Yeast starter staining to assess health
How viable is your yeast starter? What is the state of your yeast when fermentation ends (prematurely), is there any evidence of contamination? These are legitimate questions that homebrewers and professional brewers must ask themselves when they brew their most delicious beers. What can really help answer these questions are safe and cheap yeast viability stains.
Contemporary viability testing methods use a live-dead stain that selectively colours either dead or living cells. Subsequent microscopic examination of stained samples in which you count both living (L) and dead (D) cells can establish the proportion of viable cells. You can use the percentage of viable cells as a measure of yeast starter quality or vitality. The formula:
Viability (%) = L / T * 100, where T is the sum of L (living cells) and D (dead cells).
A selection of affordable stains to test yeast starters
Brewers have long used Methylene blue for their yeast viability stains. Like many other viability stains, Methylene blue penetrates the yeast cell membrane to stain cells blue. Living cells, however, convert this substance into a colourless compound. The inability of dead cells to convert methylene blue will ensure that they stain blue. Methylene blue also fluoresces when it remains in the cell. To detect MB, you can excite it with an intense light beam (laser) with a 665 nm wavelength and detect the light emitted at 690 nm. Methylene blue, however, may be carcinogenic.
Erythrosine B specifications
While Erythrosine B serves the same purpose as Methylene blue, it is quite different. Similar to MB, Erythrosine stains cells by entering the yeast cell, colouring cells pink. Living cells remain colourless, indicating the breakdown of EB. A critical difference, between MB and EB, besides its structure and colour, is safety. The food industry commonly uses Erythrosine B as a food colourant. EB is thus a safe and cheap yeast viability stain to use in a homebrewery setting and easy to dispose of as waste.
Erythrosine B staining protocol.
Stock solution: 5-10 mg/mL (1000x) in dH2O
Working solution: 5-10 μg/mL
Storage: room temp (solid), -20 oC in liquid. Photo protect.
Directions: Prepare a stock solution by measuring 10 mg (suggested) Erythrocine B in 1 mL (sterile) water. Make sure that the compound dissolves completely.
Application:
Mix your stain solution (1000 x stock) with 1/1000 of your cell suspension. This will ensure that your stain is at working strength in your sample. Incubate the solution for a few minutes before examination under the microscope. Using a hemacytometer, you can count the number of living (whitish) and dead cells (light to dark pink) per volume and estimate the % of viable cells.
Notes:
Before embarking on a serious analysis of your samples, try to play around with pretty young or viable cells and cultures that are quite old (slurry from a brew) and train your eyes to spot (and count) dead and viable cells quickly.
We hope you found our article on cheap yeast viability stains of good use. If you are seriously thinking about viability stains, make sure to check out our article on microscope types and selection criteria. Please get in touch with us by email or leave a comment if you have any questions.
Brew on!
The BrewingBrowser Team
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Photo by Alex Kondratiev on Unsplash