (2) and the cover (3) are made of plastic, the plastic preferably being from the group consisting of polylactic acid (PLA), glycolized polyethylene terephthalate (PETG), acrylonitrile butadiene styrene (ABS) or acrylonitrile styrene acrylate (ASA) is selected. 4. Incubation chamber according to one of claims 1 or 2, characterized in that the base plate (1), the stamp plate (2) and the cover (3) made of a material from the group consisting of polypropylene (PP), polyetheretherketone (PEEK) or Includes glass-ceramics is chosen. 5. Incubation chamber according to one of claims 1 or 2, characterized in that the base plate (1), the stamp plate (2) and the cover (3) are selected from a material from the group comprising polyphenylsulfone (PSU) or photopolymeric synthetic resins will. 6. Incubation chamber according to one of claims 1 to 5, characterized in that the base plate (1), the stamp plate (2) and the cover (3) are made of autoclavable plastic or synthetic resin. 7. Incubation chamber according to one of claims 1 to 6, characterized in that the cover (3) has support corners (3'). 8. incubation chamber according to any one of claims 1 to 7, characterized in that the cover (3) is designed to be substantially transparent. 9. Incubation chamber according to one of claims 1 to 8, characterized in that the cover (3) comprises light filters. 10. Incubation chamber according to one of claims 1 to 9, characterized by microchannels and/or diffusion barriers. 11. Incubation chamber according to one of claims 1 to 10, characterized in that the surface of the base plate (1) has a non-stick coating. 12. A method for incubating sample cultures with an incubation chamber according to any one of claims 1 to 11, comprising the steps • Insertion of a microscopy cover glass (4) in the base plate (1), • Pouring a nutrient medium onto the microscopy cover slip (4), preferably between 10 and 30 mL being poured in, • Application of a test culture, preferably a test co-culture, to the solidified nutrient medium, • Place the lid (3) on the base plate (1) and incubate the sample culture, preferably a sample co-culture, at the desired temperature, humidity and light. 13. Method for microscopy of a sample culture incubated in an incubation chamber according to one of claims 1 to 11, wherein the sample culture is deposited on a solid nutrient medium on a microscopy cover glass (3) which is in the chamber (5) formed by the base plate. lies, is applied, with the sample culture including microscopy 15 Cover glass (4) and culture medium is pushed up with the lid (3) open using the stamp plate (2) and can be removed in this way, whereby the microscopy cover glass (4) can be placed on a microscope table and examined under the microscope after removal. 14. The method according to claim 13, characterized by the application of an additional, substantially dimensionally identical microscopy cover glass (4) after removal of the sample culture together with the microscopy cover glass (4) from the incubation chamber.