Microbiology

Yeast–10 Page 1 of 2

DIFFERENTIATION OF ALE AND LAGER YEAST

A. BY X-α-GAL MEDIUM

X-α-gal is designed to be a simple and accurate test medium to differentiate ale from lager yeast (3). The method can be used to monitor yeast culture purity (1–3). The test differentiates α-galactosidase (melibiase)-secreting (lager) yeast from nonsecreting (ale) yeast. This colony color method is based on the ability to differentiate yeasts that secrete α-galactosidase by addition of the chromogenic substrate 5-bromo-4-chloro-3-indolyl-α-galactoside (X-α-gal) to a selective agar medium. This specific medium is designed to provide conditions for inducing α-galactosidase secretion. In the presence of secreted α-galactosidase, X-α-gal, an analogue of melibiose, is enzymatically cleaved. The indol released by this reaction oxidatively polymerizes to give an insoluble blue-green dye that does not diffuse in agar. Hence, when grown on this medium, ale yeast colonies remain cream-white, whereas lager yeast colonies turn blue-green.

Reagents (a) N,N'-Dimethylformamide or dimethyl sulfoxide. (b) 5-Bromo-4-chloro-3-indolyl-α-D-galactoside (X-α-

gal) stock solution, Boehringer Mannheim (917591) or equivalent. Prepare stock solution by dissolving 25 mg of X-α-gal with 1.25 mL of N,N'-dimethyl-formamide or dimethyl sulfoxide in the reagent vial. Store at 4°C in the dark.

(c) Basal medium agar plates. Dissolve the following ingredients in distilled water and make to 1 L: glyc-erol, 20 g; D-galactose, 2 g; yeast extract, 10 g; Bac-topeptone, 20 g; agar, 20 g. Mix on a stir plate in a 2-L flask. Autoclave at 121°C, 15 psi, for 15 min. Add 30 g ethanol. Allow to cool to approximately 50–55°C. Pour approximately 20 mL per plate and allow solidification at room temperature in a sterile environment. Store at 4°C.

Apparatus (a) Test tubes, 16 × 125 mm. (b) Hemocytometer. (c) Petri dishes, 100 × 15 mm. (d) Inoculating loops, 10 µL. (e) Micropipette, capable of measuring 100 µL.

Method Before the assay, add 100 µL of X-α-gal stock

solution to each agar plate, using a micropipette, and spread evenly with an inoculating loop. Allow to stand in the dark for 30–60 min. Determine yeast cell count via Yeast-4. Dilute sample to give 100–200 colonies per

100 µL, and spread evenly on the agar plate with an inoculating loop. Incubate plates at 25–27°C in the dark for 6 days. Count the colonies, and report the number of blue-green colonies (lager yeast) and cream-white colonies (ale yeast) per plate.

Note N,N'-dimethylformamide is a possible carcinogen and

should be handled with appropriate care.

References 1. American Society of Brewing Chemists. Report of Subcommit-

tee on X-α-Gal Medium. Journal 50:150, 1992. 2. American Society of Brewing Chemists. Report of Subcommit-

tee on X-α-Gal Medium. Journal 51:185, 1993. 3. Tubb, R. S., and Liljestrom, P. L. J. Inst. Brew. 92:588, 1986.

1993, rev. 2011

B. BY GROWTH AT 37°C

Growth at 37°C is a test traditionally used to differentiate lager yeasts from ale yeasts. Character-istically, ale yeast can grow at 37°C, whereas lager yeast cannot (1–3).

Reagents (a) Bacto YM agar. Prepare by following manufac-

turer’s instructions (Difco or equivalent). (b) Normal saline solution. Dissolve 9 g of NaCl in 1 L

of distilled water and sterilize at 121°C and 15 lb/in.2 for 20 min.

Apparatus (a) Petri dishes, 100 × 15 mm. (b) Incubator, 25°C, accurate to ± 0.5°C. (c) Incubator, 37°C, accurate to ± 0.5°C. (d) Sterile centrifuge tubes, 50-mL or equivalent. (e) Centrifuge.

Method Preparation of yeast inoculum

Streak the test yeast cultures onto plates of YM agar and incubate for 48 hr at 25°C. Suspend culture with normal saline to a concentration of 106–107 cells/mL, and smear a loop of yeast suspension onto a plate of YM agar. Incubate at 25°C for 48 h.

Determination of growth at 37°C Wash cells off YM agar plates using sterile normal

saline, transfer the culture to sterile centrifuge tubes, and centrifuge at 4000 rpm for 10 min. Decant the supernatant without disturbing the packed yeast pellet. Repeat the washing procedure two additional times.

doi: 10.1094/ASBCMOA-Yeast-10

Microbiology

Yeast–10 Page 2 of 2

Suspend the washed cells in saline to approximately 25 × 107 cells/mL, i.e., approximately 500 µg dry weight of yeast per milliliter (A640 ≈1.0). Smear loops of yeast suspension onto YM agar plates and immediately place in separate 25 and 37°C incubators. Incubate for five days and observe for growth. Record results for growth, or no growth, at both temperatures.

References 1. American Society of Brewing Chemists. Report of Subcommit-

tee on Differentiation of Ale and Lager Yeast. Journal 52:184, 1994.

2. Barnett, J. A., Payne, R. W., and Yarrow, D., Eds. Yeasts: Char-acteristics and Identification. Cambridge University Press, Cambridge, U.K., 1983.

3. Campbell, I., and Brudzynski, A. J. Inst. Brew. 72:556, 1966.

1994, rev. 2011

C. BY GROWTH ON MELIBIOSE

Lager yeast strains of Saccharomyces pastorianus are able to assimilate and ferment the disaccharide sugar melibiose, 6-O-α-D-galactopyranosyl-D-glucose, and utilize this sugar as a sole source of carbohydrate (2). Ale yeast strains of Saccharomyces cerevisiae, however, can-not assimilate or ferment melibiose (2). By culturing a brewer’s yeast strain in a fermentation broth containing melibiose as the sole carbohydrate, one can measure the yeast’s ability to ferment melibiose by the change of a pH indicator (indicating acid production) and by the production of gas that becomes trapped in an inverted Durham tube (1). If acid and gas production are both positive, the strain is considered to be a lager yeast. If they are both negative, the strain is assumed to be an ale yeast.

Reagents (a) Malt extract-yeast extract agar slants. Dissolve 3 g

malt extract, 3 g yeast extract, 5 g peptone, 10 g glu-cose, and 20 g agar in 1 L distilled water (pH is between 5 and 6 without adjustment). Sterilize at 121°C and 15 lb/in.2 for 15 min.

(b) Yeast extract-peptone fermentation broth. Dissolve 4.5 g yeast extract, 7.5 g peptone, and 0.05 g bromothymol blue in 1 L distilled water. Dispense 2-mL aliquots into 150- × 12-mm screw cap test tubes each containing an inverted 60- × 5-mm Durham tube. Sterilize at 121°C and 15 lb/in.2 for 15 min.

(c) 12% (w/v) melibiose solution. Dissolve 12 g meli-biose in 100 mL distilled water. Filter sterilize.

(d) 6% (w/v) glucose solution. Dissolve 6 g glucose in 100 mL distilled water. Filter sterilize.

(e) 4% (w/v) melibiose fermentation broth tubes. Pre-pare by aseptically adding 1 mL of the 12% meli

biose solution to the yeast extract-peptone fermentation broth tubes.

(f) 2% (w/v) glucose fermentation broth tubes. Prepare by aseptically adding 1 mL of the 6% glucose solu-tion to the yeast extract-peptone fermentation broth tubes.

(g) Control fermentation broth tubes. Prepare by adding 1 mL sterile water to the yeast extract–peptone fermentation broth tubes.

(h) Sterile water.

Apparatus (a) Screw cap test tubes, 150 × 12 mm. (b) Durham tubes, 50 × 6 mm. (c) Incubator, 28°C, accurate to ± 0.5°C. (d) Balance, accurate to ± 0.1 g. (e) Membrane filters, 0.45-μm pore size. (f) Autoclave. (g) Sterile inoculating loop. (h) Pipettes. (i) Sterile centrifuge tubes, 50-mL or equivalent. (j) Centrifuge.

Method Preparation of yeast inoculum

Streak the test cultures onto fresh malt extract-yeast extract slant cultures in 4.5 mL of sterile water. Decant washed cells into a sterile centrifuge tube and centrifuge at 4000 rpm for 10 min. Decant the supernatant without disturbing the packed yeast pellet. Repeat the washing procedure two additional times.

Determination of melibiose fermentation test Inoculate 0.1 mL of the yeast suspension into

melibiose fermentation broth, glucose fermentation broth (positive control), and control fermentation broth tubes (negative control). Incubate at 28°C for 1, 2, 3, and 7 days, and score positive samples as a change of the indicator dye to yellow (i.e., acid production) and the production of gas in the Durham tube. The negative control should show no color change and no gas production. The positive (glucose) control should show a distinct change to yellow (acid production) and gas production in the Durham tube.

References 1. American Society of Brewing Chemists. Report of Subcommit-

tee on Differentiation of Ale and Lager Yeasts by Melibiose. Journal 53:219, 1995.

2. Wickerham, L. J. Technical Bulletin No. 1029. Taxonomy of yeasts, p. 9. United States Department of Agriculture, Washington, DC, 1951.

1995, rev. 2011