John Chavez

  1. Stirring Cells or Particles in 50ml Tubes with New Stir Elements

    V&P Scientific has recently added a new line of stir elements which are capable of re-suspending cells or particles and keeping cells or particles in suspension without causing cell damage.

     The VP 772FN-25-55TF-150 {Tuning Fork} and the VP 772FN-20-21CV-150 {Torpedo-Cross} were both designed to be autoclavable. They fit inside 50ml centrifuge tubes to mix the constituents. The outer shape of both stir elements matches the inside topography of the tube.  Both designs allow you to take samples while the tube is being stirred. The Torpedo-Cross design has a lower profile and therefore requires less dead-volume than the tuning fork design. Both stir elements use two VP 782N-12-150  Neodymium Iron Boron magnetic discs.

    These stir elements have optimal performance with our VP 710D2 Multi Stirrus™. Both designs are well suited for mixing different solutions, resuspension of cells, or dissolution of powders. When these stir elements are used in conjunction with the VP 710D2 Multi Stirrus™ more than 24 50ml conical tubes can be mixed simultaneously. The stirring action does not shear or damage fragile cells even after continuous mixing.

     In a test experiment using human promyelocytic leukemia cells, 35ml samples were removed from a tissue culture flask and placed into three different 50ml conical tubes.

    The 1st tube was allowed to sit at room temperature with no stirring. The 2nd tube was stirred at 350 RPM with the Tuning Fork design{VP 772FN-25-55TF-150}. The 3rd tube was stirred at 350 RPM with the new Torpedo-Cross design {VP 772FN-20-21CV-150}.

    The cells in each tube were counted within 5 minutes of being removed from the tissue culture flask. They all had comparable cell counts with percent viability greater than 98%, as measured by Trypan blue exclusion. The three tubes were then allowed to incubate at room temperature for approximately one hour, with 2 tubes being continuously stirred at 350 RPM during the incubation.

     The cell counts taken from the tubes being stirred remained consistent with the counts that were taken within the first 5 minutes of removal from the tissue culture flask. The counts from the tube with no stir element showed a drop of 40% due to the settling of the cells. 

     The three tubes were then centrifuged for 5 minutes at 500 RPM to pellet the cells. The 2 tubes with stir elements were returned to the stir device and stirred for 5 minutes in an attempt to resuspend the pelleted cells. The counts from the tube with no stir device dropped by over 99%, consistent with the pelleting of the cells. The tubes being stirred returned to the counts comparable to those taken within the first 5 minutes of removal from the tissue culture flask. There was no evidence of cell damage seen in any tube as measured by Trypan blue dye exclusion; the percent viability remained greater than 98% in all three tubes.

     In a second test experiment, both stir elements were used to mix heavy glitter particulates. The Torpedo-Cross style element was superior in keeping heavy particulates in suspension. When very light particulates {fine glitter} were tested, the Tuning Fork design was equally effective. The Torpedo-Cross stir element, being a two piece design, is more expensive than the single piece Tuning Fork stir element, but offers a greater performance when stirring heavy particulates.

     In summation, both the Torpedo-Cross and the Tuning Fork style stir elements provide a safe and effective way of keeping cells or particulates in a uniform suspension throughout a 50ml conical tube, all the while allowing you to take samples while the tube is being stirred. The Torpedo-Cross is superior when using heavy particulates or clumping cells.

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  2. Keeping Magnetic Beads, cells and other Particulates in Suspension while Pipetting

    V&P Scientific invented Bubble Paddle Reservoirs in response to customers’ requests for a simple method to keep particulates in uniform suspension for both manual and robotic pipetting with minimum dead volume loss. We found that by designing Bubble Paddles with notches large enough between the Bubbles that would accommodate pipet tips we could keep the paddles spinning and creating up welling currents even while removing suspended particulates from the bottom of the reservoir. We also sculpted the bottom of the reservoirs to mimic the paddle shapes which enhances the turbulence efficacy and leaves space to accommodate the pipet tips.

    In the photo above, the blue dye in the reservoir highlights the contours we have machined into the bottom of the stirrer.  This design creates very powerful up welling fluid currents to keep very dense particulates like glass YSi SPA beads in uniform suspension.  Another  unique feature of the Bubble Paddle Reservoirs is their exclusive design that eliminates "fall out zones" where particulates fall out of solution that are so prevalent with other mixing reservoirs. 

    Endorsement

    Here is what one of our customer’s at a major Pharmaceutical company had to say about our 384 Bubble Paddle Reservoir system  "The reservoir is really the best thing that I have found for YSi SPA beads which immediately fall out of solution when not mixed" ..... The bubble paddle is essential for YSi beads."

     

     Although the sculptured bottom design is essential for keeping very dense particulates in uniform suspension we have found that the shear forces that this design creates, especially at high speed can damage fragile cells or resins.  We have solved this problem by creating a more open design in the reservoir bottoms.  These open designs work very well in keeping lighter particulates uniformly suspended. See these products for examples of our open designs, VP 750-ECON-6, VP 750-ECON-6

    A, VP 750AL, VP 750AL-1, VP 750AL-2, VP 751-D-1-SBS.  The photo below illustrates 6 “U” channels instead of individual Bubble Paddle sculpting used in the VP 751-D-SBS.

    We have 3 different methods for powering the spinning of our Bubble Paddles, the following describes and compares the applications and advantages of these different methods:

     1. Direct magnetic coupling is a method where one of our VP 710 series Vertical Tumble Stirrer’s magnetic cylinder is used under or to the side of the reservoir to magnetically couple to the magnetic Stainless Steel Bubble Paddles and spin the paddles at a high rate of speed.   Initially this method was used with all particulates except paramagnetic beads.  Later we discovered if we used parylene encapsulated Bubble Paddles that the paramagnetic beads are suspended uniformly because they are only weakly attracted to the parylene encapsulated Stainless Steel Bubble Paddles and they are easily dislodged from the parylene encapsulated stainless steel at even low speeds.  A recent modification of this method was the VP 754V-50 made to adapt to the Formulatrix Mantis dispensing system see this video https://youtu.be/kaL5wfZ700U to see how our Vertical Bubble Paddle system works in a 50 ml tube and is powered by a VP 710D2.   The following video illustrates the Mantis at work dispensing paramagnetic beads from a VP 754V-50 reservoir to microplates – see https://youtu.be/7kE8AdssJA4

        The direct magnetic coupling method is the most cost effective way for keeping all particulates in uniform suspension.

     

    2. Dual magnetic clutch coupling through the reservoir’s wall to a motor’s drive shaft.  The Magnetic clutch versions are used in applications where Z height or space is limited by a robotic platform’s configuration. This method can NOT be used with paramagnetic beads because the paramagnetic beads are attracted to strong NdFeB magnets used in the magnetic clutches.

    3. Direct mechanical coupling of an aluminum (non-magnetic) Bubble Paddle to a motor’s drive shaft through a sealed bearing is another way to safely keep paramagnetic beads in suspension.  It also has the advantage of being able to stir very viscous solutions or slurries that are too viscous for other magnetically coupled methods to stir.

     

    We have helped many customers with many different uniform dispensing applications, please free to contact us with your particular needs.  The chances are that we have already solved your application problem. For example a customer came to us with unique application to suspend cells in molten agarose.  The VP 750-13-AL below was our solution.  It is made from anodized aluminum and is used to keep the contents warm and suspended.  This reservoir coupled with a heating block and a Vertical Tumble Stirrer was the solution.  The aluminum's superior heat conductive properties are very useful in keeping  molten agarose containing cells in a liquid state prior to dispensing to a another cooler media where the agarose will solidify around the cells.  

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  3. Mixing E-Liquid or E-Liquid for Vaping

    There has been an increasing amount of customers purchasing our stirrers and stir bars for mixing E-Juice. E-Juice consists of a few simple ingredients which include, Vegetable Glycerine, Propylene Glycol, Nicotine and Flavoring. Vegetable glycerin, which serves as the base of E-Liquid, has a viscosity of 1412cp. For perspective, water has a viscosity of 1cp, while honey has viscosity of 10,000cp. Magnetic mixing is an effective and efficient way to mix E-Liquid, which results in faster steeping times and less work.

     

    Standard stir plates are not magnetically strong enough to mix such a viscous liquid in larger quantities. There are many YouTube videos demonstrating magnetic mixing with homemade stirrers and/or standard stir plates. However, these smaller stir plates only work for small volumes. Our customers are not making E-Liquid for themselves, rather are producing large quantities and batches to sell.

     

    V&P Scientific collaborated with San Diego based Vape Corner. Vape Corner is locally owned and has two locations in San Diego. As their business has grown, owner Gino Sugapong has researched other methods of mixing his E-Liquid. Currently they have been mixing their E-Liquid by hand. This yields a rather physical task and a 4 week steeping time.

     

    Because the ingredients in the E-Liquid have different viscosities, they separate from each other during the initial week of “steeping”. It’s important that each batch is mixed every day so that the entire mixture is homogenous. We aimed to compare stirring magnetically vs stirring manually - shaking by hand .

    We mixed 3 – one gallon batches of the same E-Liquid. Each bottle contains: Propylene glycol, Vegetable glycerine, flavoring, nicotine

    Bottle 1.            Control: Shaking by Hand, once a day for a week

    Bottle 2.            Magnetic mixing for 30 min, once a day for a week - 210 minutes of mixing total in a week

    Bottle 3.            Magnetic mixing 24hrs every day for a week - 10,080 minutes in a week

    We used the following V&P Products:

    VP 710C5 - Stir Base

    VP 772M-N52-27-111-PTFE - Stir Paddle

       

    We know that it takes up to 4 weeks for the E-Liquid to steep if shaken by hand. Our expectations were that Sample 3 - magnetic mixing 24/7-  would yield the best results in regards to taste and decreased steeping time.


    Day 1

    Bottle 1: Hand mixed for 30 min

    Bottle 2: Mixed magnetically with paddle for 30 min.

    Bottle 3: Mixed magnetically with paddle 24 hrs.

    Notes: After 15 min. of mixing, Bottles 2 and 3,  fully emulsified. Visually, a milky color due to the introduction of air during mixing. See Photo below:

    From left to right, Bottle 1, 2, 3 after 30min


    Day 3

     

     

    Bottle 1: Bottle is slightly lighter than the rest.

    Bottle 2: Looks similar to bottle 3

    Bottle 3: Still being mixed, looks similar to bottle 2

    Notes: It appears that bottles 2 & 3 are slightly darker in color than bottle 1.

     

     See Photo Below:

    From left to right, Bottle 1, 2, 3 after 72hrs

     


    Day 4-7

    Nothing changed visually from day 3. We could arguably see there was a slight difference in color. Both bottle 2 and 3, the magnetically stirred bottles were darker. The difference was slight and could be light reflections. At day 7, we took samples from each bottle and issued a blind taste test to the employees at Vape Corner.

     

    Results:

    Bottle 1 - Hand Shake- : Flavor has not yet steeped. Still needs more time to steep.

    Bottle 2 - Magnetically Stirred for 30min/day for 7 days- : Steeping time was indistinguishable to a properly steeped batch. Flavor was  optimal.

    Bottle 3 - Magnetically Stirred 24/7 for 7 days- : Flavor was less than that of the other two bottles. However, the noticeable difference was that the Vape of this bottle was smoother the other two.

     


    Summary:

    Surprisingly enough, bottle 2 was the best outcome. We were able to achieve an appropriate steeping in one week by mixing the E-Liquid 30min/day for 7days. The benefit of this is that you won’t have to plan so far in advance for your stock of E-Liquid. This is especially true of stores, like Vape Corner, that carry a large variety of flavors. You can stock a fixed amount of any flavor, and if that begins to sell out quickly, you can make more in 1 week. This is a better scenario than having to plan 4 weeks in advance and having to tell customers you’re out of stock of their favorite flavor.

    Consequently, bottle 3 - magnetically stirred 24/7-  was smoother to inhale, though the flavor was decreasing. It is interesting to find that the increased stirring would diminish the flavor. If a smoother vape is preferable, it’s quite easy to adjust the recipe with more flavoring to compensate for the effect of the magnetic stirrer.

     

    Alternatively perhaps we should have magnetically mixed for 210 minutes to achieve the optimal flavor and done the taste test at this time point.  This would have significantly reduced the time to make a new batch.

    Using the VP 710C5 and the VP 772M-N52-27-111-PTFE, you can mix multiple bottles at the same time. Using our Patented Stirring Technology, you can quickly and easily mix viscous solutions such as E-Liquid. We were able to subjectively identify improved taste and quality of the E-Liquid with magnetic mixing versus mixing by hand.  

    Click here for videos of magnetic stirrers mixing e-liquid.

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