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Thread: Methylene Blue Staining

  1. #1
    Join Date
    Dec 2017
    Location
    Lafayette, CO, USA
    Posts
    3

    Methylene Blue Staining

    I just got a new microscope and am trying to implement viability staining into my cell counting routine, but I'm running into some confusion. Depending on the focus of my microscope, even if I adjust it ever so slightly, my cells look either all clear or all dark blue! I know that there's no way I have 100% viability, or 100% dead cells. Has anyone else ever run into this problem?

    I am using a 1:1000 yeast dilution (made via serial dilutions), a 0.1% MB dilution (made from 1 mL of 1% MB into 99 mL distilled water), and mixing 1 mL of the diluted yeast solution with 1 mL of the MB for about 2 minutes. I've also tried a 0.01% MB solution, with the same problem.

    Thanks!

  2. #2
    Join Date
    Oct 2010
    Posts
    105
    I've run into the same issue. Try adjusting focus so that the blue is only around the cell walls, see what you get that way.

    Cheers,

    Rich DeLano
    rich@thebrewinglair.com

  3. #3
    Join Date
    Oct 2013
    Location
    Cincinnati, OH
    Posts
    384
    I have not run into this situation before, but generally I focus based on the sharpness of the hemocytometer lines. Then you can adjust your light filter if needed to get the right amount of light diffusion (this may be what you mean by focus). That should help the shades of blue that you see. My final concentration of MB is 0.05%. I use the same dilution as mentioned, except for the fact your math shown is wrong.

    1ml of 1% solution into 99ml of water equals 0.01%, not the 0.1% mentioned. If you made a typo this correction wont help, but if you have been doing the math wrong, then your solutions were probably 0.01% and 0.001%, then cut in half when doing the 1ml to 1ml final dilution of yeast sample to MB solution (0.005% or 0.0005%). Probably not dark enough to get proper contrast.

    A 0.1% solution made from 1% would be 1 ml (methylene blue @ 1%) to 9 ml water. Equation as follows, C1*V1=C2*V2 --- C = concentration, V = volume --- 1%*1ml=0.1%*x ---- x=(1%*1ml)/0.1% = 10ml, then subtract the initial volume to calculate the addition of water required, 10ml-1ml= 9ml water to be added to 1ml of 1% solution for a total of 10ml of 0.1% solution.

    Your serial dilution of yeast after stain solution (and any H2SO4, EDTA, ect) is added should leave you with about 100 cells in the microscope field at 400x. You can also try Trypan Blue instead.

  4. #4
    Join Date
    Nov 2016
    Location
    Sweden
    Posts
    12
    Here is photos (with a mounted mobile to the ocular), the only changes between images
    are fine zoom levels. Stained with Trypan Blue, 1:100. Which ones of these are the most
    correct?

    Name:  20180312_162512.jpg
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Size:  80.0 KBName:  20180312_162523.jpg
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Size:  72.4 KBName:  20180312_162532.jpg
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Size:  85.1 KB

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