ARTIFICIAL SEAWATER MEDIA (ASW)
FROM: Goldman & McCarthy, 1978. L&O 23:695-703
McLachlan,
1964. Can. J. Microbiol. 10:769-782
Typically, we make up 8 liter batches of media
at a time. A mark or piece of tape on
the carboy should indicate the required volume
1. Add A. and B. to Distilled/Deionized Water
mix and bring to volume then autoclave:
A.
"Dry" components:
|
Mineral
Salts |
Conc. |
MW |
grams/8
liters |
|
NaCl |
400
mM |
58.45 |
187 |
|
MgSO4-7H2O |
20
mM |
246.48 |
39.4 |
|
CaCl2-2H2O |
10
mM |
110.99 |
8.88 |
|
KBr |
1.7
mM |
119.01 |
1.62 |
|
KCl |
10
mM |
74.56 |
5.96 |
|
MgCl2-6H2O |
20
mM |
203.3 |
32.5 |
B.
"Wet" components:
|
Solution |
Concentration |
ml
per 8 liters |
|
H3BO3 |
0.2
mM (can be doubled for alkalinity determinations) |
8
ml (of 12.36 g/l stock) |
|
NaNO3 |
f/2 |
8
ml |
|
Vitamin
Mix |
f/2 |
4
ml (in freezer) |
|
TRIZMA |
5%,
pH 7.8 |
5
ml/liter |
2.
Autoclave separately:
A. f/2 trace metals ( 1 ml per liter of media) For recipe see f/2 media
B. f/2 phosphate ( 1 ml per liter of media)
f/2 media
C. f/2 silicate ( 1 ml per liter
of media) if needed see f/2 media....
3. To
make complete media:
A. Allow 1 and 2 to cool to room
temperature.
B. Add items 1 & 2 together (sterile
procedure)
C. Prepare 1.34 g of NaHCO3 in approx 30-40 ml water; Filter sterilize through sterile filter disc
(Gelman Acrodisc; 0.2 um).
D. It is recommended to "bubble" the media for 12 hours with air. Typically, our media bottles have caps having two long tubes attached, one for sampling and one for bubbling with air...the air (from an aquarium pump) passes through a bacterial air vent/filter. A short tube (above the media) attached to the bottle cap allows the air to vent from the bottle. If no bacterial air vents are available, sterile glass wool will do.
4. For Iron limitation work, wash all glassware
with 10% HCL and rinse well with distilled water.
Follow
above recipe, BUT
A.
Make up f/2 trace metals
WITHOUT iron. ie add 1 ml of each of
the primary trace metal stocks (see f/2
media) but no ferric sequestrene and bring to 1 liter for stock solution. This solution replaces the trace metal
solution in part 2A.
B.
If you need
to add iron later, make up a 0.5 mM FeCL3 and EDTA stock solution
and add 4 ml per liter of media...
(0.186 g Na2EDTA and 0.135 g FeCl3
6H2O per liter for stock solution)
5. More
Notes
A. The TRIZMA solution above should not be pH'd by HCL titration...use the proper proportions of TRIZMA-HCL and TRIZMA-Base.
(18.22 g HCL and 6.78 g Base in 500 ml dist. H2O )
B. We
also add one ml per liter of media of 10 uM Selenous acid for some
species.
6. Preparing
Iron Limited cultures
B.
Grow to approx. 106 cells/ml and add
one liter culture to 8 liters of –Fe media for five days. Do cell counts, chl determinations and FRR
measurements to determine degree of Fe limitation. To test iron limitation, take 100 ml of iron limited culture and
add to sterile Erlenmeyer flask. Add 50
ul of 0.5 mM FeCl3-EDTA solution.
Incubate overnight and the next day check cell count, chlorophyll and
FRR measurements… you should see recovery from iron limitation.