Jan 03, 2024

Public workspaceM9 minimal medium

DOI
dx.doi.org/10.17504/protocols.io.8epv5x5j4g1b/v1
  • 1LabTecLibres
Isaac Sir Núñez
Open access
DOI: dx.doi.org/10.17504/protocols.io.8epv5x5j4g1b/v1
Protocol Citation: Isaac Sir Núñez, Tamara Matute 2024. M9 minimal medium. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x5j4g1b/v1
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: January 02, 2024
Last Modified: January 03, 2024
Protocol Integer ID: 92877
Abstract
M9 minimal medium is a classical bacterial growth broth (Miller, et al., 1972) characterized by its low autofluorescence.
As it is minimal, it can be supplemented with defined carbon sources or amino acids acord the custom experimental setups.


Recipe
Recipe
This is the recipe for 500 mL total medium.
It is assembled from sterile reagents in sterile conditions (in a laminar flow or flame, and using a sterille glass bottle). They have to be added in this order and mixed well after incopore each component.

Amount250 mL Sterile Molecular grade water
Amount100 mL 5X M9 Salt Mix
Amount1 mL MgSO4:7H2O (1M)
Amount10 mL Glucose (20% w/v) --> 0.4 w/v final concentration in the medium
Amount50 µL CaCl2 (1M)
Amount100 mL Casaminoacids (1%)
complete with ~Amount39 mL Sterile Molecular grade water

In case of solid media preparation:
Amount39 mL Sterile Molecular grade water
Amount100 mL 5X M9 Salt Mix
Amount1 mL MgSO4:7H2O (1M)
Amount10 mL Glucose (20% w/v) --> 0.4 w/v final concentration in the medium
Amount50 µL CaCl2 (1M)
Amount100 mL Casaminoacids (1%)

melt Agar 3% in a microwave or hot bath and complete the media with Amount250 mL of it.
It should be added quite hot to allow you mix it well and give you enough time to do the plates before it gets solid.

Antibiotic supplementation can be done after adding the Agar.

Plates could be stores at 4°C and darkness for a couple of days but sometimes they form precipitate.

*** You can replace the carbon source or aminoacids for any other(s) accord your experimental setup ***
Reagents preparation
Reagents preparation
5X M9 Salts (600 mL)

Weight these amounts of each salt and add to a glass bottle:

Amount38.4 g Na2HPO4:7H2O
Amount9 g KH2PO4
Amount1.5 g NaCl
Amount3 g NH4Cl

Then, add Amount600 mL Molecular grade or deionized water and dissolve completely.

Sterilize by autoclaving

MgSO4:7H2O (1M) - 20 mL
Weight Amount4.930 g of MgSO4:7H2O
Complete to Amount20 mL with Molecular grade or deionized water and dissolve completely.

Sterilize by autoclaving

Glucose (20% w/v) - 100 mL
Weight Amount20 g of Glucose.
Complete to Amount100 mL with Molecular grade or deionized water and dissolve completely.

Sterilize by filtering with 0.22 µm syringe filter.

CaCl2 (1M) - 5 mL
Weight Amount0.555 g of CaCl2.
Complete to Amount5 mL with Molecular grade or deionized water and dissolve completely.

Sterilize by filtering with 0.22 µm syringe filter.
Casaminoacids (1%) - 100 mL
Weight Amount1 g of Casaminoacids.
Complete to Amount100 mL with Molecular grade or deionized water and dissolve completely.

Sterilize by autoclaving.

Agar 3% - 500 mL
Weight Amount15 g of Agar (molecular biology grade).
Complete to Amount500 mL with deionized water and dissolve completely.

Sterilize by autoclaving.