微藻养殖培养基介绍-精品

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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. 1 2

NaNO3 (Fisher 3

BP360-500) Na2HPO4·7H2O 4

(Sigma S-9390) 5 6

3 L

36 mL/3 L 10 mL/3 L 10 mL/3 L 150 mL/3 L 3 mL/3 L

0.7 M 0.02 M

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. 1 2

900 mL

NaCl (Fisher S271-23 g/L

500)

5 g/L

393.8 mM

24.6 mM

6H2O 3 MgCl2·

(Fisher M 33) Na2SO4 (Sigma S 6264)

4 g/L

28.2 mM

4

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

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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 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

\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 2 3

Na2EDTA·2H2O (Sigma ED255) FeCl3·6H2O (Sigma F-1513) MnCl2·4H2O (Baker 2540) ZnCl2 (Sigma Z-0152) CoCl2·6H2O (Sigma C-3169) Na2MoO4·2H2O

0.75 g/L 0.097 g/L 0.041 g/L 0.005 g/L 0.002 g/L 0.004 g/L

2 mM 0.36 mM 0.21 mM 0.037 mM 0.0084 mM 0.018 mM

4 5 6

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(J.T. Baker 3764) 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. 1 tsp/200 mL dH20 1 0.05 mM 2 CaCO3 (optional) 1 mg/200 mL (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 \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 3文档收集于互联网，如有不妥请联系删除.

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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.

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. HEPES buffer pH 1 7.8 (Sigma H-3375)

Vitamin B12

2 (cyanocobalamin, (Sigma V-6629)

2.4 g/200 mL dH2O 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

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Proteose Peptone

1 g/L

(BD 211684)

2

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 2 3

NaNO3 (Fisher

10 mL/L

BP360-500) CaCl2·2H2O

10 mL/L

(Sigma C-3881) MgSO4·7H2O

10 mL/L

(Sigma 230391) K2HPO4 (Sigma P

10 mL/L

3786)

KH2PO4 (Sigma P

10 mL/L

0662)

NaCl (Fisher

10 mL/L

S271-500)

10 g/400mL dH2O

2.94 mM

1 g/400mL dH2O 0.17 mM 3 g/400mL dH2O 0.3 mM 3 g/400mL dH2O 0.43 mM 7 g/400mL dH2O 1.29 mM 1 g/400mL dH2O 0.43 mM

4 5 6

(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. Ca(NO3)2·4H2O (Sigma C

1 mL/L

5676) 2 MgSO4·7H2O (Sigma 1 mL/L 1

11.8 g/100 mL

0.5 mM

dH20

4 g/100 mL 0.16 mM

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230391)

Na2glycerophosphate.5H2O 3 1 mL/L (Sigma G 6501 ) 4 KCl (Fisher P 217) 5 MES (Sigma M-8250) 6

1 mL/L 1.95 g/L 6 mL/L

dH20

5 g/100 mL dH20

5 g/100 mL dH20

0.16 mM 0.67 mM 10 mM

7 NH4Cl (Fisher A 649-500) 1 mL/L 8 9

2.67 g/100 mL

0.5 mM

dH20

1 mL/L 1 mL/L

a) Biotin Vitamin Solution

Directions

For 200 mL Total

1. Prepare 200 mL of HEPES buffer (50 mM). 2. Adjust the pH to 7.8.

3. Add biotin (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. HEPES buffer pH 1 7.8 (Sigma H-3375)

Biotin 2

(Sigma B-4639)

2.4 g/200 mL dH2O 0.005 g/200 mL dH2O

(NO.4)Enriched Seawater Medium UTEX LB 1926

Directions

Modification of L. Provasoli's ES-enrichment for seawater PES (Bold & Wynne 1978). General purpose marine medium for xenic cultures.

1. Aseptically add 20 mL of sterile ES Enrichment Solution per liter of Pasteurized Seawater (30 ppt).

2. Store at refrigerator temperature. 1 2

1) Enrichment Solution for Seawater Medium

1 L

20 mL/L

Directions

For 2 L Total

1. To approximately 1 L of dH2O, add each of the components in the order specified (except vitamins) while stirring continuously. 2. Adjust the pH to 7.8.

3. Bring the total volume to 2 L with dH2O.

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4. Cover and autoclave medium. 5. When cooled add vitamins.

6. Store at refrigerator temperature. 1 NaNO3 (Fisher BP360-500) 4.7 g/2 L

27.65 mM

Na2glycerophosphate.5H2O 2 0.7 g/2 L (Sigma G 6501 ) 3 4

325 mL/2 L

1.6 mM

14 mM

325 mL/2 L

5

HEPES buffer (Sigma H-3375)

6.5 g/2 L

3 mL/2 L

6 7 8

3 mL/2 L

3 mL/2 L

a) ES Fe Solution

Directions For 2 L Total

1. To approximately 1.5 L of dH2O, add the following components in the order listed while stirring continuously.

2. Bring total volume to 2 L with dH2O. 3. Store at refrigerator temperature. Fe(NH4)2(SO4)2·6H2O

1.4 g/2 L

(Sigma F-1513) Na2EDTA·2H2O 2 1.2 g/2 L (Sigma ED255) 1

1.8 mM 1.6 mM

b) P-II Metal Solution

Directions

For 100 mL Total

1. To approximately 70 mL of dH2O, add each of the components in the order specified while stirring continuously.

2. Bring total volume to 100 mL with dH2O. 3. Store at refrigerator temperature.

Note: CoCL2 can be interchancable with CoSO4. Na2EDTA·2H2O

0.1 g/100 mL

(Sigma ED255) 2 H3BO3 (Baker 0.114 g/100 mL 1

0.27 mM 1.8 mM

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0084) FeCl3·6H2O (Sigma F-1513) MnSO4·H2O (Sigma M8179) ZnSO4·7H2O (Sigma Z 0251) CoCl2·6H2O (Sigma C-3169)

3 4 5 6

4.9 mg/100 mL 16.4 mg/100 mL 2.2 mg/100 mL 0.48 mg/100 mL

0.018 mM 0.097 mM 0.007 mM 0.002 mM

c) Thiamine Vitamin Solution

Directions

For 50 mL Total

1. Prepare 50 mL of HEPES buffer (50 mM). 2. Adjust the pH to 7.8.

3. Add Thiamine (6.5 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. HEPES buffer pH 1 7.8 (Sigma H-3375)

Thiamine (Sigma 2

T-1270)

1.2 g/100 mL dH20 0.11 g/100 mL dH20

(NO.5)Soil Extract + Sodium Metasilicate UTEX 640

Directions

Modification of Soil Extract Medium for diatoms. For 1 L Total

1. Prepare 1 L of Bristol Medium and thoroughly mix. 2. Discard 40 mL of the Bristol Medium.

3. Add 40 mL of previously prepared GR+ Medium [Note: The GR+ should be filtered to remove soil particles].

* or 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 filter sterilized sodium metasilicate.

*For agar medium, add sodium metasilicate, mix, and dispense before agar solidifies. 6. Store at refrigerator temperature. 1 2 3

Sodium Metasilicate 960 mL

40 mL of supernatant 1 mL

200 mM

200 μM

(NO.6)

Erdschreiber's Medium UTEX LB

1002 and 2307 Directions

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Modified from the original Plymouth seawater recipe. General purpose marine medium for xenic cultures [for bacteria-free cultures see reprints in Rosowski & Parker (1971)].

For 3 L Total

1. To 3 L of pasteurized filtered seawater (30 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 2

NaNO3

(autoclave before 3

adding) (Fisher BP360-500) Na2HPO4·7H2O

(autoclave before 4

adding) (Sigma S-9390) 5 6

3 L

36 mL/3 L 10 mL/3 L

0.7 M

2.3 mM

10 mL/3 L 150 mL/3 L 3 mL/3 L

0.02 M 0.067 mM

【关键字】微藻

(NO.7)TAP medium (original )

from Gorman, D.S., and R.P. Levine (1965) Proc. Natl. Acad. Sci. USA 54, 1665-1669. This is probably the most widely-used medium at present for experimental work.

Make the following stock solutions:

1. TAP salts NH4Cl

MgSO4 . 7H2O

15.0 g 4.0 g

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CaCl2 . 2H2O water to 1 liter

2.0 g

2. phosphate solution K2HPO4 KH2PO4

water to 100 ml

28.8 g 14.4 g

3. Hutner's trace elements (follow this )

To make the final medium, mix the following:

2.42 g Tris

25 ml solution #1 (salts)

0.375 ml solution #2 (phosphate) 1.0 ml solution #3 (trace elements) 1.0 ml glacial acetic acid water to 1 liter

For solid medium, add 15 g agar per liter

Autoclave.

For Tris-minimal medium omit the acetic acid and titrate the final solution to pH 7.0 with HCl

(1) Hutner's trace elements

Hutner et al. (1950) Proc. Am. Philos. Soc. 94, 152-170 This mixture is used both in and in the medium.

For a detailed analysis of how well this trace elements solution meets the nutritional requirements of C. reinhardtii, see Merchant et al. (2006) Biochim. Biophys. Acta 1763, 578-594.

For 1 liter final mix, dissolve each compound in the volume of water indicated.

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The EDTA should be dissolved in boiling water, and the FeSO4 should be prepared last to avoid oxidation.

compound amount water EDTA disodium salt 50 g 250 ml ZnSO4 . 7 H2O 22 g 100 ml H3BO3 11.4 g 200 ml MnCl2 . 4 H2O 5.06 g 50 ml CoCl2. 6 H2O 1.61 g 50 ml CuSO4 . 5 H2O 1.57 g 50 ml (NH4)6Mo7O24. 4 H2O 1.10 g 50 ml FeSO4. 7 H2O 4.99 g 50 ml Mix all solutions except EDTA. Bring to boil, then add EDTA solution. The mixture should turn green. When everything is dissolved, cool to 70 degrees C. Keeping temperature at 70, add 85 ml hot 20% KOH solution (20 grams / 100 ml final volume). Do NOT use NaOH to adjust the pH.

Bring the final solution to 1 liter total volume. It should be clear green initially. Stopper the flask with a cotton plug and let it stand for 1-2 weeks, shaking it once a day. The solution should eventually turn

purple and leave a rust-brown precipitate, which can be removed by filtering through two layers of Whatman#1 filter paper, repeating the filtration if necessary until the solution is clear. Store

refrigerated or frozen convenient aliquots. Some people shorten the time for formation of the

precipiate by bubbling the solution with filtered air. If no precipitate forms, the solution is still usable. However, you might want to check the pH in this case and adjust it to around 7.0 using either KOH or HCl as needed.

To prepare sulfur-free trace elements for hydrogen generation, the sulfate salts can be replaced with equimolar chloride salts (ZnCl2 10.0 g; CuCl2 . 2 H2O 1.00 g; FeCl2 . 4 H2O, 3.60 g). .

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