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Here are recipes of some media and solutions often used in C. elegans research.

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[Bio101] Common Worm Media and Buffers

[Abstract] Here are recipes of some media and solutions often used in C. elegans research.

Materials and Reagents

  1. Agar, peptone (BD Biosciences)
  2. Cholesterol (Sigma-Aldrich)
  3. Streptomycin (Sigma-Aldrich)
  4. Nystatin (Life Technologies, Gibco®)
  5. Bleach (Clorox)
  6. Potassium phosphate
  7. Clorox bleach
  8. NaCl
  9. CaCl2
  10. MgSO4
  11. EtOH
  12. FeSO4.7H2O
  13. Na2EDTA
  14. MnCl2.4H2O
  15. ZnSO4.7H2O
  16. CuSO4.5H2O
  17. KH2PO4
  18. Na2HPO4

Equipment

  1. 60 x 15 mm plate
  2. Plastic boxes

Recipes

  1. Nematode growth medium (NGM) agar: For the maintenance of worms.
    For 1 liter medium
    3 g NaCl
    17 g agar
    2.5 g peptone
    1 ml cholesterol (5 mg ml-1 in 95% EtOH)
    975 ml H2O
    Autoclave, and then add the following sterile solution (autoclaved)
    1 ml 1 M CaCl2
    1 ml 1 M MgSO4
    25 ml 1 M potassium phosphate (pH 6) (to avoid precipitation, mix between addition of MgSO4 and potassium phosphate)
    To make 1 M potassium phosphate (pH 6): For 1 liter, dissolve 136.1 g KH2PO4 in about 800 ml dH2O, then adjust to pH 6.0 with solid KOH (approx 15 g) before bringing up to volume. Make 100 ml aliquots and autoclave.
    Need to add streptomycin (300 ng ml-1) if plate is used for seeding bacterial food E coli OP50-1. Typically pour 60 x 15 mm plate and store NGM plates in plastic boxes with covers at room temperature.
  2. S-basal medium (adapted from the Kim Lab at Stanford) : For liquid culture of worms.
    For 1 liter medium
    5.8 g NaCl
    50 mL 25 ml 1 M potassium phosphate (pH 6)
    1 ml cholesterol (5 mg ml-1 in 95% EtOH)
    950 ml dH2O
    Autoclave, and then add the following sterile solution (autoclaved)
    3 ml 1 M CaCl2
    3 ml 1 M MgSO4
    10 ml trace metals solution
    10 ml 1 M potassium citrate (pH 6.0)
    10 ml 100x Nystatin (antifungal agent, keep in freezer; do not have to add it all the time).
    To make 500 ml trace metals solution
    0.346 g FeSO4.7H2O
    0.930 g Na2EDTA
    0.098 g MnCl2.4H2O
    0.144 g ZnSO4.7H2O
    0.012 g CuSO4.5H2O
    Sterilize by autoclaving. Keep in dark (wrap in foil).
    To make 100 ml of 1 M potassium citrate: dissolve 21.02 g citric acid, monohydrate in 80 ml and adjust to pH 6.0 with solid KOH (approx 17g) before bringing up to volume.
  3. Worm M9 buffer
    3 g KH2PO4
    6 g Na2HPO4
    5 g NaCl
    Add H2O to 1 liter. Sterilize by autoclaving.
    After solution cools down, add 1 ml autoclaved/sterile 1 M MgSO4.
  4. 100 ml 2x worm lysis solution: For worm egg prep
    50 ml ddH2O
    10 ml 10 M NaOH
    40 ml Clorox bleach
    Make fresh and store at 4 °C up to one week.


How to cite: He, F. (2011). Common Worm Media and Buffers. Bio-protocol Bio101: e55. DOI: 10.21769/BioProtoc.55; Full Text



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7/22/2016 12:22:53 PM  

Norah Althobaiti
Queens university Belfast

Hi there,
Has anyone done chemosensory assay for c.elegans using Diacetyl 0.1% as an attractant?
Any help,please
Thanks
Norah

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3/14/2016 11:16:15 AM  

Robert Lofaro
Brentwood Schools

I wish to evaluate the effects of a neurotoxin on C. elegans. The exposure time will be over a 24 h period. Would you recommend exposing the worms to the S-buffer as the control and the treatment solutions in various concentrations of toxin in S-Buffer?

4/12/2016 7:00:50 PM  

Peichuan Zhang
UCSF

I do not really know the answer to your question. You might try the neurotoxin in M9 as well, just in case that cholesterol in the S-basal buffer may affect neurotoxin's effects.

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2/16/2016 8:16:32 AM  

Mike Morris
Clayton State University

I am a Biology Research Student working on lifespan research. I am using FuDR and it appears toxic during research experiments. Do you have any advice in reference to using FuDR? I am applying the FuDR in with the agar after it cools to an appropriate temperature. Thanks

4/12/2016 7:00:19 PM  

Peichuan Zhang
UCSF

It has been shown that FuDR by itself can have effects on lifespan. For example, a recent work by Anne Hart and co-workers has shown that FuDR induces stress response and it, in combination with hypertonic stress, can extend the lifespan of wild-type C. elegans by 30%.
http://www.ncbi.nlm.nih.gov/pubmed/?term=26854551

The Hekimi lab also showed that FuDR can extend the lifespan of gas-1 mitochondrial mutants, which are relatively short-lived, by ~2-fold.
http://www.ncbi.nlm.nih.gov/pubmed/?term=21893079

In this regard, I will recommend using FuDR at the final concentration of ~25uM to 40uM. It'd be better to add FuDR (diluted in 500ul M9) directly onto the seeded lawn of bacteria and wait for at least one day before using the plate for any experiment.

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6/9/2015 6:11:06 AM  

Sri hari laskhmi Ramamoorthy
Alagappa University

Sir,

1.Why that buffer is said to be M9 why not 10 or 11 and what is that M stands for ?
2.Why do we need to add Mgso4 after sterilization why not before?
3.I have a doubts in the role of Na2Hpo4 ,KH2PO4,Nacl ?

I have used only distilled water for washing sir,, I didnot find anything wrong, Iam in confusion, MAKE ME UNDERSTAND
THANK YOU

6/19/2015 12:31:02 PM  

Lingbo Li
Stanford university

Dear Sri Hari Laskhmi,

Regarding your questions:
1. Why that buffer is said to be M9 why not 10 or 11 and what is that M stands for ?
The short answer is that "M" stands for Medium. 9 stands for the ninth recipe that worked relatively well in the maintenance of C.elegans culture over time.
The longer answer is: During the early days when the scientists were first trying to culture C.elegans in a well-controlled laboratory setting to study , they tested a large number of growth medium in order to make growing and culturing an easy & consistent job. The 9th version (although it is not clear how they counted it) worked pretty well, so all the following scientists adopted it. The original researchers did come up with a 10th modification of the recipe by adding saccharose, which they called M10, but in the end it was dropped probably because of the effects are not as good as M9.
A brief explanation about this question and its history can be found here: http://www.wormbook.org/wli/wbg3.2p16/.

2.Why do we need to add MgSO4 after sterilization why not before?
It is because of the physical properties of MgSO4. Because it decomposes at a temperature higher than 150°C, which is the typical temperature of autoclave condition, MgSO4 can not be autoclaved in general.

3.I have a doubts in the role of Na2Hpo4 ,KH2PO4,Nacl ?
Again, the general purpose for an good culture medium is the appropriate composition of salt, nutrient, osmotic pressure, and so on. The reason the initial pioneers came up with a certain concentration of all these components is that after trying a great number of times, this composition gives the best, consistent growth conditions. In theory, those components are not the only way to reach the same culturing results for C.elegans, but one has to spend a huge amount of time to try out.
Why reinvent the wheel, right?

4. I have used only distilled water for washing.
Distilled water should work, too.

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6/9/2015 6:10:47 AM  

Sri hari laskhmi Ramamoorthy
Alagappa University

Sir,

1.Why that buffer is said to be M9 why not 10 or 11 and what is that M stands for ?
2.Why do we need to add Mgso4 after sterilization why not before?
3.I have a doubts in the role of Na2Hpo4 ,KH2PO4,Nacl ?

I have used only distilled water for washing sir,, I didnot find anything wrong, Iam in confusion, MAKE ME UNDERSTAND
THANK YOU

Reply

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4/20/2015 12:21:15 AM  

Manoranjan Kumar
Central university of south Bihar

what is the role of cholesterol in ethanol,cacl2, mgso4,kpo4 in NGM media.

4/25/2015 10:26:49 AM  

Fanglian He (Author)
Carnegie Institution for Science at Stanford

Cholesterol is an important component of animal membranes. Worms could not make it by themselves. That is why NGM contains cholesterol. Regarding CaCl2, MgSO4, KPO4, they are also important for worm growth.

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12/6/2013 10:12:24 PM  

lipika parida
iit-kgp

Thank you Fanglian for the reply. But my question is why M9 is used for transferring C. elegans. We can transfer them by using distilled water too.

12/16/2013 10:05:50 AM  

Fanglian He (Author)
Carnegie Institution for Science at Stanford

It depends on the subsequent experiments. Sometime, sterile DI water is fine for resuspending worms, like during egg prep for removing bacteria/fungi contamination.

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11/29/2013 1:48:14 AM  

lipika parida
iit-kgp

why m9 buffer is used for transferring c.elegans or as a media for c.elegans?

12/6/2013 2:43:28 PM  

Fanglian He (Author)
Carnegie Institution for Science at Stanford

M9 buffer is used for transferring C. elegans, but not used as medium for the worm.

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