Neidhardt EZ Rich Defined

Background

This media is a slight variant on that defined by Neidhardt et al. The modifications were made by Blattner et al. This is a rich defined medium which should allow repeatable high growth rates. The recipe from below is edited from the E. Coli Genome Project. It would be our intention to use this as a standard media for all characterization work as a replacement for supplemented M9 media.

EZ Media has the following advantages -

• Concentrations of the major nutrients can be varied independently (C, P, N, S)
• Concentrations of all constituents are defined
• Growth rate should be similar to that of 'E. coli' in rich media such as LB
• Supports a high density of cell growth
• Stable and gives reproducible results
• Low background fluorescence, can be used for fluorescent measurements
• Excellent pH buffering capacity
• The levels of micronutrients (eg. copper, manganese etc.) are set to a level that is saturating for growth but also sufficiently low that there is no possibility of growth inhibition. Variance in the levels of these micronutrients due to contamination of glassware or water source should not move the concentrations out of this optimum range.

It also has the following disadvantages -

• It is expensive if bought from a commercial supplier, and it is slow to make up on-site.
• Some of the constituents and their levels are arbitrarily chosen. For example, NaCl is added to the medium, yet it function in aiding bacterial growth was unknown at the time of development.
• Due to the heat lability of the MOPS buffer, it cannot be autoclaved.

Endy Lab Materials Sourcing & Usage

• We buy the two of the ingredients (10X ACGU and the 5X Supplement EZ) from Teknova because they are tedious to make. The Endy Lab has an account with Teknova (see Barry for details).
• The kitchen makes up batches of 10X MOPS Mixture for the us.
• The kitchen also makes K₂HPO₄ for the us.
• Currently, because some of the ingredients are stored in our freezer we are making up the final media mix. In the future we may get the kitchen to make up the whole thing except for the carbon source so people can choose what they want to use. As listed in the protocol section, the carbon source should be glycerol to conform to the working SBWG media standards.
• I have 4 L of the final media made up so people can test their experiments in this media before switching over entirely. Please see me if you want to get some --Bcanton 14:16, 7 Jun 2005 (EDT)
• As the kitchen will be making the solutions on demand, please see me if you want more media made up.
• Please feel free to share any feedback on this media via the discussion page or in person.

Protocol

1. The following protocol makes 1 L of Media
2. All of the ingredients have already been filter sterilized or autoclaved as appropriate.
3. To a 1 L flask add each of the following ingredients listed in the table below.
4. To conform to the working SBWG standardization, the 100x carbon source should be 40% Glycerol.
5. Filter Sterilize with a .2μm filter.

1	10X MOPS Mixture	100 mL
2	0.132M K2HPO4(Dibasic)	10 mL
3	10X ACGU	100 mL
4	5X Supplement EZ	200 mL
5	Sterile H2O	580 mL
6	100x Carbon Source	10 mL
7	TOTAL	1 L

Notes

• Tricine is added to the MOPS mixture to chelate Fe ions to act as an Fe reservoir.

References

• F. C. Neidhardt, P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. J Bacteriol 119(3): 736-747