Neidhardt EZ Rich Defined

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This media is a slight variant on that defined by [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=4604283 Neidhardt ''et al'']. The modifications were made by [http://www.genome.wisc.edu/ Blattner ''et al''.]  This is a rich defined medium which should allow repeatable high growth rates.  The recipe from below is edited from the [http://www.genome.wisc.edu/resources/protocols/ezmedium.htm 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.
This media is a slight variant on that defined by [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=4604283 Neidhardt ''et al'']. The modifications were made by [http://www.genome.wisc.edu/ Blattner ''et al''.]  This is a rich defined medium which should allow repeatable high growth rates.  The recipe from below is edited from the [http://www.genome.wisc.edu/resources/protocols/ezmedium.htm 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.
-
==Materials Sourcing & Usage==
+
EZ Media has the following advantages -
-
*We buy the two of the ingredients (10X ACGU and the 5X Supplement EZ) from [http://www.teknova.com Teknova] because they are tedious to make.  The Endy Lab has an account with [http://www.teknova.com Teknova] (see Barry for details).
+
*Concentrations of the major nutrients can be varied independently (C, P, N, S)
-
*The kitchen makes up batches of 10X MOPS Mixture for us.
+
*Concentrations of all constituents are defined
-
*The kitchen also makes K<sub>2</sub>HPO<sub>4</sub> for us.
+
*Growth rate should be similar to that of ''E. coli'' in rich media such as LB
-
*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.
+
*Supports a high density of cell growth
-
*I have 4L 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 --[[User:Bcanton|Bcanton]] 14:16, 7 Jun 2005 (EDT)
+
*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 [http://www.teknova.com Teknova] because they are tedious to make.  The Endy Lab has an account with [http://www.teknova.com Teknova] (see [[Barry Canton | Barry]] for details).
 +
*The kitchen makes up batches of 10X MOPS Mixture for the us with some assistance.  Here is the [[Endy:Neidhardt EZ Rich Defined|recipe]] we use to make the 10X MOPS mixture.
 +
*The kitchen also makes K<sub>2</sub>HPO<sub>4</sub> 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 --[[User:Bcanton|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.
*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.
*Please feel free to share any feedback on this media via the discussion page or in person.
==Protocol==
==Protocol==
-
#The following protocol makes 1l of Media
+
#The following protocol makes 1L of Media
#All of the ingredients have already been filter sterilized or autoclaved as appropriate.
#All of the ingredients have already been filter sterilized or autoclaved as appropriate.
-
#To a 1l flask add each of the following ingredients listed in the table below.
+
#To a 1 L flask add each of the following ingredients listed in the table below.
#'''To conform to the working SBWG standardization, the 100x carbon source should be 40% Glycerol.'''
#'''To conform to the working SBWG standardization, the 100x carbon source should be 40% Glycerol.'''
#Filter Sterilize with a .2&mu;m filter.
#Filter Sterilize with a .2&mu;m filter.
Line 20: Line 35:
|-
|-
! 1
! 1
-
| 10X MOPS Mixture || 100ml
+
| 10X MOPS Mixture || 100 mL
|-
|-
! 2
! 2
-
| 0.132M K<sub>2</sub>HPO<sub>4</sub>(Dibasic) || 10ml
+
| 0.132M K<sub>2</sub>HPO<sub>4</sub>(Dibasic) || 10 mL
|-
|-
! 3
! 3
-
| 10X ACGU || 100ml
+
| 10X ACGU || 100 mL
|-
|-
! 4
! 4
-
| 5X Supplement EZ || 200ml
+
| 5X Supplement EZ || 200 mL
|-
|-
! 5
! 5
-
| Sterile H<sub>2</sub>O || 580ml
+
| Sterile H<sub>2</sub>O || 580 mL
|-
|-
! 6
! 6
-
|100x Carbon Source || 10ml
+
|100x Carbon Source || 10 mL
|-
|-
! 7
! 7
-
| TOTAL || 1l
+
| TOTAL || 1 L
|}
|}
 +
 +
==Notes==
 +
*Tricine is added to the MOPS mixture to chelate Fe ions to act as an Fe reservoir.
 +
*I had been storing made up media at 4&deg;C and began to notice some brown precipitate at the bottom of the bottle.  This precipitate would not go back into solution when the media was warmed to 37&deg;C.  I contacted Teknova about this. 
 +
**'''Teknova's response''': The M2001 used in your M2006 MOPS Minimal Kit contains temperature-sensitive components that require storage at -20&deg;C. Storing this media for long term at 4&deg;C will compromise the potency and may cause subcomponents to fall out of solution, just as you've experienced. I don't believe the particles you're observing in the solution are either fungus or mold, but rather iron sulfate that has precipitated out.  To prevent this from occurring in the future, it would be advantageous to aliquot the mixed media, freeze, and thaw only the quantity you need.  In my experience, even keeping the mixed solution refrigerated for more than a couple of days will severely reduce it's potency. Unfortunately, discarding the solution may be necessary.
 +
**Note that cells still seem to be able to grow in the media, though the integrity of media is likely compromised.
 +
*You might notice a significant lag if you start cultures from diluted overnights grown in LB.  In the Niedhardt paper, there is data showing that large dilutions from log-phase cultures cause substantial lags (like hours), but these can be completely avoided by adding 10 mM NaCO<sub>3</sub>.  I have found that making the RDM with 10% LB really helps get them going.  Although this goes against the "defined" mantra, I haven't noticed an problem with reproducability when I do this. --[[User:Smoore|Smoore]] 18:17, 3 July 2008 (UTC)
==References==
==References==
* F. C. Neidhardt, P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. ''J Bacteriol'' 119(3): 736-747
* F. C. Neidhardt, P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. ''J Bacteriol'' 119(3): 736-747
 +
 +
 +
[[Category:Material]] [[Category:Defined Media]]
 +
[[Category:Escherichia coli]]

Current revision

Contents

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 with some assistance. Here is the recipe we use to make the 10X MOPS mixture.
  • 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 1L 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.
  • I had been storing made up media at 4°C and began to notice some brown precipitate at the bottom of the bottle. This precipitate would not go back into solution when the media was warmed to 37°C. I contacted Teknova about this.
    • Teknova's response: The M2001 used in your M2006 MOPS Minimal Kit contains temperature-sensitive components that require storage at -20°C. Storing this media for long term at 4°C will compromise the potency and may cause subcomponents to fall out of solution, just as you've experienced. I don't believe the particles you're observing in the solution are either fungus or mold, but rather iron sulfate that has precipitated out. To prevent this from occurring in the future, it would be advantageous to aliquot the mixed media, freeze, and thaw only the quantity you need. In my experience, even keeping the mixed solution refrigerated for more than a couple of days will severely reduce it's potency. Unfortunately, discarding the solution may be necessary.
    • Note that cells still seem to be able to grow in the media, though the integrity of media is likely compromised.
  • You might notice a significant lag if you start cultures from diluted overnights grown in LB. In the Niedhardt paper, there is data showing that large dilutions from log-phase cultures cause substantial lags (like hours), but these can be completely avoided by adding 10 mM NaCO3. I have found that making the RDM with 10% LB really helps get them going. Although this goes against the "defined" mantra, I haven't noticed an problem with reproducability when I do this. --Smoore 18:17, 3 July 2008 (UTC)

References

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