Yunke Zhao Answered May 31, 2017
University of Southern California
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
Lingbo Li Answered Jun 19, 2015
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.