Mar 11, 2022
Version 2
Minimal Basal Medium (liquid) V.2
- 1University of Georgia
Protocol Citation: Christa Smith 2022. Minimal Basal Medium (liquid). protocols.io https://dx.doi.org/10.17504/protocols.io.b58kq9uwVersion created by Christa Smith
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: March 11, 2022
Last Modified: March 11, 2022
Protocol Integer ID: 59372
Keywords: Minimal basal medium
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Abstract
A defined artificial seawater medium for growing bacteria.
Attachments
MBM.docx
19KB
Guidelines
Increase or decrease amount proportionally to prepare stock quantities as needed. Add dry reagents slowly to allow for dissolution in between each addition. Solutions will be prepared and autoclaved separately, except for carbon and vitamins which are filter sterilized through 0.2 micron PES membranes. Each autoclaved solution should be cooled completely before combining - remember to autoclave extra bottles or graduated cylinders as needed for measuring and mixing. The carbon source should only be added after autoclaved solutions have cooled; it is filter sterilized into the medium and can be added during mixing or at any time before setting up a culture. Extra solutions can be stored frozen or at 4 Celsius. This recipe is for preparing 1 L of liquid medium, but plates can be made as well with slight variation.
Before start
Acid wash all glassware with 10% HCl and rinse well with dH2O.
Sea Salt Solution
Sea Salt Solution
Add 20 g Sigma Sea Salts into a 1 L glass bottle.
Add 699 mL dH2O to sea salts in bottle and mix well to dissolve.
Note
Add a stir bar to bottle if needed to mix and dissolve salts.
Note
Remember if adding Bacto Agar that this will not dissolve until solution is autoclaved.
Set prepared sea salt solution aside until ready to autoclave.
FeEDTA Stock
FeEDTA Stock
Add 50 mg FeEDTA into a 250 mL glass bottle.
Add 100 mL dH2O to FeEDTA in bottle and mix well to dissolve (stock concentration is 1.36 mM).
Filter-sterilize FeEDTA solution using a 0.2 micron PES membrane filter.
Basal Medium
Basal Medium
Add 150 mL of 1 M Tris HCl, pH 7.5, into a 1 L glass bottle.
Note
Accurate pH is very important!
Add 0.34 g NH4Cl into the same bottle and dissolve.
Add 182.98 mg K2HPO4 into the same bottle and dissolve.
Add 375 mL dH2O to the same bottle and mix well.
Set prepared basal medium aside until ready to autoclave.
Autoclave
Autoclave
Autoclave sea salt solution and basal medium.
Note
Remember to throw in extra bottles and/or graduated cylinders if needed.
Note
Don't forget to loosen the lids before and tighten the lids after!
Note
Do not combine solutions before autoclaving.
Cool autoclaved solutions to room temperature.
Mix
Mix
Add 50 mL of FeEDTA stock to the bottle of sea salt solution and mix well.
Add 250 mL basal medium to the bottle of sea salt solution and mix well.
Add 1 mL of vitamin supplement the the bottle of sea salt solution and mix well.
Protocol
NAME
MBM Vitamin SupplementCREATED BY
Christa Smith
Measure 950 mL dH2O into a 1 L glass bottle.
Dissolve 2 mg biotin into the bottle.
Dissolve 2 mg folic acid into the bottle.
Dissolve 10 mg pyridoxine-HCl into the bottle.
Dissolve 5 mg riboflavin into the bottle.
Dissolve 5 mg thiamine into the bottle.
Dissolve 5 mg nicotinic acid into the bottle.
Dissolve 5 mg pantothenic acid into the bottle.
Dissolve 0.1 mg cyanocobalamin into the bottle.
Dissolve 5 mg p-aminobenzoic acid into the bottle.
Bring final volume to 1 L with dH2O.
Filter sterilize final vitamin solution through a 0.2 micron PES membrane.
Store frozen in 1 mL aliquots.
Finish
Finish
Store prepared medium at 4 Celsius.
Note
Prepared medium can be filtered (0.2 micron PES membrane) if needed.
Carbon source
Carbon source
A carbon source can be added to the finished minimal medium.
Note
Carbon source (type and concentration) can be varied as needed.
Note
Carbon source stock solution should be filter-sterilized through a 0.2 micron PES membrane filter and stored frozen until needed.