clumping cells - s. aureus wildtyp cells (Sep/29/2006 )
I am a beginner in working with S. aureus, that`s why i need your help:
1) my s.aureus cells (wildtyp) build always clumps. Its difficult to use a over night culture for same experiments because you get often some clumps and you can not really measure the OD.
2) my cells grow after freezing very different. They build small and larger colonies. I know this happens sometimes, but is there a trick to minimize the problem.
3) what a the normal concentrations of different antibiotics to inhibit S. aureus cells:
Thank a lot for helping me....
I can't answer the antibiotic question because I don't use them, but you may find the answer at google (possibly check ATCC's site?)
I work on S aureus too, and I will tell you two things from my experience:
1. some strains are clumpier (all that PGN and stuff) but you normally don't see this unless you are overgrowing them? what sort of media do you use and what are your growth conditions?
2. for the small/large colonies...this is not really a problem. as far as minimizing, it's almost impossible. I would recommend going to pubmed and doing a search for "staphylococcus small colony variant" and you can dig up the papers about this phenomenon. as long as the morphology is the same otherwise, I don't worry about this when I see it
one other thing, though...how is your technique? before you get all excited, are you absolutely certain your staph isn't contaminated? the excessive clumpiness, the variable colonies...perhaps you have contaminated your cultures. this needs to be ruled out for certain. after all there's a lot of staph in the environment and you have to be super-careful
thank you for answering!
first i have to say that there is no contamination (hopefully!). My mutants have a antibiotic cassette and so i can select them (it is a stable mutation). What i can say is that the cells grow more different when i select them on antibiotics. But now i see that they also grow different without antibiotics. I also checked the chromosomal dna of the frozen cultures and i can still see a shift in the agarose gel of a specific pcr-product of my mutants and wildtype.
Not only my mutants but also my wildtyp is building really thick clumps. I use normal B media which contains soy peptone (Roth), yeast extract, NaCl, K2HPO4 and 0,1% glucose. Which media do you use? Perhaps you have a minimal media? To inocculate my experiments i use a over night culture. I use 20 ml media and let them grow by 37>C.
My main problem at the moment is that i dont know which colonies i should use for my experiments? The larger ones or the smaler ones? It seems you have a lot of experience-what would you do in such a case?
I pick the larger ones. I don't think it matters, but it seems to me that they are growing fast and happy, and that's what you want to use when inoculating, right? the genotype will be the same either way, it is only a fairly minor phenotypic variance (in my understanding, anyways)
I use standard, good ol' TSA or TSB to grow my staph. I do not know your media and am not sure if that contributes to the clumping or not? I suppose a good question would be, have your strains started clumping, or have they always been clumpy?