3.8 Bacterial Doses
Preparation
for Mucosal
Administration
Day 1
1. Add 50 μL of bacteria culture in 10 mL of M17 + Glucose 0.5%
broth.
2. Incubate at 30
C, without shaking, for 20 hours.
Day 2
1. Measure the OD 600 of the bacterial culture previously grown.
2. Make a dilution for reaching the bacterial lag phase (based on
the bacteria growth curve) (see Notes 7 and 8).
3. Incubate at 30
C until the OD 600 reaches the bacteria’s exponential phase.
4. Centrifuge at 980 Â g for 10 min at 4
C.
5. Discard the supernatant.
6. Wash the pellet twice with PBS (0.01 M).
7. Repeat the steps 4 and 5.
8. Resuspend the pellet in 30 μL of cold sterile PBS (0.01 M).
9. Each microtube will contain one dose (see Note 9).
10. The dose can be used immediately after preparation or stored
for future use at À80
C for up to 5 days [5] (see Note 10).
11. Add PBS 0.01 M to the desired amount according to the
chosen form of administration.
12. All procedures should be done in a sterile laminar flow.
3.9 Animal
Restraints
One-hand restraint (Fig. 2).
1. Lift the mouse by holding the base of the tail and place it on the
wire bar cage top or any similar rough surface (see Note 11).
2. With thumb and index finger hold the tip of the mouse’s tail,
tuck the tail between the third and fourth finger and pull back
gently so the mouse grabs the surface.
3. With the second and third finger press gently the back of the
mouse against the surface and with the thumb and index finger,
grab the mouse scruff skin.
4. Be sure the grasp is firm and the mouse’s head does not
move [15].
Two-hand restraint (Fig. 3).
1. Lift the mouse by the base of the tail and place it on the wire bar
cage top or any similar rough surface.
2. With the dominant hand hold the base of the tail (see Note 11).
3. With the thumb and index finger of the nondominant hand,
grab the mouse scruff skin.
452
Viviane Lima Batista et al.
Preparation
for Mucosal
Administration
Day 1
1. Add 50 μL of bacteria culture in 10 mL of M17 + Glucose 0.5%
broth.
2. Incubate at 30
C, without shaking, for 20 hours.
Day 2
1. Measure the OD 600 of the bacterial culture previously grown.
2. Make a dilution for reaching the bacterial lag phase (based on
the bacteria growth curve) (see Notes 7 and 8).
3. Incubate at 30
C until the OD 600 reaches the bacteria’s exponential phase.
4. Centrifuge at 980 Â g for 10 min at 4
C.
5. Discard the supernatant.
6. Wash the pellet twice with PBS (0.01 M).
7. Repeat the steps 4 and 5.
8. Resuspend the pellet in 30 μL of cold sterile PBS (0.01 M).
9. Each microtube will contain one dose (see Note 9).
10. The dose can be used immediately after preparation or stored
for future use at À80
C for up to 5 days [5] (see Note 10).
11. Add PBS 0.01 M to the desired amount according to the
chosen form of administration.
12. All procedures should be done in a sterile laminar flow.
3.9 Animal
Restraints
One-hand restraint (Fig. 2).
1. Lift the mouse by holding the base of the tail and place it on the
wire bar cage top or any similar rough surface (see Note 11).
2. With thumb and index finger hold the tip of the mouse’s tail,
tuck the tail between the third and fourth finger and pull back
gently so the mouse grabs the surface.
3. With the second and third finger press gently the back of the
mouse against the surface and with the thumb and index finger,
grab the mouse scruff skin.
4. Be sure the grasp is firm and the mouse’s head does not
move [15].
Two-hand restraint (Fig. 3).
1. Lift the mouse by the base of the tail and place it on the wire bar
cage top or any similar rough surface.
2. With the dominant hand hold the base of the tail (see Note 11).
3. With the thumb and index finger of the nondominant hand,
grab the mouse scruff skin.
452
Viviane Lima Batista et al.