Friday, October 10, 2008

Staining & Yeast Viability

Above is a video of a Safale US-05 I got from the bottom of a bottle of a nice Pale Ale a friend of mine brewed. The sample was prepared using the following method and magnified 400x for your viewing pleasure.

Staining is a common practice to ensure the yeast you pitch in your starter are nice and healthy and capable of converting your hard efforts of brewing into the tasty beverage we know as beer. The idea is that healthy yeast will not absorb the stain while non-healthy (non-viable, non-metabolizing) yeast cells will.

"In the brewery industry, the most common yeast viability stain is methylene blue. In fact, methylene blue is accepted as the industry standard.1 However, in recent years, there have been reports that methylene blue stain may overestimate yeast viability.2 This stain has been reported to be inaccurate when yeast viabilities fall below 95%,3  hence, the use of an alternative stain, methylene violet, has been tested by several standard committees as a suitable alternative stain for yeast viability determination.4" - Comparison of the efficacy of various yeast viability stains by Stephen E Szabo  Ph.D.

So in this method I chose Methylene Blue, your mileage my vary in your stain of choice. It's worth noting that the stain that comes in the bottle is VERY concentrated, and in my experience is better served if you take a few drops in a clean vial and dilute it with a few ml of sterile water. This also reduces the intensity of the stain, and could help with the mentioned inaccuracies of staining viable cells.

Start with a clean slide, and a clean slide cover. Place the slide on a clean surface and put the slide cover in place. Then with your inoculating loop get a drop of the yeast slurry you want to examine and place it as indicated above (1) right where the cover and the slide meet. The sample should start to flow from right to left filing the space between the slide and the cover. Next take a dropper of your stain of choice and place it on the left side of the slide (2). The stain should then start to flow into the sample from left to right. I often do the second step while the slide is on the microscope. Watching the stain slowly flow across the sample is pretty neat.

The nice part about doing this method is that you can get a nice mix of intensities of yeast cells (more on the right) and stain (higher on the left) so you get sort of a gradient across the sample equilizing in the center.

Your results, if you have a healthy starter/sample, should be dominated with clear cells.

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