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(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. # Component 1 2
NaNO3 (Fisher 3 BP360-500)
Na2HPO4·7H2O 4
(Sigma S-9390) 5 6
Amount 3 L
36 mL/3 L 10 mL/3 L 10 mL/3 L 150 mL/3 L 3 mL/3 L
Stock Solution Concentration 0.7 M 0.02 M
Final
Concentration 2.3 mM 0.067 mM
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. # Component
Amount
Stock Solution Concentration
1
900 mL
2
NaCl (Fisher S271-23 g/L
500) MgCl2·6H2O (Fisher M 33)
5 g/L
393.8 mM
Final
Concentration
3 24.6 mM
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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 \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
\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.
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2. Bring total volume to 1 L with dH2O. 3. Store at refrigerator temperature.
# Component 1 2
Na2EDTA·2H2O (Sigma ED255) FeCl3·6H2O (Sigma F-1513) MnCl2·4H2O (Baker 2540) ZnCl2 (Sigma Z-0152) CoCl2·6H2O (Sigma C-3169) Na2MoO4·2H2O (J.T. Baker 3764)
Amount 0.75 g/L 0.097 g/L 0.041 g/L 0.005 g/L 0.002 g/L 0.004 g/L
Stock Solution Concentration
Final
Concentration 2 mM 0.36 mM 0.21 mM 0.037 mM 0.0084 mM 0.018 mM
3
4 5 6
3) Soilwater: GR+ Medium
Directions
The basic garden-type soilwater; includes a pinch of CaCO3, which is added to the soil and water prior to steaming; suitable for most phototrophic freshwater algae. Optional ingredients: add vitamin B12 to cultures of Volvox; a pinch of NH4MgPO4. 6H2O added to soilwater cultures of Botryococcus, Synechococcus and some Euglenoids enhances growth and to LB 826 Gonium pectorale increases the numbers of 16-celled colonies formed. For 200 mL Total
1. Combine all components listed.
2. Cover the medium container and steam for 2 consecutive days, 3 hours on each day. Pasteurization is a gradual rising of temperature to approximately 95°C in 15 minutes. Then increased just over 98°C for the 3 hour duration. Cooling occurs gradually at room temperature.
3. Refrigerate 24 hours or more and bring to room temperature before using.
# Component
Amount
Stock Solution Concentration
Final
Concentration
1 2
1 tsp/200 mL dH20
0.05 mM
CaCO3 (optional) 1 mg/200 mL
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(Fisher C 64)
dH2O
a)Green House Soil
Directions
Preparation of the Green house soil:
Prior to its use in soil-water media, treat soil in batches by placing it in a heat-resistant pan lined with aluminum foil, fill the soil to a so depth of ? inch, and bake at 150°C for 2 hours. After it cools, cover the pan with aluminum foil and store in darkness at room temperature. Avoid excessive moisture during storage.
Adaptation of E.G. Pringsheim's biphasic soil-water medium. Variations of this medium are suitable for xenic cultures, especially for isolation purposes and for growing algae to secure \medium, yet not all soil is suitable for culturing a broad range of algae. UTEX utilizes soil that was obtained in the early 1970s as greenhouse soil from Indiana University. For a long shelf life it must be kept dry and away from light. UTEX keeps it in sealed 5-gallon plastic containers. There is almost certainly nothing special about the particular soil used by UTEX. However, several 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.
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 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 physically 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.
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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.
# Component HEPES buffer pH 1 7.8 (Sigma H- 3375)
Vitamin B12
2 (cyanocobalamin, (Sigma V-6629)
Amount 2.4 g/200 mL dH2O 0.027 g/200 mL dH2O
Stock Solution Concentration
Final
Concentration
(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. # Component
Amount 1 L
Stock Solution Concentration
Final
Concentration
1
2
Proteose Peptone
1 g/L
(BD 211684)
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.
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微藻养殖培养基介绍
