Jun 12, 2020
A green micro-algal growth media modified for use as a stringent minimal media for bacteria.
- 1University of West Georgia
External link: https://f1000research.com/articles/9-656/v1
Protocol Citation: Mautusi Mitra 2020. A green micro-algal growth media modified for use as a stringent minimal media for bacteria.. protocols.io https://dx.doi.org/10.17504/protocols.io.bgzujx6w
Manuscript citation:
1. Mitra M, Nguyen KMAK, Box TW et al. Isolation and characterization of a novel Sphingobium yanoikuyae strain variant that uses biohazardous saturated hydrocarbons and aromatic compounds as sole carbon sources [version 1; peer review: 2 approved]. F1000Research 2020, 9:767 (https://doi.org/10.12688/f1000research.25284.1)
2. Mitra M, Nguyen KMAK, Box TW et al. Isolation and characterization of a novel bacterial strain from a Tris-Acetate-Phosphate agar medium plate of the green micro-alga Chlamydomonas reinhardtii that can utilize common environmental pollutants as a carbon source [version 1; peer review: 3 approved]. F1000Research 2020, 9:656 (https://doi.org/10.12688/f1000research.24680.1)
License: This is an open access protocol 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
Protocol status: Working
We use this protocol in our group and it is working.
Created: May 31, 2020
Last Modified: June 12, 2020
Protocol Integer ID: 37652
Keywords: TAP medium, M9 medium, Chlamydomonas reinhardtii, alternative carbon source, bacteria grown in TAP medium, TP medium, acetate-consuming bacteria, alternative carbon souces, bacterial minimal medium
Abstract
Introduction:
Chlamydomonas reinhardtii, a green micro-alga can be grown at the lab heterotrophically or photo-heterotrophically at room temperature using the Tris-Phosphate-Acetate (TAP) medium which contains 0.1 % acetate (acetic acid) as the sole carbon source. Standard TAP medium recipe can be found at the website of Chlamydomonas Resource Center: https://www.chlamycollection.org/methods/media-recipes/tap-and-tris-minimal/. Hutner’s trace element solution is an ingredient in the TAP medium. Hutner’s trace element recipe can be found at https://www.chlamycollection.org/methods/media-recipes/hutners-trace-elements/. M9 medium is the standard minimal medium for growing bacteria (http://www.thelabrat.com/protocols/m9minimal.shtml). When grown in TAP medium, Chlamydomonas can utilize the exogenous acetate in the medium to make net biosynthesis of sugar using the Glyoxylate cycle and can grow either heterotrophically or photo-heterotrophically without being dependent on photosynthesis for glucose biosynthesis. Higher green plants have the glyoxylate cycle which they use to make sugar from internal pool of acetyl CoA but cannot take up exogenous acetate like Chlamydomonas.
In our lab we use a slightly modified TAP medium recipe which has a final concentration of phosphate, nitrogen, magnesium and calcium approximately 10-fold, 2-fold, 2-fold and 2-fold higher than that in the TAP recipe described on the Chlamydomonas Resource Center website, respectively. Final concentrations of acetate and Hutner’s trace elements are same in both TAP recipes. Attached Table 1 compares the chemical ingredients in our lab’s TAP recipe (full recipe can be found on protol.io) with that present in the standard M9 medium described at http://www.thelabrat.com/protocols/m9minimal.shtml. M9 medium has a final concentration of phosphate, nitrogen, magnesium and carbon, approximately 70-fold, 2.5-fold, 4-fold and 5-fold higher than that present in our lab’s TAP medium, respectively. Additionally, M9 contains 0.05% salt (8.56 mM) (Table 1). TAP has additional trace elements like iron, zinc, copper, manganese, cobalt, boron and molybdenum which are components in the Hutner’s trace element solution (Table1). In summary, TAP medium is chemical composition wise, a more stringent minimal medium than the M9 medium used by microbiologists.
Applications:
There are many bacteria that can also utilize acetate as a carbon source. Some of these bacteria also have glyoxylate cycle like Chlamydomonas and green plants. Hence, TAP medium can be used to isolate acetate-requiring bacteria. If acetate is removed from the TAP medium, it becomes the TP medium which lacks a carbon source and will not allow Chlamydomonas or other acetate-requiring bacteria to grow. The 0.1 % acetate in the TAP medium can be substituted with alternative carbon sources (e.g. glucose, sucrose, lactose, hydrocarbons, aromatic compounds and polyhydroxyalkanoates etc.) to test physiological abilities of candidate bacteria to use the tested chemicals as the sole alternative carbon and energy source.
Attachments
Table 1.pdf
40KB
Guidelines
Do not use NaOH for pHing Hutner's trace element. Use KOH pellets and solution for pHing Hutner's trace element.
Materials
MATERIALS
Cobaltous chloride hexahydrate
Zinc sulfate heptahydrateSigma AldrichCatalog #204986
Boric acidFisher ScientificCatalog #BP1681
Manganese chlorideFisher ScientificCatalog #7773-01-5
Ammonium molybdate (VI) tetrahydrateFisher ScientificCatalog # 12054-85-2
Calcium chloride, dihydrateBio Basic Inc.Catalog #CD0050.SIZE.500g
Copper (II) sulfate pentahydrateBio Basic Inc.Catalog #CDB0063.SIZE.500g
Iron (II) sulfate, heptahydrateBio Basic Inc.Catalog #FB0461.SIZE.500g
Potassium hydroxideBio Basic Inc.Catalog #PB0441.SIZE.500g
Magnesium Sulfate Heptahydrate, ACS GradeGold BiotechnologyCatalog #M-020
Acetic acid GlacialFisher ScientificCatalog #A38-212
Tris BaseFisher ScientificCatalog #604204
Potassium phosphate Dibasic Sigma Aldrich
Potassium Phosphate, Monobasic, Molecular Biology Grade, Calbiochem™, , 1kgThermo FisherCatalog #5295681KG
Ammonium ChlorideFisher ScientificCatalog #A661-500
Before start
You can purchase all chemicals needed to make the TAP medium from any vendor that sells them and do not have to use the vendors that I have selected under "Materials" .
Preparation of TAP stock nutrients
Preparation of TAP stock nutrients
Make TAP salts (TAP stock nutrients/FBS solution [Filner’s Beijerinck solution X40]) by dissolving Ammonium chloride (16g), Magnesium sulfate (4g) and Calcium chloride (2.65g). This will give you a final concentration of: Ammonium chloride (7.48 mM ), Magnesium sulfate (16.2 mM) and Calcium chloride (18.10 mM).
Preparation of Phosphate solution
Preparation of Phosphate solution
Make Phosphate solution (1M Potassium phosphate solution, pH7) by dissolving 28.8 g of K2HPO4 and 14.4g of KH2PO4. Note: You don’t have to adjust pH for this solution as it automatically gives a pH of 7-7.2 when mixed in the above stated ratio. Phosphate solution has the final concnetration of K2HPO4 (1.65M) and KH2PO4 (1.058M).
Preparation of Hutner's trace element
Preparation of Hutner's trace element
Preparation of Hutner’s trace element (X 1000) (Note: If you do not want to make it, you can purchase this solution from Chlamydomonas Resource center at at https://www.chlamycollection.org/methods/media-recipes/hutners-trace-elements/). In order to make this solution, dissolve the following salts in order in 800 mL of E-pure water—dissolve each fully before adding the next.
| Salt | Molecular Weight | Final concentration in 1L | Amount to be added in 1L | |
| FeSO4.7H2O | 278.01 | 18 mM | 4.99 g | |
| ZnSO4 .7 H2O | 287.56 | 76.5mM | 22 g | |
| H3BO3 | 61.83 | 184 mM | 11.4 g | |
| MnCl2.4H2O | 197.91 | 25.6 mM | 5.06 g | |
| CuSO4.5H2O | 249.68 | 6.3mM | 1.57 g | |
| (NH4)6Mo7O24. 4 H2O | 1235.86 | 0.89mM | 1.10 g | |
| CoCl2.6H2O | 237.93 | 6.8mM | 1.61 g |
Table of chemical ingredients needed to make Hutner's trace element.
Bring the salt mixture to a slow boil. Add 50 grams of disodium salt of EDTA to the boiling mixture, acid form (Na2 EDTA.2H2O; molecular weight is 372.24; final concentration in the 1L solution is 134 mM).
Add KOH pellets (and not NaOH) to the boiling mixture to adjust the pH to 6.5. Make up the volume to 1L with pure water after adjusting the pH to 6.5.The solution should be clear green at this point.
Pour the green solution in a 1 L bottle. Close the cap not too tightly. Shake it occasionally every week. The color will slowly change to dark magenta/purplish color over time. If you see any brown precipitate, filter the solution through two layers of Whatman#1 filter paper, repeating the filtration if necessary until the solution is clear.
Preparation of TAP medium
Preparation of TAP medium
To make the final TAP medium, mix the following in 950 L of water:
2.42 g Tris base/Trizma , 25 ml solution #1 (salts), 0.375 ml solution #2 (phosphate), 1.0 ml solution #3 (trace elements) and 1.0 ml glacial acetic acid (0.1% in the final 1 L of TAP medium).
Check the pH of the solution before making up the volume to 1L. pH of TAP medium should be approximately between 7- 7.2 once you make it but if it is not, adjust pH with acid/base. The final concentration sof all chemical ingredients that you have added to make TAP medium is given in the attached Table 1 pdf.
Preparation of TAP-agar plates
Preparation of TAP-agar plates
For preparing solid TAP-agar media plates to maintain Chlamydomonas in lab, add 15 grams of agar per liter of the media after checking the pH, shake it and then autoclave. Note: Make sure to have a magnetic stir bar inside the agar media bottle before you autoclave the bottle. Stirring using a magnetic stirrer is required during the cooling of the hot media after autoclaving to mix agar uniformly in the media solution before pouring media plates.
Preparation of TP medium
Preparation of TP medium
TP medium is prepared exactly the same way as the TAP medium, except glacial acetic acid is not added. Hence TP medium has no carbon souce. Nothing will grow on it unless you add a carbon source to the TP medium. You will pH the TP medium like you would for making TAP medium. You can add your preferred alternative carbon source (e.g. glucose, sucrose, lactose, hydrocarbons, aromatic compounds and polyhydroxyalkanoates etc.) for testing biochemical abilites of bacteria to use exogenous carbon sources for energy production and growth.