Agitating – Stirrer within stationary mixing chamber – Magnetic stirrer
Reexamination Certificate
1999-06-15
2002-03-19
Cooley, Charles E. (Department: 1723)
Agitating
Stirrer within stationary mixing chamber
Magnetic stirrer
C422S064000
Reexamination Certificate
active
06357907
ABSTRACT:
BACKGROUND—FIELD OF INVENTION
This invention provides a method, devices and machines to uniformly and simultaneously mix in tall vessels in such a manner as to increase the aeration of the liquid. The purpose is to provide a robust method, economical devices and very simple machines to uniformly stir and aerate the contents of one tall vessel or thousands of tall vessels, vials, test tubes or deep well microplate wells (6 well, 12 well, 24 well, 48 well, 96 well, 384 well, 864 well, 1536 well, 10,000 well, “U” bottom, “V” bottom, PCR and other deep well versions of those microplates) at the same time.
BACKGROUND—Description of Prior Art
Suspending particulates in liquids, breaking up aggregates, aeration of gases in liquids, dissolving solids in liquids, emulsifying two liquids or mixing liquids together has usually been accomplished in large vessels, bottles, flasks and test tubes by magnetic horizontal spin stirring, orbital shakers, vortexing, rocking platforms, bubblers, vibrators or magnetic tumble stirring. In an effort to miniaturize many of the operations that were done in bottles, flasks and test tubes, scientists have shifted to deep well multi well microplates (24 deep well, 48 deep well, 96 deep well, and 384 deep well). Other deep well plates will undoubtedly follow. These deep wells are characterized by a tall vertical height and a very small air surface to volume ratio, thus making aeration difficult. Deep well microplates are now commonly used in a wide variety of scientific applications to act as mini-reaction or culture vessels for liquid assays. However, because of the combination of the tall height of the column of liquid, small air surface area to volume ratio and the significant attraction of liquid surface tension forces to the walls of small diameter wells (especially in the 48, 96, and 384 deep well microplates) it is not possible to adequately resuspend particulates, aerate gases into liquids, stimulate the growth of microorganisms, break up aggregates, break open cells, emulsify two liquids, mix two liquid reagents, or to dissolve a solid in a liquid by simple agitation on an orbital shaker, vortexer, rocker platform, bubbler or by vibration without generating a force so violent that it would throw the liquids out of the miniature vessels.
Magnetic horizontal spin stirring has been adapted to 96 well microplates by VARIOMAG-USA. They put spinning permanent magnet stirrers into the wells of a 96 well microplate and place the 96 well microplate on a magnetic stirring system with an individual modulated (driving) electromagnetic stir point engineered under the center of each well. The placement of the wells exactly over a stir point is critical so the spinning stirrers don't hit the well walls and lose synchronization with the driving electromagnetic field, causing them to vibrate in the wells. If they hit the well wall the whole stirrer has to be stopped repositioned and slowly started spinning again. Often when the stir point system is rapidly spinning, the stir magnets in individual wells get out of synchronization with the driving electromagnetic field for no apparent reason or due to the vibration of the stirring action moving the plate. Part of this sensitivity phenomena is due to the physics of the interaction of the magnetic fields of the driving electromagnet and the spinning magnetic stirrer which line up parallel to each other, thus resulting in a weaker magnetic coupling than if the poles were directly opposed to each other. This weak magnetic coupling also is demonstrated if there is a differential viscosity between the liquids of different wells of the same microplate, as this will slow the stirrers in those wells and throw those magnetic stirrers out of synchronization. The cost of this magnetic stir point system is $2,400.00 for each 96 well plate and the cost of the individual stirring magnets for each well is $2.45 thus bringing the total cost to stir each plate to $2,635.00. Furthermore a unique stir point configuration (6, 12, 24, 48, 96, 384, 1536, and 10000 wells) is required for each well configuration of the microplate well format used. Because of these technical and cost considerations this system has not been widely used.
In a previous patent application Ser. No. 09/170,459, now U.S. Pat. No. 6,176,609, we invented and applied “Magnetic Tumble Stirring” as a method to stir the contents of microplates. Magnetic Tumble Stirring consists of stainless steel magnetic stir discs, bars and dowels of different dimensions, shapes and with magnetic field orientations through the long axis so that they will provide a vigorous stirring action when they are tumbled end over end through the long axis in a vertical magnetic field(s). By making the magnetic stirrers nearly the same diameter or length as the diameter of the vessel they are stirring or by putting multiple magnetic stirrers into a single vessel a very significant stirring action is effected when they tumble through the magnetic axis. Magnetic Tumble Stirring in the vertical plane is produced by the magnetic poles of stirrers inside vessels being attracted to an opposite vertically oriented drive magnetic field produced by a permanent magnet or an electromagnet under the vessel. This vertical magnetic attraction causes the stirrers to stand on a polar end and then fall when either the drive magnetic field or the vessels are moved laterally in respect to each other and the stirrer can not maintain the vertical orientation because of the wall of the vessel or friction with the well bottom. Although this method works well with microplates that have short to medium columns of liquid above them, it is not as effective with the taller deep well microplates. Furthermore it is does not effectively aerate the liquid.
Another way to provide adequate mixing and aeration in microplates is by pipetting the contents of each well up and down. This can be done manually or by robotic work stations. There are several robotic work stations that will do this pipet mixing operation, but they will just mix 4, 8 or 12 wells at a time and the pipets must be washed or changed between wells. Recently Robbins Scientific introduced the “Hydra” work station which will pipet and mix 96 wells at a time but it costs ~$30,000 and is still laborious as the pipets must be washed between wells. Furthermore, it would be economically impossible to do continuous mixing of multiple microplates simultaneously by pipetting. In addition, it would be difficult to place a whole large robotic workstation in an incubator if microorganisms were the objects being mixed while culturing. Thus, the miniaturization of continuous mixing processes on a large number of microplates remains impractical until now.
OBJECTS AND ADVANTAGES
Thus it is the object of the method, devices and machines to provide a very simple process of uniformly mixing and aerating the contents of thousands of vessels, vials, test tubes and deep well microplates. We have made stainless steel magnetic stir balls, discs, bars, and dowels of different dimensions, shapes so that they will fit into any vessel and provide a vigorous stirring action when they are levitated by a magnetic field, then dropped by gravity or pulled down by the same or another magnetic field. By making the magnetic stirrers nearly the same diameter or length as the diameter of the vessel they are stirring or by putting multiple magnetic stirrers into a single vessel, a very significant stirring action is effected when they levitate up and down through the liquid. When the stirrers are levitated through the liquid meniscus it significantly increases the liquid's surface area and therefor increases aeration. We have discovered that while stainless steel is commonly thought to be non-magnetic, several stainless steels that have been “hardened”, “spring tempered” or “cold worked,” are magnetic. Because stainless steel is corrosion resistant, it maybe used as a magnetic stirrer without any protective coating, like teflon, thus making stainless steel magnetic stirrers very inexpensive.
Cleveland Patrick H.
Markle John R.
Callan Edward W.
Cooley Charles E.
Sorkin David L.
V & P Scientific, Inc.
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