Dec 19, 2019

Public workspaceSynthetic media (A1) for Aurantiochytrium limacinum (ATCC MYA-1381)

DOI
dx.doi.org/10.17504/protocols.io.bafuibnw
  • 1University of Illinois at Urbana-Champaign;
  • 2State University of New York at Stony Brook
  • Collier Lab
Anbarasu Karthikaichamy
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DOI: dx.doi.org/10.17504/protocols.io.bafuibnw
Protocol Citation: Anbarasu Karthikaichamy, Jackie Collier 2019. Synthetic media (A1) for Aurantiochytrium limacinum (ATCC MYA-1381). protocols.io https://dx.doi.org/10.17504/protocols.io.bafuibnw
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 and it's working
Created: December 12, 2019
Last Modified: December 19, 2019
Protocol Integer ID: 30932
Keywords: Aurantiochytrium, media, synthetic, protists, recipe
Abstract
Synthetic media recipe for Aurantiochytrium limacinum (ATCC MYA-1381). This media may not be suitable for Aplanochytrium and Oblongichytrium.
Guidelines
Work under sterile conditions (use flame). Use gloves, wipe the bench with 70% ethanol.
Safety warnings
The autoclaved bottles and solutions will be hot! Always wear gloves and appropriate clothing before handling autoclaved materials.
Heavy metals are toxic for the environment and need to be discarded accordingly.
Before start
Thaw the antibiotics on ice.
Autoclave empty media bottles for trace metal mix and vitamins.
Autoclaved water for preparing 1X stocks.
5x A1stock
5x A1stock
Measure and add the following components to an appropriate media bottle. Make up the solution to desired volume with water. Autoclave for Duration00:20:00 at 15 PSI and Temperature121 °C

Componentsg/L
CaCl2·2H2O2.5
Glycerol150
KCl 2
KH2PO4 20
maleic acid 29
MgCl2 4
Na2SO4 15
NaCl72
Sodium-glutamate hydrate125
Tris base30

Note
The composition is for 5X

Trace metal mix
Trace metal mix
Measure and add the following components to an amber bottle. Make up the solution to desired volume with water. Filter sterilize and refrigerate the trace metal mix.

Componentsg/L
FeSO4 · 7H2O50
CuSO4 · 5H2O3.9
ZnSO4 · 7H2O4.4
MnSO4 · H2O1.5
Na2MoO4 · 2H2O0.1
CoCl2 · 6H2O in 0.6 M HCl0.2
Note
Do not autoclave!

Vitamins
Vitamins
Measure and add the following components to individual amber bottles. Make up the solution to desired volume with water. Filter sterilize the vitamins separately and refrigerate.
Componentsg/L
Thiamine·HCl (B1)5.00E-05
Cyanocobalamin (B12)5.00E-06

Note
Do not autoclave!

Prepare working stock (1X)
Prepare working stock (1X)
For Amount1 L ,
Add the stock solutions to an autoclaved bottle.

Stock solutionVolume (mL)
A1 stock (5X )200
Vitamin B1 (1000X)1
Vitamin B12 (10000X)0.1
Trace metal mix (400X)2.5
Make up the media to Amount1 L using autoclaved Millipore water.

Note
To prevent bacterial contamination, add Amount1 mL of 100 mg/mL Ampicillin to the final media.

One of the vitamins may not be added for the A1 vitamin drop out media.

A1 plates
A1 plates
  • Prepare 1X stock of A1 media (do not add trace metal and vitamins).

  • Add 1% (w/v) agar in to the 1X A1 media.

  • Autoclave for Duration00:20:00 at 15 PSI and Temperature121 °C . Allow the solution cool down.

  • Add the trace metal mix, vitamins and appropriate antibiotics.

  • Pour the plates and store it in the refrigerator until use.