Feb 26, 2022
Version 2
MLA Medium Preparation V.2
This document is a draft, published without a DOI.
- Katherine Hunsberger1,
- Kristel Sanchez1
- 1University of Michigan - Ann Arbor
- Duffy Lab, EEB, University of Michigan
Document Citation: Katherine Hunsberger, Kristel Sanchez 2022. MLA Medium Preparation. protocols.io https://protocols.io/view/mla-medium-preparation-b5ngq5bwVersion created by Kira Monell
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: February 26, 2022
Last Modified: February 26, 2022
Document Integer ID: 58792
Abstract
This recipe is used to grow algae in the genus Anabaena, Dolichospermum, and Aphanizomenon in the Duffy lab. It can be used to grow other freshwater cyanobacteria cultures.
Written by: K. Hunsberger, February 2016
Updated by: K. Sánchez, March 2021
Based on methods from Australian National Algae Culture Collection
MLA Medium Preparation
Summary
This recipe is used to grow algae in the genus Anabaena, Dolichospermum, and Aphanizomenon in the Duffy lab. It can be used to grow other freshwater cyanobacteria cultures.
Materials
| A | B | C | |
| Apparatus & Equipment | Labware | Reagents | |
| Autoclave | 1L polyethylene bottles | Sodium nitrate, NaNO3 | |
| Aluminum foil | Graduated cylinder | Calcium chloride, CaCl2•2H2O | |
| Autoclave tape | 500 mL beakers | Magnesium Sulfate, MgSO4•7H2O | |
| Analytical balance | stirring magnet | Potassium phosphate dibasic, K2HPO4 | |
| Weight boats or paper | 5L glass aspirator bottle | Boric acid, H3BO3 | |
| Rubber cap for 5L | Selenious acid, H2SeO3 | ||
| Rubber hose | Copper (II) sulfate, CuSO4 · 5H2O | ||
| spatula | Iron (III) chloride, FeCl3 | ||
| Zinc sulfate, ZnSO4 · 7H2O | |||
| Cobalt (II) chloride, CoCl2 · 6H2O | |||
| Sodium molybdate, Na2MoO4 · 2H2O | |||
| EDTA, Na2EDTA | |||
| Sodium bicarbonate, NaHCO3 | |||
| Manganese (II) chloride, MnCl2 · 4H2O |
1. Stock Solutions Set #1: Using the analytical balance, weight the prescribed amounts of each of chemicals in the table below. Dissolve the prescribed amount of each chemical into 1 L (final volume) of MilliQ water. There will be 5 separate solutions. Store in polyethylene bottles.
2. Vitamin Solution: Use the solution in the fridge labeled VIM. (This VIM solution is from the Standard COMBO Medium Protocol and contains Vitamin B12, Biotin, and Thiamine-HCl. See protocol for specific concentrations.)
3. Micronutrient Primary Stocks: First prepare these 4 primary stocks by adding the prescribed amount to MilliQ water for a final volume of 1L. There will be 4 separate solutions.
| A | B | C | |
| Compound | Symbol | g/L MilliQ | |
| Copper (II) sulfate | CuSO4 · 5H2O | 1.0 | |
| Zinc sulfate | ZnSO4 · 7H2O | 2.2 | |
| Cobalt (II) chloride | CoCl2 · 6H2O | 1.0 | |
| Sodium molybdate | Na2MoO4 · 2H2O | 0.6 |
4. Micronutrient Stock Solutions: Dissolve the prescribed amount of the following constituents in ~800 mL of MilliQ water. Allow each to mix and dissolve completely before adding the next ingredient.
| A | B | C | |
| Compound | Symbol | g | |
| EDTA* | Na2EDTA | 4.36 | |
| Iron (III) chloride | FeCl3 · 6H2O | 1.58 | |
| Sodium bicarbonate | NaHCO3 | 0.60 | |
| Manganese (II) chloride | MnCl2 · 4H2O | 0.36 |
*Add the EDTA first and stir on low heat to fully dissolve.
Then, add 10 of each of the 4 primary stock solutions. Bring the final volume up to 1L. If a precipitate forms, increase the pH up to 7. This solution is the micronutrient stock solution.
5. Stock Solution Set #2: Dissolve the prescribed amount of each chemical into 1 L (final volume) of MilliQ water in 1L flasks. There will be 2 separate solutions.
6.
| A | B | C | |
| Compound | Symbol | g/L MilliQ | |
| Sodium bicarbonate | NaHCO3 | 16.9 | |
| Calcium chloride | CaCl2 · 2H2O | 29.4 |
Autoclave each of the compounds using the setting number 2 of the BSB autoclaves in floors 4th & 5th(or 121 °C for 15 minutes) and then store in 1L glass bottles.
7. MLA x40 Concentrated Nutrient Solution: Prepare this 1L solution by adding the following volumes of the stock ingredients to 540 mL MilliQ water.
| A | B | |
| Solution | Volume to add | |
| MgSO4 · 7H2O | 40 mL | |
| NaNO3 | 80 mL | |
| K2HPO4 | 200 mL | |
| H3BO3 | 40 mL | |
| H2SeO3 | 40 mL | |
| VIM Solution | 20 mL | |
| Micronutrient stock solution | 40 mL |
Filter sterilize using a 0.22 mm filter into a sterile 1L glass bottle.
STORE ALL OF THE ABOVE STOCK SOLUTIONS AND PRIMARY STOCK SOLUTIONS IN THE FRIDGE.
Final MLA Medium Preparation: is prepared by adding ~750 mL MilliQ water for each liter. Then, add the correct amount of each solution according to the following table. After adding all of the solutions, the volume should be increased to the specified final amount and adjusted to a pH of 7.5-8 with HCl (if necessary). Prepare final solution in a 1L or 5L aspirator bottle. Place the rubber stopper laterally (so it does not completely plug the top opening of the aspirator bottle) and completely cover the top with aluminum foil. Cover the end of the rubber hose with aluminum foil (or connect it to the second aspirator bottle if setting up a new culture). Autoclave the mixture by selecting program number 2 in the autoclaves found in the 4 or 5th floor of the BSB building (or at 121 °C for 15 min). Allow to cool in autoclave overnight, if possible. This helps to minimize amount of precipitate.
| A | B | C | |
| Solution | Vol. per 1L | Vol. per 5L | |
| Sterile MLA x40 concentrated nutrients | 25 mL | 125 mL | |
| Sterile NaHCO3 | 1 mL | 5 mL | |
| Sterile CaCl2 · 2H2O | 1 mL | 5 mL |