Goal

When lactose is converted into lactic acid by bacteria, milk is converted into yoghurt. In this practical we will isolate the most common agents: Lactobacillus and Streptococcus.

Materials

Step 1

  • 50 mL Ringer’s Solution
  • 5 test tubes
  • 6 MRS Agar plates
  • 1 Malt Agar plate
  • pH indicator paper
  • commercial yoghurt, such as Actimel
  • 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 yoghurt 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 MRS 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 yoghurt and streak it on the MRS plate.
  12. Once more, heat up the inoculation loop to red hot, let it cool down, dip it in the yoghurt and streak it on the MA plate.
  13. Incubate plates at 30° C.
  14. Measure the pH of the yoghurt 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 bacterial colony on each fresh MRS plate.
  5. Streak a yeast contamination on a clean MA 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