38
25X CONCENTRATED PSS:
Chemical Mol.Wt
mM
g/0.5L
g/L
g/2L
g/4L
NaCl
(58.45)
3250
94.98
189.96
379.92
759.84
KCl
(74.557)
117.5
4.375
8.75
17.5
35.0
KH
2
PO
4
(136.09)
29.5
2.0
4.0
8.0
16.0
MgSO
4
7H
2
O
(246.498)
29.25
3.625
7.25
14.5
29.0
NaHCO
3
(84.01)
14.9
0.625
1.25
2.50
5.00
Glucose
(180.16)
5.5
0.5
1.00
2.00
4.00
EDTA
(380)
0.65
0.125
0.25
0.50
1.0
CaCI
2
(110.99)
40
20mL
40mL
80mL
160mL
(1.0 M solution)
1. Make a 1.0M solution of CaCl
2
(110.99)
in double-distilled H
2
O. Filter-sterilize the
calcium solution through a 0.22 μm filter.
The sterilized solution can be stored in the
refrigerator for up to 3 months.
2. Dissolve all the chemicals except the CaCl
2
in approximately 80% of the desired final
volume of double distilled H
2
O while being
constantly stirred. For example, if 1 liter
of PSS is to be made, then dissolve all the
chemicals in 800mL of double distilled H
2
O.
3. Add the appropriate volume of 1.0M CaCl
2
for the total volume of PSS being made (for
example, 1.6mL of 1.0M CaCl
2
for 1 liter of
buffer). Continue to stir the PSS while the
CaCl
2
is being added.
4. Bring the solution up to the final volume with
double-distilled H
2
O. Continue to stir the
solution until the EDTA is fully dissolved. This
takes about 15 minutes at room temperature.
Before use:
5. Dilute the 25 x PSS stock solution 1:25 using
double distilled H
2
O.
6. Add:
1.091 g/L Glucose
2.100 g/L NaHCO
3
7. Aerate the solution with carbogen (95% O
2
+
5% CO
2
) for at least 20 minutes. If necessary
wait further for the pH of the buffer to reach
pH 7.4.
