3.2 M17 Agar
Preparation (1 L)
1. Prepare the solution as detailed above.
2. Before sterilizing add 15 g of agar.
3. Mix it well.
4. Sterilize it by autoclavation.
5. After autoclavation, let cool to approximately 50
C.
6. Add the glucose to 0.5%.
7. Pour the liquid in the petri dishes inside a laminar flow cabinet
(20–25 mL of medium per 90-mm petri dish).
8. Let the agar solidify and then close the petri dishes, turn them
upside-down and store at 2–8
C.
3.3 Glucose Solution
50% Preparation
(100 mL)
This solution (stock solution) will be used by being added to the
medium to reach the concentration of 0.5%.
1. Dissolve 50 g of glucose in 100 mL of water (with the help of a
magnetic stirring bar and magnetic mixer).
2. Sterilize by filtering (0.22 μm) inside a laminar flow cabinet (see
Note 1).
3. Store at 2–8
C.
3.4
Phosphate-Buffered
Saline 0.01 M (PBS 1Â)
Preparation (1 L)
1. Dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na 2 HPO 4 , and
0.24 g of KH 2 PO 4 in 800 mL of distilled water.
2. Add water q.s. for 1 L.
3. Adjust the pH to 7.4 with HCl 1 M solution.
4. Sterilize by autoclaving.
3.5 Preparation
of the Bacterial Strain:
Lactococcus Lactis
1. Inoculate 50 μL of bacteria suspension in 30 mL of M17 broth
supplemented with glucose to 0.5% (bacteria stored at À80
C
with 25% glycerol).
2. Incubate the culture at 30
C, without shaking, for 20 h.
3.6 Quantitative
Growth Evaluation
of Bacterial Culture
1. Measure the OD 600 of the bacterial culture previously grown.
2. To measure the OD 600 adjust the spectrophotometer to a
wavelength reading of 600 nm.
3. Calibrate the instrument using the noninoculated broth as a
blank.
4. Measure the absorbance of the overnight cultured sample.
5. Dilute the bacterial culture in M17 + Glucose 0.5% broth for
OD 600 ¼ 0.04 (bacterial lag phase).
6. Incubate the bacteria at 30
C without shaking.
7. Measure the OD 600 of the growing bacterial cultures hourly
(until reaching the stationary phase).
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Viviane Lima Batista et al.
Preparation (1 L)
1. Prepare the solution as detailed above.
2. Before sterilizing add 15 g of agar.
3. Mix it well.
4. Sterilize it by autoclavation.
5. After autoclavation, let cool to approximately 50
C.
6. Add the glucose to 0.5%.
7. Pour the liquid in the petri dishes inside a laminar flow cabinet
(20–25 mL of medium per 90-mm petri dish).
8. Let the agar solidify and then close the petri dishes, turn them
upside-down and store at 2–8
C.
3.3 Glucose Solution
50% Preparation
(100 mL)
This solution (stock solution) will be used by being added to the
medium to reach the concentration of 0.5%.
1. Dissolve 50 g of glucose in 100 mL of water (with the help of a
magnetic stirring bar and magnetic mixer).
2. Sterilize by filtering (0.22 μm) inside a laminar flow cabinet (see
Note 1).
3. Store at 2–8
C.
3.4
Phosphate-Buffered
Saline 0.01 M (PBS 1Â)
Preparation (1 L)
1. Dissolve 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na 2 HPO 4 , and
0.24 g of KH 2 PO 4 in 800 mL of distilled water.
2. Add water q.s. for 1 L.
3. Adjust the pH to 7.4 with HCl 1 M solution.
4. Sterilize by autoclaving.
3.5 Preparation
of the Bacterial Strain:
Lactococcus Lactis
1. Inoculate 50 μL of bacteria suspension in 30 mL of M17 broth
supplemented with glucose to 0.5% (bacteria stored at À80
C
with 25% glycerol).
2. Incubate the culture at 30
C, without shaking, for 20 h.
3.6 Quantitative
Growth Evaluation
of Bacterial Culture
1. Measure the OD 600 of the bacterial culture previously grown.
2. To measure the OD 600 adjust the spectrophotometer to a
wavelength reading of 600 nm.
3. Calibrate the instrument using the noninoculated broth as a
blank.
4. Measure the absorbance of the overnight cultured sample.
5. Dilute the bacterial culture in M17 + Glucose 0.5% broth for
OD 600 ¼ 0.04 (bacterial lag phase).
6. Incubate the bacteria at 30
C without shaking.
7. Measure the OD 600 of the growing bacterial cultures hourly
(until reaching the stationary phase).
450
Viviane Lima Batista et al.