CONTAINERS FOR IMPROVED GRINDING AND HOMOGENIZATION, AND MIXING

Non-cylindrical and non-standard shaped containers for improved grinding and homogenization with grinding beads of biological samples for DNA, RNA, Protein and Lipid Extraction, and mixing of biological samples.

PATENT (PCT) APPLICATION

Inventor: Kosier; Dr. Branko Bob (Hermann-Dorfner-Stra e la, 92253 Schnaittenbach, Germany - formerly Lemiers, NL)

Applicant: Kosier; Dr. Branko Bob (Hermann-Dorfner-Stra e la, 92253 Schnaittenbach, Germany - formerly Lemiers, NL)

Appl. No.:

DESCRIPTION of the INVENTION:

[0001] Conventional containers have an interior space with a circular cross-section. Filled with liquid or particles such containers when subjected to a rotational movement cause the liquid to form a vortex with a funnel-shaped centre i.e. a whirlpool or plughole vortex forms. The individual sample components within the liquid move in unison so that the sample particles or sample components collide or impact with each other with reduced energy. In the case of grinding beads the beads act like pearls on a string and there is minimal contact among the beads and sample. (Fig. 1A).

[0002] Without providing scientific proof for the beneficial effects of the vessels according to the invention, it is assumed here that the asymmetric interior design causes a turbulent flow which transfers the motion and energy to the mixing or grinding and pulverizing of the sample material (Fig. IB).

[0003] For the invention to be effective the lower, that is, bottom-side region of the vessel must have a non-circular inner cross-section the minimum being at least one corner or angle. This design is such that when a circular horizontal movement is applied to the upright vessel the contents contained in the vessel are set in motion in a turbulent manner.

This is the area which corresponds to the mixing or crushing area or function of the asymmetric vessel shape when filled with samples and liquid in the upright position of the vessel

[0004] The dimensions (size and length) of the asymmetric region can be selected to correspond to the level of contents normally used to fill the vessel.

[0005] In one embodiment, the asymmetric region comprises at least 15%, preferably at least 33% and in particular preferably at least 99% of the vessel length.

[0006] In the case of the most commonly used vessels with a volume of 1-3 ml, the lower bottom region has a non-circular cross-sectional area with a length of at least 5 mm, preferably at least 10.5 mm and in particular preferably at least 40.5 mm.

[0007] The non-circular cross section interior space of the vessel can be realized by various forms.

Worthy to be noted here is that a circular cross-section is the maximal shape of symmetry. Any deviation from this form towards a less symmetrical shape is possible in the invention and results in an improved mixing and grinding.

[0008]. In one embodiment of the invention, the vessel has an interior and in particular a lower or bottom region constructed for stirring, mixing or grinding purposes.

[0009]. In an alternative embodiment of the invention, the vessel has no internal and in particular no attachments or devices for stirring, mixing or grinding at the bottom of the container.

[0010]. In one embodiment of the invention, the non-circular inner tube cross-section is not realized by an interior thickening of the vessel wall and in particular is not configured or constructed in the form of ribs.

[0011]. Preferably, the inventive non-circular cross-section is realized solely by the shape of the vessel wall with uniform thickness. In this embodiment, therefore, the vessel has a substantially constant vessel wall thickness in the range of the non-circular inner cross-section.

[0012]. In one embodiment of the invention, the vessel according to the invention has a non-circular cross section and this shape can be formed from or is comprised of the following group of shapes:

a. N-angular / angles /corners with N = 1 to 12 and in this case preferably with rounded corners. b. Circular shape with one, two or three rounded corners; and

c. Elliptical.

[0013]. Preferred is a triangular, square or pentagonal configuration of the non-circular cross-section, but a six- seven-, eight-, nine-, ten-, eleven- or twelve-sided design is also possible.

[0014]. Significantly, however, the ability to induce a turbulent flow is reduced with increasing number of corners, but the cross-section volume and thereby the filling capacity or volume increases. In particular, a preferred embodiment therefore is to use a square configuration as this provides an adequate filling volume with thorough mixing and grinding capability.

[0015]. Each corner of the listed N-squared configuration is preferably rounded. This is easier to manufacture when plastic is used and is associated with an increased stability of the vessel.

[0016]. In a further embodiment, the inner cross-section edges are rounded. The then preferably rounded corners disrupt the circular symmetry of the vessel in such a decisive way to promote mixing and grinding of the contents. In this embodiment, either two or three corners can be incorporated. Just as easily realizable is an elliptical shape.

[0017]. In a preferred embodiment of the invention, the tubular container has circular cross section at least in the upper region of the vessel. Thereby the vessel of the invention is still compatible with standard laboratory equipment such as centrifuges for circular tubes and also allows for an easy and secure closure of the vessel with normal circular lids.

[0018]. In a preferred embodiment, the vessel is designed so that it comprises an upper portion having a circular outer cross-section and a lower portion having a non-circular outer cross-section, whereby the lower non-circular portion does not exceed the diameter of the upper circular area cross-sectional sides. This allows the vessel of the invention to be used in standard laboratory instruments and devices for round vessels, such as tube racks, centrifuges, thermal blocks and water baths. [0019]. In one embodiment of the invention, the vessel has a nominal volume of 0.2 ml to 20 litres, preferably from 1 ml to 50 ml, and especially 1.5 ml, 2 ml, 2.5 ml, 3 ml, 5 ml, 15 ml, 20 ml or 50 ml.

[0020]. The container of the present invention can be manufactured from the normally deployed laboratory materials such as glass, ceramics, metal or plastic. However, the preferred material is plastic.

[0021]. To manufacture the vessel different plastic polymers can be deployed. An injection moulding specialist is able to select a suitable plastic polymer depending on the application and purpose of the vessel. Preferably, the plastic is a polyolefin, and in particular, a plastic polymer selected from the group consisting of polypropylene, polyethylene, polyamide, polyvinyl chloride, polytetrafluoroethylene, polymethylpentene, polybutylene, styrene acrylonitrile and polycarbonate.

[0022]. According to one embodiment of the invention a container made of plastic is manufactured preferably by injection moulding.

[0023]. In another embodiment, the vessel opening is made with a screw thread either on the outer or inner wall to enable closure of the vessel with a screw cap. A screw thread and cap provide a very tight seal, which is particularly advantageous with the high mechanical stress subjected to the vessel during the rotary, oscillatory or shaking operation.

[0024]. In a preferred embodiment of the invention, the vessel has a lid which is connected by means of a flat hinge with the vessel in the area of the vessel opening. This embodiment has the advantage that the closing and opening of the vessels with such a lid can be carried out easily and quickly.

[0025]. In a preferred embodiment, the tube and lid geometry of the vessel is designed to minimize sample loss, provide secure lid sealing during the phase of mechanical stress but to enable easy closing and opening of the lid by an operator.

[0026]. In a second aspect, the invention relates to the use of the closed vessel for the mixing or grinding of samples, and especially for the disruption of biological cell samples, in particular by shaking or rotating mixing imparting a rapid circular horizontal movement to the vessel.

[0027]. In one embodiment, the sample in the vessel with the addition of grinding media is crushed or broken up by the shaking or rotating motion applied to it. For this purpose, commercially available "Bead- Mixer" devices can be used, such as FastPrep (MP Biomedicals), Precellys 24 (Fa. VWR International GmbH, Eriangen, Germany), oscillating mill MM400 (Retsch GmbH, Haan, Germany) or Qiagen's mixer Mill (Qiagen, Hilden, Germany), Vortex or Vibrax shaker ^® and mixers (IKA ^® -Werke GmbH & CO. KG, Staufen Germany)

[0028]. In a preferred embodiment, the grinding media are selected from the group consisting of sand, silica particles, glass particles, metal particles and ceramic particles, the particles preferably being balls or beads. Glass beads, ceramic beads or metal balls are most commonly used. For optimal sample disruption, the grinding media should be inert, unbreakable and hard.

[0029]. In a third aspect, the invention relates to a method for grinding biological samples, the steps comprising of:

a. filling a vessel of the invention with a biological sample and with grinding media, and optionally with a solvent.

b. optimal closing and sealing of the vessel; c. disrupting and grinding the sample by shaking, oscillating or rotating the prepared vessel according to steps a and b, preferably using a vortex-rotary mixer, an oscillating mill, a Vibrax ^® mixers (IKA ^® -Werke GmbH & CO. KG, Staufen Germany) or a "bead beater".

[0030]. The disruption and grinding process of the invention can be carried out without a solvent, but also with such a liquid. Commonly used solvents are buffered solutions such as phosphate-buffered saline (PBS) or any other lysis or extraction buffer.

[0031]. According to one embodiment, the grinding process may be carried out with a frozen sample. For this purpose, the sample is preferably placed into the vessel and then sample and vessel frozen in liquid nitrogen. Due to the rapid freezing the soft, hydrated biological samples become so brittle that they are very quickly and effectively ground by the grinding media. In addition, the cellular structures are broken by the shock freezing.

[0032]. In a fourth aspect, the invention provides a kit for the disruption of biological samples, comprising of:

a. at least one vessel of the present invention having one of the available nominal volumes.

b. grinding media; and

c. optionally a lysis and extraction buffer (exclusively for DNA or NA isolation).

[0033]. In another embodiment of the present invention, the lower region of the vessel tapers from the rim of the circular opening to the bottom closed end and thereby have non-parallel lengthwise-walls. That is the walls of the vessel start to taper from the rim of the opening to the closed bottom. The diameter of the bottom closed end (D _bottom ) is less than the diameter of top open end (D _top ) and can vary between 0.5 - 99% of the open end's diameter (D _bottom = 0.5 to 99%* D _top ). This design is such that when a back & forth movement is applied to the closed vessel the contents contained in the vessel are set in motion in a turbulent manner (Fig. 5).

[0034]. These and other aspects of the invention are described in detail in the examples and in the figures.

BACKGROUND of the INVENTION:

[0002] The use of plastic containers sealable with screw caps or with snap lock lids is well known as a state of the art laboratory consumable or disposable product. These vessels are found in a wider form in the laboratory and are in biological, biochemical, medical, analytical or chemical areas essential for all laboratory work dealing with sample analysis. As light, transparent and resistant disposable vessels, they allow not only the storage of samples, but also the processing of samples in numerous applications, such as enzymatic reactions (both digestive and cloning), the polymerase chain reaction, and incubation, mixing of samples and centrifuging to separate solids from liquids and the disruption and homogenization of biological samples with a small pestle or steel, glass or ceramic balls. Sample disruption is the breaking up the cellular material of biological samples to release the cell components such as proteins, DNA or NA, lipids etc. To disrupt and homogenize biological samples, various techniques have been established. One technique is to homogenize the sample by introducing an ultrasonic rod (so-called "sonifier"). This method when used for multiple samples brings a high contamination risk with it and is not suitable for the disruption of harder material (such as some parts of plants, seeds, some yeasts or bone tissue). An alternative method is bead beating, in which the sample is placed in a closable container together with milling media. Bead beating or grinding is accomplished by rapidly agitating a sample with grinding material (beads, balls or sand) in a closed container using preferably a bead beater (any device that shakes or oscillates such containers). The grinding material (beads, balls or sand) can be made of glass, ceramic, metal or any naturally occurring hard material such as garnet.

A variety of devices are used to disrupt samples using beads in a container with vortexers being the simplest and least effective bead beater. More effective and therefore more expensive bead beating devices include MP Biomedical's FastPrep ^® Instruments; Roche's MagNA Lyser, AnalytikJena's SpeedMill PLUS, Omni's Prep Multi-Sample Homogenizer, HT Mini and 24; 1600 Mini G; SPEX SamplePrep ^' s

GenoGrinders; Precellys24/24-Dual Homogenizer, OJagen's TissueLyser II = MixerMill MM30-1/2/3 = Retsch's MixerMill MM3-/4-00. These high throughput homogenizers utilize either a figure-eight or a backwards and forwards i.e. linear oscillating motion to impart a high kinetic energy to the beads or balls which is focussed on the sample to disrupt it

A vortexer (vortex mixer) works by swirling the sample and the grinding material (beads or balls) in a motion that causes disruption of the sample. However, this swirling action is imparted onto the sample and grinding materials within the container so that both have the same motion and there is very little impact of the grinding material upon the sample

Here the limiting factor is the length and speed of motion that is imparted to the grinding material by the beating devices or mills and the yield is not ideal, as it is accompanied by a degradation of the biological material due to excessive shear forces and or elevated temperatures caused by friction.

REFERENCES CITED BY THE EXAMINER:

[0003] GB 2432667A: Apparatus and method for the separation of material from biological samples.

This patent is for an appliance (Fig. 2/GB-Patent) in which a vessel has many chambers of the same design (chamber 7) with a long channel, not round, at the bottom of the chamber, and cannot be employed in standard laboratory equipment. The appliance is not used to disrupt and homogenize biological samples

[0004] DE 690273A: Zerkleinerungsvorrichtung / Crushing appliance

This patent is for an appliance which has many chambers of the same design either parallel slots (Abb. 1 and Abb. 2/DE-Patent) or round concentric circles (Abb. 3 and Abb. 4/DE-Patent) in which there is a certain ratio between width of the slots or the circles and the diameter of the grinding balls. The slots are long but the width is relatively small depending on the above ratio. The device then in a back and forth motion transverse to the parallel walls causes the grinding beads to form a long chain like a string of pearls which then grind the sample on the parallel walls of the chamber. The grinding of the samples (in this case; spices) takes place between the walls and the grinding balls. The shape and size makes this apparatus unsuitable to be used as a laboratory vessel

[0005] US 2,847,169 A: Grinding Charge for Ball Mills

This patent describes new grinding elements for use in a TUBULAR horizontal revolving mill with a noncircular cross-section (Fig. 2/US-Patent). The mechanism of the mill is depicted in Fig. 2/US-Patent (a diagrammatic transverse section), whereby the arrow shows the revolving direction and the position of the grinding charge (4) within the mill. Figure 1 is a diagrammatic longitudinal section of a revolving ball mill of the kind referred to in the patent and has two (2) openings at each end of the mill (1 and 2 in Fig 1 US-Patent). This makes the mill unsuitable for sample preparation in the laboratory as vessels with one opening are standardly employed.

[0006] There is therefore a need for improved processing of laboratory samples and in particular of fresh biological samples.

SUMMARY of the INVENTION:

[0007] It is the objective of the present invention to provide an improved vessel or container for the processing of laboratory samples.

[0008] According to the invention this is achieved by a vessel described in claim 1. Specific embodiments of the invention are the subject of additional dependent or independent claims. [0009] In a first aspect, the invention provides a vessel for the mechanical disruption of biological samples prepared with a tubular vessel having a closed bevelled or conical or round or flat bottom at one end and an opening at the other end. The tubular vessel has a circular tube cross-section towards the upper open region and has a non-circular tube cross-section towards the lower closed region which is designed so that the biological sample contained in the vessel is displaced in a horizontal circular turbulent movement of the upright vessel.

[0010] The vessel according to the invention has numerous advantages over the containers known from the prior art and commercially available.

[0011] As the inventor has determined, the tube or container of the present invention allows for a better mixing of samples and particularly for an improved and efficient disruption and homogenization of biological samples.

[0012] Due to the non-circular inner cross-section in the lower region of the tube or container, a turbulent and chaotic motion is generated in the mixing of liquids and contents. This causes a more effective mixing of the sample and contents in the tube particularly during the commonly used rotating movement of shakers, vortexers and mixers.

[0013] Due to the non-circular inner cross-section in the lower region of the tube or vessel, the kinetic energy that is normally used to form a vortex is converted to a chaotic motion which increases the movement and interaction of liquid or particles of sample and contents in the vessel.

[0014] This is the decisive advantage when disrupting and homogenizing samples, as the grinding charge (balls, beads, sand, garnets, etc.) move chaotically through the interior of the vessel caused by the asymmetric design of the vessel, thus enabling increased and effective collisions between the grinding material and sample to be disrupted and homogenized. This leads to increased shearing and a very quick and efficient disruption of the samples.

[0015] It was found that using non-cylindrically shaped containers with a plurality of grinding material according to the present invention is effective in a rapid, safe, and efficient manner facilitating the release of cellular components, including nucleic acids, proteins and lipids. This enables a quicker downstream isolation and processing of the desired cellular components without compromising the quality of the analysis. As the inventor was able to show, disrupting and homogenizing biological material according to the present invention with a vessel of the present invention yielded DNA and NA of high quantity and quality.

[0016] By a preferred embodiment of the vessel with an upper circular area these vessels can be used in conjunction with the usual laboratory equipment such as tube racks, centrifuges, water baths, heating blocks, etc.

[0017] The vessels can be manufactured with conventional plastic materials in a simple manner and therefore inexpensive to make. By choosing a suitable plastic material, the vessels can be specifically adapted for particular applications. An injection mould should therefore be relatively inexpensive to precision-machine.

[0018] In summary, vessels or containers of the present invention allow a quicker and better processing of all samples in a simple and cost-effective manner resulting in higher quantity and purer quality of the desired product. DESCRIPTION of the INVENTION:

[0019] Conventional containers have an interior space with a circular cross-section. Filled with liquid or particles such containers when subjected to a rotational movement cause the liquid to form a vortex with a funnel-shaped centre i.e. a whirlpool or plughole vortex forms. The individual sample components within the liquid move in unison so that the sample particles or sample components collide or impact with each other with reduced energy. In the case of grinding beads the beads act like pearls on a string and there is minimal contact among the beads and sample. (Fig. 1A).

[0020] Without providing scientific proof for the beneficial effects of the vessels according to the invention, it is assumed here that the asymmetric interior design causes a turbulent flow which transfers the motion and energy to the mixing or grinding and pulverizing of the sample material (Fig. IB).

[0021] For the invention to be effective the lower, that is, bottom-side region of the vessel must have a non-circular inner cross-section the minimum being at least one corner or angle. This design is such that when a circular horizontal movement is applied to the upright vessel the contents contained in the vessel are set in motion in a turbulent manner.

This is the area which corresponds to the mixing or crushing area or function of the asymmetric vessel shape when filled with samples and liquid in the upright position of the vessel

[0022] The dimensions (size and length) of the asymmetric region can be selected to correspond to the level of contents normally used to fill the vessel.

[0023] In one embodiment, the asymmetric region comprises at least 15%, preferably at least 33% and in particular preferably at least 100% of the vessel length.

[0024] In the case of the most commonly used vessels with a volume of 1-3 ml, the lower bottom region has a non-circular cross-sectional area with a length of at least 5 mm, preferably at least 10.5 mm and in particular preferably at least 40.5 mm.

[0025] The non-circular cross section interior space of the vessel can be realized by various forms.

Worthy to be noted here is that a circular cross-section is the maximal shape of symmetry.

Any deviation from this form towards a less symmetrical shape is possible in the invention and results in an improved mixing and grinding.

[0026]. In one embodiment of the invention, the vessel has an interior and in particular a lower or bottom region constructed for stirring, mixing or grinding purposes.

[0027]. In an alternative embodiment of the invention, the vessel has no internal and in particular no attachments or devices for stirring, mixing or grinding at the bottom of the container.

[0028]. In one embodiment of the invention, the non-circular inner tube cross-section is not realized by an interior thickening of the vessel wall and in particular is not configured or constructed in the form of ribs.

[0029]. Preferably, the inventive non-circular cross-section is realized solely by the shape of the vessel wall with uniform thickness. In this embodiment, therefore, the vessel has a substantially constant vessel wall thickness in the range of the non-circular inner cross-section. [0030]. In one embodiment of the invention, the vessel according to the invention has a non-circular cross section and this shape can be formed from or is comprised of the following group of shapes:

a. N-angular / angles /corners with N = 1 to 12 and in this case preferably with rounded corners. b. Circular shape with one, two or three rounded corners; and

c. Elliptical.

[0031]. Preferred is a triangular, square or pentagonal configuration of the non-circular cross-section, but a six- seven-, eight-, nine-, ten-, eleven- or twelve-sided design is also possible.

[0032]. Significantly, however, the ability to induce a turbulent flow is reduced with increasing number of corners, but the cross-section volume and thereby the filling capacity or volume increases. In particular, a preferred embodiment therefore is to use a square configuration as this provides an adequate filling volume with thorough mixing and grinding capability.

[0033]. Each corner of the listed N-squared configuration is preferably rounded. This is easier to manufacture when plastic is used and is associated with an increased stability of the vessel.

[0034]. In a further embodiment, the inner cross-section is circular made or designed with edges. The then preferably rounded corners disrupt the circular symmetry of the vessel in such a decisive way to promote mixing and grinding of the contents. In this embodiment, either two or three corners can be incorporated. Just as easily realized is an elliptical shape.

[0035]. In a preferred embodiment of the invention, the tubular container has circular cross section at least in the upper region of the vessel. Thereby the vessel of the invention is still compatible with standard laboratory equipment such as centrifuges for circular tubes and also allows for an easy and secure closure of the vessel with normal circular lids.

[0036]. In a preferred embodiment, the vessel is designed so that it comprises an upper portion having a circular outer cross-section and a lower portion having a non-circular outer cross-section, whereby the lower non-circular portion does not exceed the diameter of the upper circular area cross-sectional sides. This allows the vessel of the invention to be used in standard laboratory instruments and devices for round vessels, such as tube racks, centrifuges, thermal blocks and water baths.

[0037]. In one embodiment of the invention, the vessel has a nominal volume of 0.2 ml to 20 litres, preferably from 1 ml to 50 ml, and especially 1.5 ml, 2 ml, 2.5 ml, 3 ml, 5 ml, 15 ml, 20 ml or 50 ml.

[0038]. The container of the present invention can be manufactured from the normally deployed laboratory materials such as glass, ceramics, metal or plastic. However, the preferred material is plastic.

[0039]. To manufacture the vessel different plastic polymers can be deployed. An injection moulding specialist is able to select a suitable plastic polymer depending on the application and purpose of the vessel. Preferably, the plastic is a polyolefin, and in particular, a plastic polymer selected from the group consisting of polypropylene, polyethylene, polyamide, polyvinyl chloride, polytetrafluoroethylene, polymethylpentene, polybutylene, styrene acrylonitrile and polycarbonate.

[0040]. According to one embodiment of the invention a container made of plastic is manufactured preferably by injection moulding. [0041]. In another embodiment, the vessel opening is made with a screw thread either on the outer or inner wall to enable closure of the vessel with a screw cap. A screw thread and cap provide a very tight seal, which is particularly advantageous with the high mechanical stress subjected to the vessel during the rotary, oscillatory or shaking operation.

[0042]. In a preferred embodiment of the invention, the vessel has a lid which is connected by means of a flat hinge with the vessel in the area of the vessel opening. This embodiment has the advantage that the closing and opening of the vessels with such a lid can be carried out easily and quickly.

[0043]. In a preferred embodiment, the tube and lid geometry of the vessel is designed to minimize sample loss, provide secure lid sealing during the phase of mechanical stress but to enable easy closing and opening of the lid by the operator.

[0044]. In a second aspect, the invention relates to the use of the closed vessel for the mixing or grinding of samples, and especially for the disruption of biological cell samples, in particular by shaking or rotation mixing imparting a rapid circular horizontal movement to the vessel.

[0045]. In one embodiment, the sample in the vessel with the addition of grinding media is crushed or broken up by the shaking or rotating motion applied to it. For this purpose, commercially available "Bead- Mixer" devices can be used, such as FastPrep (MP Biomedicals), Precellys 24 (Fa. VWR International GmbH, Eriangen, Germany), oscillating mill MM400 (Retsch GmbH, Haan, Germany) or Qiagen's mixer Mill (Qiagen, Hilden, Germany), Vortex or Vibrax shaker ^® and mixers (IKA ^® -Werke GmbH & CO. KG, Staufen Germany)

[0046]. In a preferred embodiment, the grinding media are selected from the group consisting of sand, silica particles, glass particles, metal particles and ceramic particles, the particles preferably being balls or beads. Glass beads, ceramic beads or metal balls are most commonly used. For optimal sample disruption, the grinding media should be inert, unbreakable and hard.

[0047]. In a third aspect, the invention relates to a method for grinding biological samples, the steps comprising of:

a. filling a vessel of the invention with a biological sample and with grinding media, and optionally with a solvent.

b. optimal closing and sealing of the vessel;

c. disrupting and grinding the sample by shaking, oscillating or rotating the prepared vessel

according to steps a and b, preferably using a vortex-rotary mixer, an oscillating mill, a Vibrax ^® mixers (IKA ^® -Werke GmbH & CO. KG, Staufen Germany) or a "bead beater".

[0048]. The disruption and grinding process of the invention can be carried out without a solvent, but also with such a liquid. Commonly used solvents are buffered solutions such as phosphate-buffered saline (PBS) or any other lysis or extraction buffer.

[0049]. According to one embodiment, the grinding process may be carried out with a frozen sample. For this purpose, the sample is preferably placed into the vessel and then sample and vessel frozen in liquid nitrogen. Due to the rapid freezing the soft, hydrated biological samples become so brittle that they are very quickly and effectively ground by the grinding media. In addition, the cellular structures are broken by the shock freezing. [0050]. In a fourth aspect, the invention provides a kit for the disruption of biological samples, comprising of: a. at least one vessel of the present invention having one of the available nominal volumes.

b. grinding media; and

c. optionally a lysis and extraction buffer (exclusively for DNA or NA isolation).

[0051]. In another embodiment of the present invention, the lower region of the vessel tapers from the rim of the circular opening to the bottom closed end and thereby have non-parallel lengthwise-walls. That is the walls of the vessel start to taper from the rim of the opening to the closed bottom. The diameter of the bottom closed end (D _bottom ) is less than the diameter of top open end (D _top ) and can vary between 0.5 - 99% of the open end's diameter (D _bottom = 0.5 to 99%* D _top ). This design is such that when a back & forth movement is applied to the closed vessel the contents contained in the vessel are set in motion in a turbulent manner (Fig. 5).

[0052]. These and other aspects of the invention are described in detail in the examples and in the figures.

EXAMPLES:

1. Disruption & homogenization of tobacco leaves to release DNA for isolation & purification processes

1.1 Issue

[0053]. In this experiment, a well-established grinding method using liquid nitrogen and a mortar and pestle is compared with the disruption & homogenization (=grinding) using the non-circular container of the present invention and steel balls as grinding material. Tobacco leaves were ground to release DNA for isolation & purification processes. As fresh tobacco leaves are used, the material represents a difficult substrate to grind for DNA isolation.

1.2 Materials and Methods

[0054]. Starting material for each preparation and method was approximately lg tobacco leaves (fresh weight). In the first method, the leaves are snap-frozen in liquid nitrogen and ground in a mortar using a pestle. Ground leaf powder was transferred into a 50 mL Falcon tube and 5 ml of either DEB12 or Shorty extraction buffer was added. DEB12 is a proprietary DNA Extraction Buffer. The shorty buffer contains 200 mM Tris, 400 mM LiCI, 25 mM EDTA, 1% SDS and the pH was adjusted with NaOH to pH 9.0.

In the second method according to the present invention lg of tobacco leaf is placed in a 50 ml plastic container with a square internal cross-section, 5 ml of extraction buffer (EB) and 6 x 4mm and 3 X 10mm steel balls are added and for vortexed for 1 - 2 min.

The tobacco leaves disrupted by the two methods were then further processed to isolate and purify DNA according to the following procedure:

• incubate at 65° C for 10 minutes

• centrifuge at 4000 rpm for 10 minutes

• pipette 1 ml of the clear supernatant into a 2 ml microcentrifuge tube

• add of 0.6 ml CHCI ₃ to the supernatant and mix gently with inversion for 1 min.

• separate the phases by centrifuging at 13,000 rpm for 7 minutes

• pipette the upper aqueous phase into a new 2 ml microcentrifuge tube without disturbing the interphase • add 600 μΙ isopropanol to the aqueous solution

• incubate at 4° C or T for 10 minutes

• centrifuge at 13000 rpm for 15 minutes to pellet the DNA

• remove the supernatant and wash the pellet with 1 ml of 70% ethanol

• centrifuge at 13,000 rpm for 7 minutes

• remove the supernatant

• resuspend the precipitate in ΙΟΟμΙ of sterile deionized water or 2.5mM TE buffer pH 8.0

• incubate at 65 ° C for 10 to 30 minutes with the tube lid open

• assess the quality of the extracted DNA (8μΙ) in an agarose gel (1% agarose, 7 volts / cm, 45 min)

• spectrometric analysis of an aliquot (ΙμΙ) using a NanoDrop UV/Vis spectrophotometer

1.3 Results and Discussion

[0055]. The four different approaches with the leaf tissue quantities used are summarized in the following table:

[0056]. Figure 3A documents the genomic DNA isolated from Nicotiana tabacum L. cv Petit Havana SRI resolved by electrophoresis. The gel electrophoretic analysis showed that the tobacco leaf disruption with the vessels or containers according to the invention leads to a qualitatively and quantitatively superior DNA (and RNA).

[0057]. The spectrophotometric profile and yield analysis of the DNA isolated (Fig. 3B) confirmed the high quality of DNA disrupted and isolated using the containers according to the invention. In addition, the method of the present invention resulted in an increased recovery of DNA, which was 32% and 158% higher than the yield of DNA obtained with the comparison method.

2. Disruption & homogenization of tobacco leaves to release RNA for isolation & purification processes

2.1 Issue

[0058]. In this experiment, the disruption and homogenization with milling media of tobacco leaves in conventional round containers was compared to that of in non-circular containers of the present invention. As fresh tobacco leaves are used, the material represents a difficult substrate to grind for RNA isolation of high quality and stability.

2.2 Materials and Methods [0059]. In both approaches ca.lg fresh tobacco leaf was placed in either 50 ml conventional FALCON- plastic containers with round internal cross section ("O" or "rund") or in 50 ml plastic square containers of the present invention ("SQ"), together with 5 ml of NA extraction buffer (REB14 or REB15) and stainless steel balls (6 balls of 4 mm diameter and 3 balls of 10mm diameter) and vortexed for 1 - 3 minutes. The tobacco leaves disrupted by the two methods were then further processed to isolate and purify RNA according to the following procedure:

• incubate at 65° C for 10 minutes

• centrifuge at 4000 rpm for 10 minutes

• pipette 1 ml of the clear supernatant into a 2 ml microcentrifuge tube

• add of 0.6 ml CHCI ₃ to the supernatant and mix gently with inversion for 1 min.

• separate the phases by centrifuging at 13,000 rpm for 7 minutes

• pipette the upper aqueous phase into a new 2 ml microcentrifuge tube without disturbing the interphase

• add 600 μΙ isopropanol to the aqueous solution

• incubate at 4° C for 10 minutes

• centrifuge at 13000 rpm for 15 minutes to pellet the RNA

• remove the supernatant and wash the precipitate with 1 ml of 70% ethanol

• centrifuge at 13,000 rpm for 7 minutes

• remove the supernatant

• resuspend the pellet in ΙΟΟμΙ of sterile deionized water

• incubate at 65 ° C for 10 minutes with the tube lid open

• assess the quality of the extracted RNA (8μΙ) in an agarose gel (1% agarose, 7 volts / cm, 45 min)

• spectrometric analysis of an aliquot (ΙμΙ) using a NanoDrop UV/Vis spectrophotometer

2.3 Results and Discussion

[0060]. The four different approaches with the leaf tissue quantities used are summarized in the following table:

[0061]. The disruption using containers of the present invention resulted in a high grade and uniform grinding of tobacco leaves with tissue particles being less than 1 mm in diameter.

[0062]. The documented gel electrophoretic analysis (Figure 4A) shows that the sample disruption with the vessels according to the invention results in RNA of outstanding quality.

[0063].The spectrophotometric analysis of RNA samples (Fig. 4B) confirmed the high quality of the RNA isolated using the containers according to the invention (A260 / A280 ratio ~ 2.0). In addition, the method using containers of the present invention resulted in RNA yields, which were 128% and 114% higher than the corresponding yields of RNA obtained with the conventional round 50ml Falcon tubes.

[0064]. Other variants and embodiments of the present invention can be realized by experts skilled in the methods of the preceding disclosures, figures and claims.

Such a variant is the use of e.g. 1.5 or 2ml containers with tapering walls starting directly below the rim of the opening (Fig 5). The tapering walls are in contrast to the conventional 1.5 or 2ml tubes used in bead beating appliances which have parallel walls for 47 - 99 % of the tube length. The variant of the present invention has tapering walls or conical geometry for more than 75 % of the tube length. The tubes end in a closed bottom which is usually conical or rounded but can also be bevelled or flat. The variant of the container of the invention uses the same principle in that application of rapidly oscillating reciprocal mechanical energy is imparted to the container via a shaker or preferably a bead beating machine eg. FastPrep (MP Biomedicals), Precellys 24 (Fa. VWR International GmbH, Eriangen, Germany), oscillating mill MM400 (Retsch GmbH, Haan, Germany) or Qiagen's mixer Mill (Qiagen, Hilden, Germany). The application of said energy is conducted to the material in the tube causing it to oscillate and the motion of the beads or grinding material instead of oscillating in a parallel to the longitudinal axis and uniform manner, the beads or grinding material and sample move in a chaotic and turbulent manner, caused by colliding against the inner tapering and conical walls.

3. Definitions

[0065]. According to the invention, the cross section of the interior of the tubular vessel is (synonymously referred to as "tube inner cross-section") that section of the tubular vessel defined which is perpendicular to its longitudinal axis which extends from the vessel opening to the bottom of the vessel and is usually rotationally symmetrical and represents the axis of rotation at the round containers.

[0066]. In the context of the present invention, a "turbulent or chaotic flow/motion" is defined as a flow/motion in which the formation of a simple or complex vortex is prevented and a swirling or turbulence may occur over a broad magnitude range. This type of flow/motion is characterized by a generally three-dimensional flow field with a temporally and spatially seemingly randomly varying component.

[0067]. In the present invention, "disruption of biological samples" means a crushing or rupturing of the sample, especially the cell walls to release the cell contents. "Homogenization of biological samples" means to reduce the particles of the sample especially the cell walls and cellular organelles so that they are uniformly small and evenly distributed.

[0068]. In the context of the present invention, under laboratory use of any technical or scientific applications includes applications which are accompanied by controlled reaction conditions, thus includes the use in research and development (R&D) as well as for analysis, diagnosis, quality assurance, and pathological or forensic analysis. The laboratory use of the invention comprises the storage of biological material of any kind, including viruses, phages and bacteria. Furthermore, this also includes processes such as cryopreservation, freezing samples, for the mixture of stock solutions, the production of aliquots etc. Finally, laboratory use of the invention also includes the mixing of liquids, and in particular the mixing of highly viscous liquids, or solubilizing of solids (eg. powder or (salt) crystals in liquids).

[0069]. According to the invention, "sample" is defined as any material that can be used in the laboratory. According to the invention this includes both inorganic and organic samples and in particular biological samples such as tissue samples, bone samples, plant samples or bacterial samples.

[0071]. In the patent claims, the terms used such as "comprise", "encompass", "contain" and "include" and the like does not exclude additional elements or steps. The use of the indefinite article does not exclude a plurality. A single device can perform the functions of several units or devices mentioned in the claims. In the claims, reference numerals indicated are not intended to be limitations on the means and steps employed. FIGURES

Fig. 1 shows the motion of beads as grinding media in a vessel with a circular cross-section (A) or in a vessel with four rounded corners (B) using a vortex shaker.

Fig. 1 A Conventional containers have an interior space with a circular cross-section. Filled with liquid or particles such containers when subjected to a rotational movement cause the liquid to form a vortex with a funnel-shaped centre i.e. a whirlpool or plughole vortex forms. The individual sample components within the liquid move in unison so that the sample particles or sample components collide or impact with each other with reduced energy. In the case of grinding beads the beads act like pearls on a string and there is minimal contact among the beads and sample.

Fig. 1 B The beneficial effects of the vessels according to the invention due to the asymmetric interior design causes a turbulent flow or chaotic motion which transfers the motion and energy to the beads which collide with each other and the sample thereby mixing and grinding and pulverizing of the sample material.

Fig. 2 A shows a schematic representation of a vessel according to the invention with screw cap lid (top) and with snap lid (bottom) in a side view (left) and in cross-section (right). The diagrams are not drawn to scale and are valid for vessels ranging in volume from 0.2 ml to 20 L.

Fig. 2 B depicts the cross-sections of a conventional vessel (i) and two exemplary cross-sectional configurations of the vessels of the invention (ii).

Fig. 3 A documents the genomic DNA and NA isolated from Nicotiana tabacum L.cv Petit Havana SRI resolved by gel electrophoresis. The tobacco leaves were either ground frozen with conventional mortar and pestle method (samples 1 and 2: DEB12 N ₂ and Shorty N ₂ ) or ground with vessels of the invention (samples 3 and 4: DEB12-SQ and Shorty-SQ). Shorty is a quick and rapid extraction buffer modified from Edwards et al. Nucleic Acids Research. 1991, page 1349. The gel electrophoretic analysis showed that the tobacco leaf disruption with the vessels or containers according to the invention leads to a qualitatively superior DNA (and RNA).

Fig. 3 B. The results of spectrophotometric measurement of DNA samples 1 to 4 are summarized in tabular form (top) and the respective absorption curves overlaid in the graph as a comparison (bottom). The spectrophotometric profile and yield analysis of the DNA isolated (Fig. 3B) confirmed the high quality of DNA disrupted and isolated using the containers according to the invention. In addition, the inventive method resulted in an increased recovery of DNA, which was 32% and 158% higher than the yield of DNA obtained with the comparison method.

Fig. 4A shows a gel electrophoretic separation of RNA from Nicotiana tabacum L.cv Petit Havana SRI leaves, isolated either with conventional round tubes (Samples 1 and 2: REB14 and REB15 rund or 0) or isolated with vessels of the invention (Samples 3 and 4: REB14 and REB15 Square). All other parameters are the same. The documented gel electrophoretic analysis shows that the sample disruption with the vessels according to the invention results in RNA of high quality and quantity.

Fig. 4B. The results of spectrophotometric measurement of RNA samples 1 to 4 are summarized in tabular form (top) and the respective absorption curves overlaid in the graph as a comparison (bottom). The spectrophotometric analysis of RNA samples confirmed the high quality of the RNA isolated using the containers according to the invention (A260 / A280 ratio ~ 2.0). In addition, the inventive method resulted in increased RNA yield, which was 128% and 114% higher than the yield of RNA obtained with the conventional round 50ml Falcon tubes. Fig. 5. In another embodiment of the present invention, the lower region of the vessel from just below the rim of the opening (a) to the bottom closed end tapers resulting in walls that have a non-parallel longitudinal -section (b). The diameter of the bottom closed end is less than the top open end and can vary between 1 - 99% of the open end's diameter. This design is such that when a back & forth movement along the L axis is applied to the vessel the contents contained in the vessel are set in motion in a turbulent manner by colliding with the tapering walls causing multiple collisions between the grinding media and the sample.