Goal

Especially famous Belgian beers continue to ferment in the bottle. During fermentation the sugars are converted into ethanol. In this practical we will isolate the yeasts.

Materials

Step 1

  • 50 mL Ringer’s Solution
  • 5 test tubes
  • 1 MRS Agar plate
  • 6 Malt Agar plate
  • pH indicator paper
  • Beer, for example Duvel or Grimbergen
  • Glass spatula
  • Inoculation loop
  • 100 mL beaker glass with some 95% ethanol
  • Gas burner

Step 2

Step 3

  • Microscope
  • Microscope slides and cover slides
  • Gram and Loeffler staining

Method Step 1

  1. Label the 5 test tubes: -1, -3, -5, -7 and -9
  2. Add 9 mL Ringer’s solution to tube -1
  3. Add 9.9 mL Ringer’s solution to tube -3, -5, -7 and -9
  4. Add 1 g of beer to tube -1 and vortex/mix
  5. With the use of sterile pipette transfer 0.1 mL from the -1 tube to the -3 tube. Vortex / mix.
  6. Transfer 0.1 mL from the -3 tube to the -5 tube. Vortex / mix.
  7. Transfer 0.1 mL from the -5 tube to the -7 tube. Vortex / mix.
  8. Transfer 0.1 mL from the -7 tube to the -9 tube. Vortex / mix.
  9. Label 5 MA plates with the same labels: -1, -3, -5, -7 and -9
  10. Starting with the largest dilution (-9) transfer 0.25 mL from the test tube to the -9 plate and spread it using the glass spatula that has been sterilized by dipping it in alcohol and torching it. Continue with the next tube (-7) and so on.
  11. Heat up the inoculation loop to red hot, let it cool down, dip it in the beer and streak it on the MA plate.
  12. Once more, heat up the inoculation loop to red hot, let it cool down, dip it in the beer and streak it on the MRS plate.
  13. Incubate plates at 30° C.
  14. Measure the pH of the beer using the pH indicator paper.

Method Step 2

After about one week:

  1. Take a look at your plates.
  2. Select a plate with clear individual colonies.
  3. Count the colonies.
  4. Streak one yeast colony on each fresh MA plate.
  5. Streak a bacterial contamination on a clean MRS plate.
  6. Incubate the plates at 30° C.

Method Step 3

After about one more week:

  1. Perform a Gram staining on the bacteria and a Loeffler staining on the yeast.
  2. Take a look at the cultures under the microscope.

Back to Biofactory - Class 1: Incubator