Abstract
Here are recipes of some media and solutions often used in C. elegans research.
Materials and Reagents
Equipment
Recipes
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after adding cholesterol to ethanol place it on vertex shaker for 15 mnts then it will dissolve and add chol. to ngm at 55degress
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
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
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.
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=26854551The 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=21893079In 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.
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?
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.
Hi Dr. Li,Regarding the M9 buffer, I have a question about the buffers used in different egg prep protocol.People use egg buffer(Required osmolality) or M9 buffer (Seems much simplier)to stop the bleach and wash the egg~Which is better for synchronize the worm cultures?Thank you
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.
It depends on the subsequent experiments. Sometime, sterile DI water is fine for resuspending worms, like during egg prep for removing bacteria/fungi contamination.
M9 buffer is used for transferring C. elegans, but not used as medium for the worm.