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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
[Bio101] 蠕虫常用培养基和溶液

作者: Fanglian He
4/5/2011, 23603 views, 10 Q&A
DOI: https://doi.org/10.21769/BioProtoc.55

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

[Abstract] 这里介绍的是在C.线虫中常用的培养基和溶液的配制方法。

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.

材料和试剂

  1. 琼脂,蛋白胨(BD Biosciences)
  2. 胆固醇(Sigma-Aldrich)
  3. 链霉素(Sigma-Aldrich)
  4. 制霉菌素(Life Technologies,Gibco )
  5. 漂白剂(Clorox)
  6. 磷酸钾
  7. Clorox漂白剂
  8. NaCl
  9. CaCl <2>
  10. MgSO 4 4 /
  11. EtOH
  12. FeSO 4 47H 2 O
  13. Na 2 EDTA
  14. MnCl 2 2·4H 2 O·m/2
  15. ZnSO 4·7H 2 O·m/2
  16. CuSO 4 H 5 H 2 O·m/2
  17. KH 2 PO 4
  18. Na HPO 4

设备

  1. 60 x 15 mm板
  2. 塑料盒

食谱

  1. 线虫生长培养基(NGM)琼脂:用于维持蠕虫 对于1升介质
    3克NaCl
    17克琼脂 2.5克蛋白胨 1ml胆固醇(5mg/ml在95%EtOH中) 975ml H 2 O 2 / 高压灭菌,然后加入以下无菌溶液(高压灭菌)
    1ml 1M CaCl 2 1ml 1M MgSO 4 25ml 1M磷酸钾(pH6)(为了避免沉淀,在加入MgSO 4和磷酸钾之间混合)
    制备1M磷酸钾(pH6):对于1升,将136.1g KH 2 PO 4溶解在约800ml dH 2 O中,然后在升至体积之前用固体KOH(约15g)调节至pH 6.0。取100毫升等分试样并高压灭菌 如果平板用于接种细菌性食物大肠杆菌OP50-1,则需要加入链霉素(300ng ml -1 -1 )。通常倾倒60 x 15 mm板,并在室温下将NGM板存放在带盖的塑料盒中
  2. S基础培养基(改编自Stanford的Kim实验室):用于蠕虫的液体培养 对于1升介质
    5.8克NaCl 50 mL 25 ml 1M磷酸钾(pH 6)
    1ml胆固醇(5mg/ml在95%EtOH中) 950ml dH 2 O
    高压灭菌,然后加入以下无菌溶液(高压灭菌)
    3ml 1M CaCl 2 3ml 1M MgSO 4 10ml痕量金属溶液
    10ml 1M柠檬酸钾(pH6.0) 10 ml 100x制霉菌素(抗真菌剂,保存在冰箱中;不必一直添加)。
    制备500ml痕量金属溶液
    0.346g FeSO 4·7H 2 O·h/v 0.930g Na 2 EDTA 0.098g MnCl 2·4H 2 O 2·h/v 0.144g ZnSO 4·7H 2 O·dm / 0.012g CuSO 4·5H 2 O 2·h/v 通过高压灭菌消毒。 保持在黑暗中(用箔包装)。
    制备100毫升1M柠檬酸钾:将21.02克柠檬酸一水合物溶解在80毫升中,用固体KOH(约17克)调节至pH6.0,然后升至体积。
  3. 蠕虫M9缓冲区
    3g KH sub 2 PO 4 sub
    6g Na 2 HPO 4
    5克NaCl
    将H 2 O加至1升。 通过高压灭菌消毒。
    溶液冷却后,加入1ml高压灭菌的/无菌的1M MgSO 4
  4. 100 ml 2x蠕虫溶解液:用于蠕虫蛋制备
    50ml ddH 2 O 2·h/v 10ml 10M NaOH
    40 ml Clorox漂白剂
    新鲜,在4°C下储存一周。
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How to cite this protocol: He, F. (2011). Common Worm Media and Buffers. Bio-protocol Bio101: e55. DOI: 10.21769/BioProtoc.55; Full Text



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2/9/2017 5:29:58 AM  

Kazuko Collins
Gurdon Institute University of Cambridge

I have a problem whist preparing NGM plates. The NGM solution becomes cloudy. Can anyone help, please.

2/14/2017 1:14:05 AM  

Fanglian He (Author)
Department of Biology, University of Pennsylvania, USA

Hi, Kazuko,

The cloudy solution is very likely due to the formation of CaSO4. When you add CaCl2 or MgSO4 to the solution, you need to make sure that the solution is mixed well before adding the 2nd one.

Hope this helps.

Fanglian

2/19/2017 8:39:53 PM  

Kazuko Collins
Gurdon Institute University of Cambridge

Hi Fanglian,

Thank you for your help.

However, I should have provided more information in my first question.

We've been very careful with mixing solutions (time and tempareture). We use "Fisher Scientific" NaCl, "BD" Pepton and "Melford" Agar. We used to use "Formedium" Agar for many years but it caused cloudy NGM. So we switched to use "Melford" agar (as suggested by one of PI). To start with it was very clear and everyone's happy. But last few weeks, we noticed that NGM is cloudy again and it's before anything added. It is clear just out of autoclave but it becomes cloudy when it's ready for the additives.

Hope I explained better this time.

Kazuko

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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
Department of Biology, The Pennsylvania State University, USA

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
Department of Biology, The Pennsylvania State University, USA

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.

12/18/2016 4:35:20 AM  

Evrim Çelebi
Ankara university

Hello,

Mr. Peichuan ı would to know any alternative for plate which seeded FuDR such as liquid culture. if answer is yes how can I perform with liquid culture for lifespan 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)
Department of Biology, University of Pennsylvania, USA

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)
Department of Biology, University of Pennsylvania, USA

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)
Department of Biology, University of Pennsylvania, USA

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

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