Snow Algae

From Protists

Contents 1 Background 2 Snow Algae Media Recipe 2.1 Chemical Stock Solution Instructions 2.2 Trace Metal Solution Instructions 3 Snow Algae Agar Plates

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. See directions below on how to make Snow Algae Plates

Chemical Stock Solution Instructions

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)

Trace Metal Solution Instructions

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
CoCl2 x 6H2O	0.05g
BaCl2 x 2H2O	0.01g
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 media bottle using a LIVE graduated cylinder
• add 10.5g of LB (? check original paper recipe) 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
• Pour contents of media bottles into bottom half of petri dishes (~3/4 full) when media is cool enough (but not hardening!)
• Store plates upside-down in petri dish bags (taped closed and labeled as Snow Algae Plates) in the walk in cooler (~5C)

Alternatively, could do 500mL Snow Algae Media with 8g of LB agar as the original recipe specified, but then agar not as firm.