(NO.1) 2X Erdschreiber's Medium For UTEX LB 2538
Directions
Erdschreiber's Medium made with Supplemented Seawater instead of Pasteurized Seawater. For 3 L Total
1. To 3 L of pasteurized supplemented seawater (60 ppt) aseptically add each of the sterile components in the order specified.
2. Vigorously swirl the contents of the flask to mix thoroughly.
3. Store at refrigerator temperature.
1 3 L
vrml2 36 mL/
3 L
3 NaNO3 (Fisher
BP360-500)
10 mL/3 L 0.7 M 2.3 mM
4 Na2HPO4·7H2O
(Sigma S-9390)
10 mL/3 L 0.02 M 0.067 mM
5 150 mL/3 L
6 3 mL/3 L
1)Supplemented Seawater
Directions
For 1 L Total
1.To approximately 900 mL of Pasteurized Seawater, add the following components in the order specified while stirring continuously.
2. Bring total volume to 1 L with Pasteurized Seawater.
1 900 mL
2 NaCl (Fisher S271-
500)
23 g/L 393.8 mM
3 MgCl2·6H2O
(Fisher M 33)
5 g/L 24.
6 mM
4 Na2SO4 (Sigma S
6264)
4 g/L 28.2 mM
a）Pasteurized Seawater
Directions
Most salt-water media used by UTEX includes natural seawater collected off-shore from Port Aransas, Texas in the Gulf of Mexico. Seawater having a salinity of at least 30 ppt is collected and pre-filtered, then stored undisturbed within polyethylene carboys at ambient temperature.
Seawater is diluted to 30 ppt with double distilled water immediately prior to
pasteurization. A three-liter batch of seawater at 30 ppt in a 4-liter Erlenmeyer flask is covered with a small inverted glass petri plate and an inverted 250-ml beaker, then "pasteurized" in a steamer for 45 minutes. The pasteurized content of the flask is allowed to cool and left undisturbed at ambient temperature for approximately 24 hr. It is then again steam-pasteurized for 45 min., as on the previous day. After the flask cools the second time, the inverted-petri-plate lid is sealed in place with Parafilm and the flask is stored at refrigerator temperature until it is used to prepare culture medium. This pasteurized seawater may used immediately or may be stored for several months prior to use.
Procedure:
In various laboratories marine algae are cultured in media prepared from "pasteurized" seawater that is assumed to be heated to exactly 73 degrees C. The procedure described here heats 3-L batches of seawater to over 95 degrees C for two consecutive days, although it does not reach boiling temperature.
This procedure generally does not cause precipitation of seawater, although excessive agitation of flasks, the use of scratched or etched flasks, or pasteurization of seawater at higher salinity may result in salt precipitation during heating.
Pasteurized seawater prepared as described above appears to be nearly sterile, although it is not used to culture axenic UTEX cultures without further heating in agar. Liquid unialgal cultures grown in media prepared from seawater that has been pasteurized by this method can be sub-cultured for many years without the introduction of invasive contamination.
2) P-IV Metal Solution
Directions
For 1 L Total
Note final concentration listed is for the stock solution.
1.To approximately 950 mL of dH2O, add the nutrients in the order listed while stirring continuously.
Note: The Na2EDTA should be fully dissolved before adding other components.
2. Bring total volume to 1 L with dH2O.
3. Store at refrigerator temperature.
1 Na2EDTA·2H2O
(Sigma ED255)
0.75 g/L 2 mM
2 FeCl3·6H2O
(Sigma F-1513)
0.097 g/L 0.36 mM
3 MnCl2·4H2O
(Baker 2540)
0.041 g/L 0.21 mM
4 ZnCl2 (Sigma Z-
0152)
0.005 g/L 0.037 mM
5 CoCl2·6H2O
(Sigma C-3169)
0.002 g/L 0.0084 mM
6 Na2MoO4·2H2O 0.004 g/L 0.018 mM
considerations are probably important, including the following:
1. The soil should be a loam, with a mixture of particle sizes
(sand, silt, clay).
2. It should contain a moderate amount (15 - 20%) of very-well-decomposed organic matter.
3. It must not contain pesticides, especially herbicides.
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4. It should be soil that has been aged (preferably for 6 months
or more) under moist conditions and not, for example, fresh
potting soil, soil that contains fresh manure, or soil to which a
民事案件案由规定commercial fertilizer was recently applied. 5. A slightly acidic soil derived from granite or other igneous
rock is preferable to soil obtained from calcareous soils. Calcium carbonate can be added to the soilwater medium when it is
prepared if a slightly alkaline medium is required.
6. Particulate matter in the soil such as gravel, Perlite, or
vermiculite are not necessarily damaging but can be of
considerable nuisance when wishing to quantitate the amount of
soil used in the medium or when handling algae that are道州论坛 摩托罗拉a768physically associated with the soil. Particulate organic matter,
such as compost that is only partially degraded, should be
avoided altogether.
4) Vitamin B12
Directions
For 200 mL Total
1. Prepare 200 mL of HEPES buffer (50 mM).
2. Adjust the pH to 7.8.
3. Add Vitamin B12 (0.1 mM) wait until fully dissolved.
4. Sterilize by 0.45 µm Millipore filter. Store in dark at freezer temperature. ** The amount of vitamins added can vary from medium to medium so the final concentration is not listed.
1 HEPES buffer pH
7.8 (Sigma H-
3375)
2.4 g/200 mL
dH2O
2 Vitamin B12 (cyanocobalamin,
(Sigma V-6629)
0.027 g/200 mL
dH2O
(NO.2)Proteose Medium UTEX 32
Directions
General purpose freshwater medium suitable for axenic cultures. Modified bristol's medium.
For 1 L Total pH ~6.8
1. Add proteose peptone to Bristol Medium.
*For 1.5% agar medium add 15 g of agar into the flask; do not mix.
2. Cover and autoclave medium.
1 1 L
2 Proteose Peptone
(BD 211684)
1 g/L
1)Bristol Medium
Directions
H.C. Bold's modification of Bristol's recipe (Bold 1949). General purpose freshwater medium and as bristol's solution, an essential component of other media--see Bold 1NV, Bold 3N, Bristol-NaCl, LDM, Proteose, Soil extract, and Trebouxia.
For 1 L Total
1. To approximately 900 mL of dH2O add each of the components in the order specified while stirring continuously.
2. Bring total volume to 1 L with dH2O.
*For 1.5% agar medium add 15 g of agar into the flask; do not mix.
3. Cover and autoclave medium.
4. Store at refrigerator temperature.
1 NaNO3 (Fisher
BP360-500)
10 mL/L
10 g/400mL
dH2O
2.94 mM
2 CaCl2·2H2O
(Sigma C-3881)
10 mL/L 1 g/400mL dH2O 0.17 mM
流体中文网3 MgSO4·7H2O
(Sigma 230391)
10 mL/L 3 g/400mL dH2O 0.3 mM
4 K2HPO4 (Sigma P
3786)
10 mL/L 3 g/400mL dH2O 0.43 mM
5 KH2PO4 (Sigma P
0662)
10 mL/L 7 g/400mL dH2O 1.29 mM
6 NaCl (Fisher
S271-500)
10 mL/L 1 g/400mL dH2O 0.43 mM
(NO.3) MES-volvox Medium UTEX 2505
Directions
General purpose medium for freshwater strains, especially those requiring ammonium. Suitable for xenic and axenic cultures. Modified volvox medium. For 1 L Total
1. To approximately 950 mL of dH2O, add each of the components in the order specified (except vitamins) while stirring continuously.
2. Adjust the pH to 6.7.
3. Bring the total volume to 1 L with dH2O.
*For 1.5% agar medium add 15 g of agar into the flask; do not mix.
4. Cover and autoclave medium.
5. When cooled add vitamins.
*For agar medium add vitamins, mix, and dispense before agar solidifies.
6. Store at refrigerator temperature.
1 Ca(NO3)2·4H2O (Sigma C
5676)
1 mL/L
11.8 g/100 mL
dH20
0.5 mM
2 MgSO4·7H2O (Sigma 1 mL/L 4 g/100 mL 0.16 mM
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