With Tg
0
M , the glass transition temperature of the maximally freeze-concentrated phase of the multi-component solution and Tg
0
i , and C i , the Tg
0 value, and the concentration of
component i, respectively.
5. Select the PS with a Tg
0 higher than “À25-X + Y
C.”
6. Mix cell pellets (CP) with the protective solution (PS) at a ratio
of 1:2 (1 g of cells and 2 g of protective solution). Suspend cells
thoroughly by using a spatula and a vortex mix until complete
cell resuspension.
7. Determine the collapse temperature of the protected bacterial
suspension by using freeze-drying microscopy.
8. If the collapse temperature is higher than À25
C, the protective solution can be selected for the freeze-drying process (see
Note 5).
9. If the collapse temperature is lower than À25
C, two options
can be examined: (a) the modification of the composition of
the protective solution by addition of molecules exhibiting
higher collapse temperature or (b) the addition of a washing
step of the bacterial pellet (CP) with saline solution before
adding the protective solution (see Note 6).
3.3 Freeze-Drying
Protocol
1. Prepare the protective solution at a total solute concentration
of 400 g/L in saline solution, and autoclave the solution at
120
C for 20 min. Store the protective solution at 4
C.
2. Prepare an antioxidant solution (sodium ascorbate) at a concentration of 10 g/L in saline solution, and filter-sterilize the
solution by passing it through a 0.2 μm sterile membrane filter.
Protect the solution from light (in a dark flask and/or cover
with aluminum foil), and use immediately or store at 4
C for
up to 1 day.
3. Aseptically mix equal volumes of protective and antioxidant
solutions to obtain a protective solution with the following
composition: 200 g/L total protective molecules, 9 g/L
sodium chloride, and 5 g/L sodium ascorbate (see Note 7).
Use immediately or store at 4
C for up to 1 day, protected
from light.
4. Perform bacterial growth according to your production protocol or the protocol described by Fonseca et al. [15].
5. Aseptically transfer the cell suspension to sterile centrifugation
flasks (see Note 8).
6. Centrifuge (17,000 Â g, 4
C, 30 min), and remove the supernatant (see Note 3).
Physical Properties and Freeze-Drying of Bacteria
713
0
M , the glass transition temperature of the maximally freeze-concentrated phase of the multi-component solution and Tg
0
i , and C i , the Tg
0 value, and the concentration of
component i, respectively.
5. Select the PS with a Tg
0 higher than “À25-X + Y
C.”
6. Mix cell pellets (CP) with the protective solution (PS) at a ratio
of 1:2 (1 g of cells and 2 g of protective solution). Suspend cells
thoroughly by using a spatula and a vortex mix until complete
cell resuspension.
7. Determine the collapse temperature of the protected bacterial
suspension by using freeze-drying microscopy.
8. If the collapse temperature is higher than À25
C, the protective solution can be selected for the freeze-drying process (see
Note 5).
9. If the collapse temperature is lower than À25
C, two options
can be examined: (a) the modification of the composition of
the protective solution by addition of molecules exhibiting
higher collapse temperature or (b) the addition of a washing
step of the bacterial pellet (CP) with saline solution before
adding the protective solution (see Note 6).
3.3 Freeze-Drying
Protocol
1. Prepare the protective solution at a total solute concentration
of 400 g/L in saline solution, and autoclave the solution at
120
C for 20 min. Store the protective solution at 4
C.
2. Prepare an antioxidant solution (sodium ascorbate) at a concentration of 10 g/L in saline solution, and filter-sterilize the
solution by passing it through a 0.2 μm sterile membrane filter.
Protect the solution from light (in a dark flask and/or cover
with aluminum foil), and use immediately or store at 4
C for
up to 1 day.
3. Aseptically mix equal volumes of protective and antioxidant
solutions to obtain a protective solution with the following
composition: 200 g/L total protective molecules, 9 g/L
sodium chloride, and 5 g/L sodium ascorbate (see Note 7).
Use immediately or store at 4
C for up to 1 day, protected
from light.
4. Perform bacterial growth according to your production protocol or the protocol described by Fonseca et al. [15].
5. Aseptically transfer the cell suspension to sterile centrifugation
flasks (see Note 8).
6. Centrifuge (17,000 Â g, 4
C, 30 min), and remove the supernatant (see Note 3).
Physical Properties and Freeze-Drying of Bacteria
713