Alkaline Methylene Blue SOLUTION

Alkaline Methylene Blue
What you need:

Methylene Blue Solution
Sterile Pipets
Citric Acid
Distilled Water
0.1M Glycine Buffer (pH 10.6)                            Mechanical Pipeter
2 small, clean (100-125ml) Glass Bottles
Foil

Procedure:

1.  Weigh 0.1g Methylene Blue and place in small glass bottle.

2.  Add distilled water up to 100ml mark on bottle.

3.  Swirl bottle to dissolve media. (This is your 0.1% Methylene Blue stock solution.)

4.  Place 10ml of the stock solution into a new glass bottle.

5.   Dilute stock Methylene Blue solution up to 100ml mark with the 0.1M glycine buffer.  This solution is ready to use for vitality tests.

6.   Place 500ml of the “ready to use” methylene blue solution into 1.5ml eppendorf tubes for cell & vitality counting.

Note: Discard solution after 6 months.

The following is a published description of a White Labs test involving the use of the methylene blue solution described above:

"A methylene blue stock solution (0.1%) was diluted 10-fold with a 0.1M glycine buffer solution, pH 10.6.  0.5ml of yeast suspension (1 x 107 cells/ml) was added to 0.5ml of alkaline methylene blue staining solution, mixed, and incubated for 15 minutes at room temperature.  Yeast cells were microscopically examined and recorded as dead cells were medium to dark blue in color. Pale blue and unstained cells were counted as living. The viability was tested in triplicate. The viability counts were averaged."

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