Snow Algae

From Protists

(Difference between revisions)
(→Snow Algae Media Recipe)
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== Background ==
== Background ==
-
These cultures were originally from Ron Hoham and are currently in the UTEX culture collection.  We were storing them and maintaining them for use in a collaboration between Ron Hoham (now retired from Colgate) and Dr. Jim Leebens-Mack (plant systematist at UGA).  However, most were contaminated with fungi and/or lost because they were killed when the walk-in broke over the summer of 2007 and/or they were not transferred often enough and the agar dried out.
+
These cultures were originally from Dr. Ron Hoham and are currently in the UTEX culture collection.  We were storing them and maintaining them for use in a collaboration between [http://www.colgate.edu/desktopdefault1.aspx?tabid=2353 Dr. Ron Hoham] (now retired from Colgate) and [http://www.plantbio.uga.edu/~jleebensmack/JLMmain.html Dr. Jim Leebens-Mack] (plant systematist at UGA).  However, most cultures were contaminated with fungi and/or lost because they were killed when the walk-in cooler broke over the summer of 2007 and/or they were not transferred often enough and the agar dried out.
 +
 
 +
Also, the original recipe is in a journal article that Dr. Hoham is a co-author on.  However, this recipe has several typos (e.g. 1N Bromate instead of 0.1N Bromate).
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#Measure 500mL DI H<sub>2</sub>O in a [[LIVE]] graduated cylinder
#Measure 500mL DI H<sub>2</sub>O in a [[LIVE]] graduated cylinder
-
#Add appropriate amount of all chemical stock solutions (Table 1) to the graduated cylinder
+
#Add appropriate amount of all chemical stock solutions (Table 1; stored in plastic bottles in the walk in cooler) to the graduated cylinder
#Mix until all chemicals are dissolved in the solution (should be yellow-golden color)
#Mix until all chemicals are dissolved in the solution (should be yellow-golden color)
#Bring volume in graduated cylinder up to 1000mL with DI H<sub>2</sub>O
#Bring volume in graduated cylinder up to 1000mL with DI H<sub>2</sub>O
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! Volume of Stock Solution to Add (mL)
! Volume of Stock Solution to Add (mL)
|-  
|-  
-
| CaCl<sub>2</sub>
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| <center>CaCl<sub>2</sub></center>
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| 0.5g per 100mL
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| <center>0.5g per 100mL</center>
-
| 1
+
| <center>1</center>
|-  
|-  
-
| NaNO<sub>3</sub>
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| <center>NaNO<sub>3</sub></center>
-
| 2.5g per 100mL
+
| <center>2.5g per 100mL</center>
-
| 1
+
| <center>1</center>
|-  
|-  
-
| NH<sub>4</sub>Cl
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| <center>NH<sub>4</sub>Cl</center>
-
| 0.5g per 100mL
+
| <center>0.5g per 100mL</center>
-
| 1
+
| <center>1</center>
|-  
|-  
-
| CaSO<sub>4</sub> x 2H<sub>2</sub>O
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| <center>CaSO<sub>4</sub> x 2H<sub>2</sub>O</center>
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| 0.4g per 100mL
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| <center>0.4g per 100mL</center>
-
| 1
+
| <center>1</center>
|-  
|-  
-
| MgSO<sub>4</sub> x 7H<sub>2</sub>O
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| <center>MgSO<sub>4</sub> x 7H<sub>2</sub>O</center>
-
| 0.5g per 100mL
+
| <center>0.5g per 100mL</center>
-
| 1
+
| <center>1</center>
|-  
|-  
-
| NaSiO<sub>3</sub> x 9H<sub>2</sub>O
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| <center>NaSiO<sub>3</sub> x 9H<sub>2</sub>O</center>
-
| 0.2g per 100mL
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| <center>0.2g per 100mL</center>
-
| 1
+
| <center>1</center>
|-  
|-  
-
| Fe (as EDTA)
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| <center>Fe (as EDTA)</center>
-
| see notes below
+
| <center>see notes below</center>
-
| 250
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| <center>250</center>
|-  
|-  
-
| Trace Elements
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| <center>Trace Elements</center>
-
| see below (Table 2 and notes)
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| <center>see below (Table 2 and notes)</center>
-
| 10
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| <center>10</center>
|-  
|-  
-
| K<sub>2</sub>HPO<sub>4</sub>
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| <center>K<sub>2</sub>HPO<sub>4</sub></center>
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| 3.48g per 100mL (0.2M)
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| <center>3.48g per 100mL (0.2M)</center>
-
| 2
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| <center>2</center>
|-  
|-  
-
| KH<sub>2</sub>PO<sub>4</sub>
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| <center>KH<sub>2</sub>PO<sub>4</sub></center>
-
| 2.72g per 100mL
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| <center>2.72g per 100mL</center>
-
| 98
+
| <center>98</center>
|-
|-
|}
|}
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 +
 +
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Stock Solution of Fe (as EDTA) - use a 1L [[LIVE]] media bottle:
 +
#Dissolve 660mg (0.660g) of Na<sub>2</sub>EDTA in 700mL DI H<sub>2</sub>O and adjust with NaOH or HCl to obtain a pH of 7.5 with the pH meter
 +
#Dissolve 702mg (0.702g) of Fe(NH<sub>4</sub>)<sub>2</sub>(SO<sub>4</sub>)<sub>2</sub> x 6H<sub>2</sub>O in 100mL of DI H<sub>2</sub>O then add this to the Na<sub>2</sub>EDTA solution from step 1.
 +
#Use DI H<sub>2</sub>O to bring final solution volume to 1000mL (1L).
 +
#Use the pH meter to check that the final solution pH is ~3.3-3.5 (adjust with NaOH and/or HCl as necessary)
 +
 +
 +
 +
Stock Solution of Trace Elements - store in a 1L [[LIVE]] media bottle:
 +
#Measure 800mL DI H<sub>2</sub>O in a [[LIVE]] graduated cylinder
 +
#Add appropriate amount of all trace elements (Table 2) to the graduated cylinder
 +
#Mix until all chemicals are dissolved in the solution
 +
#Bring the solution up to a final volume of 1000mL (1L) using DI Hsub>2</sub>O
 +
#Store final solution in labeled media bottle in the walk in cooler (~4C)
 +
 +
 +
 +
 +
<center>'''Table 2.'''  Trace metal ingredients and amounts to make solution.</center>
 +
{| border="1" cellspacing="0" cellpadding="5" align="center"
 +
! Chemical Compound
 +
! Amount to Add to Solution
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|-
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| <center>CuSO<sub>4</sub> x 5H<sub>2</sub>O</center>
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| <center>0.1g</center>
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|-
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| <center>MnCl<sub>2</sub> x 4H<sub>2</sub>O</center>
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| <center>0.1g</center>
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|-
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| <center>Br (0.1 N Solution)</center>
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| <center>1mL</center>
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|-
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| <center>ZnSO<sub>4</sub> x 7H<sub>2</sub>O</center>
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| <center>0.1g per 100mL</center>
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|-
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| <center>CoCl<sub>2</sub> x 6H<sub>2</sub>O</center>
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| <center>0.05g per 100mL</center>
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|-
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| <center>BaCl<sub>2</sub> x 2H<sub>2</sub>O</center>
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| <center>0.01g per 100mL</center>
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|-
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| <center>H<sub>3</sub>BO<sub>3</sub></center>
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| <center>0.1g</center>
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|-
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| <center>FeCl<sub>3</sub> x 6H<sub>2</sub>O</center>
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| <center>0.1g</center>
 +
|-
 +
| <center>Na<sub>2</sub>MoO<sub>4</sub> x 2H<sub>2</sub>O</center>
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| <center>0.05g</center>
 +
|-
 +
|}
 +
 +
 +
 +
== Snow Algae Agar Plates ==
== Snow Algae Agar Plates ==

Revision as of 20:26, 16 October 2007

Background

These cultures were originally from Dr. Ron Hoham and are currently in the UTEX culture collection. We were storing them and maintaining them for use in a collaboration between Dr. Ron Hoham (now retired from Colgate) and Dr. Jim Leebens-Mack (plant systematist at UGA). However, most cultures were contaminated with fungi and/or lost because they were killed when the walk-in cooler broke over the summer of 2007 and/or they were not transferred often enough and the agar dried out.

Also, the original recipe is in a journal article that Dr. Hoham is a co-author on. However, this recipe has several typos (e.g. 1N Bromate instead of 0.1N Bromate).



Snow Algae Media Recipe

  1. Measure 500mL DI H2O in a LIVE graduated cylinder
  2. Add appropriate amount of all chemical stock solutions (Table 1; stored in plastic bottles in the walk in cooler) to the graduated cylinder
  3. Mix until all chemicals are dissolved in the solution (should be yellow-golden color)
  4. Bring volume in graduated cylinder up to 1000mL with DI H2O
  5. Check pH with pH meter and adjust solution to pH ~5.1-5.3 (using HCl or NaOH as needed)
  6. Dispense 500mL into two 1L LIVE media bottles and add 8g of LB Agar to each bottle
  7. Autoclave media bottles on the liquid cycle with at least 30 minutes of sterilization time
  8. Pour contents of media bottles into bottom half of petri dishes (~3/4 full) when media is cool enough (but not hardening!)
  9. Store plates upside-down petri dish bags (taped closed and labeled as Snow Algae Plates) in the walk in cooler (~5C)


Table 1. Chemical stock solutions to be added to snow algae and how to make the stock solutions.
Chemical Compound Stock Solution Composition Volume of Stock Solution to Add (mL)
CaCl2
0.5g per 100mL
1
NaNO3
2.5g per 100mL
1
NH4Cl
0.5g per 100mL
1
CaSO4 x 2H2O
0.4g per 100mL
1
MgSO4 x 7H2O
0.5g per 100mL
1
NaSiO3 x 9H2O
0.2g per 100mL
1
Fe (as EDTA)
see notes below
250
Trace Elements
see below (Table 2 and notes)
10
K2HPO4
3.48g per 100mL (0.2M)
2
KH2PO4
2.72g per 100mL
98


Stock Solution of Fe (as EDTA) - use a 1L LIVE media bottle:

  1. Dissolve 660mg (0.660g) of Na2EDTA in 700mL DI H2O and adjust with NaOH or HCl to obtain a pH of 7.5 with the pH meter
  2. Dissolve 702mg (0.702g) of Fe(NH4)2(SO4)2 x 6H2O in 100mL of DI H2O then add this to the Na2EDTA solution from step 1.
  3. Use DI H2O to bring final solution volume to 1000mL (1L).
  4. Use the pH meter to check that the final solution pH is ~3.3-3.5 (adjust with NaOH and/or HCl as necessary)


Stock Solution of Trace Elements - store in a 1L LIVE media bottle:

  1. Measure 800mL DI H2O in a LIVE graduated cylinder
  2. Add appropriate amount of all trace elements (Table 2) to the graduated cylinder
  3. Mix until all chemicals are dissolved in the solution
  4. Bring the solution up to a final volume of 1000mL (1L) using DI Hsub>2</sub>O
  5. Store final solution in labeled media bottle in the walk in cooler (~4C)



Table 2. Trace metal ingredients and amounts to make solution.
Chemical Compound Amount to Add to Solution
CuSO4 x 5H2O
0.1g
MnCl2 x 4H2O
0.1g
Br (0.1 N Solution)
1mL
ZnSO4 x 7H2O
0.1g per 100mL
CoCl2 x 6H2O
0.05g per 100mL
BaCl2 x 2H2O
0.01g per 100mL
H3BO3
0.1g
FeCl3 x 6H2O
0.1g
Na2MoO4 x 2H2O
0.05g



Snow Algae Agar Plates

  • measure out 525mL Snow Algae Media (above) into a LIVE graduated cylinder
  • add 10.5g of Agar (not agarose!!) to make a 2% weight/volume agar concentration
  • distribute the mixture into a LIVE media bottle(s)
  • heat the mixture on the LIVE hot/stir plate to dissolve the agar completely
  • Autoclave the mixture in the bottles on the liquid cycle for the appropriate time
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