My colleague and I are working with immortalized mammalian smooth muscle cells. We are re-using a modified 96-well plate. Thus far, this plate has shown no signs of contamination. However, to reuse the plate, we have to remove the cells that are currently growing in the wells. This was accomplished by aspirating out the old media, adding 0.1% (w/v) SDS to each of the wells, incubating the plate at 37 C for a bit, and then rigorously rinsing the wells with sterile water. Unfortunately, we neglected to filter-sterilize the 25% (w/v) SDS stock prior to making up the 0.1% SDS solution.
My question is this: What are the odds (qualitatively speaking) that some microbe--capable of surviving in 25% SDS--is going to destroy our cell culture?
Intuitively, I think the odds are pretty low, since everything else was filter or autoclave sterilized, since we have not had any contamination up to this point, and since 25% SDS is a pretty inhospitable environment.
But if I trusted my intuition alone, I would not be asking this question, right? I am new to this field. Have other people ever encountered this situation before? Has it led to problems?
Thank you for any advice you can provide!
It's very unlikely that any bacteria might survive in 25% SDS solution. It's not just inhospitable, it's absolutely killing environment for any microorganisms because this detergent at 25% concentration will dissolve any bacterial cell membrane immediately. That's why people usually keep their 10% SDS stock solution for SDS-gels for many years at room t°C without any bacterial growth. If you spotted any contamination of your cells that might have happened at some other step of your procedure.
dear Darmian,
I totally agree with Viktor; is IMPOSSIBLE that some bacteria can grow inside your 25% SDS solution.
however my suggestion to solve your contamination problem is only one and very easy : THROW ALL THE OLD SOLUTIONS and make a new sterilization of the incubator.
then if you are curios and you want to understand what is the contaminated solution, take some microliters of yours solutions and put it in LB and let it growth ON on shaking at 37C. if there is some bacteria, in this way, they will grow and you'll observe the change of the turbidity of the solution.
regards
Francesco
I did some research and found that SDS is classified as bacteristatic and not bactericid. Also look at this publication:
http://www.ema.europa.eu/docs/en_GB/document_library/Report/2015/08/WC500191475.pdf
As a surfactant, SLS (another name for SDS) has bacteriostatic properties given its pore-forming ability in lipid membranes. It displays some action against Gram
-positive bacteria, although it is ineffective against many Gram-negative microorganisms. It therefore is used in skin cleansing and medicated shampoo products, although SLES is more often used due to its better foaming properties. SLS potentiates the fungicidal activity of certain substances such as sulfanilamide and sulfathiazole, and has been demonstrated to exert microbicidal activity against Human Immunodeficiency Virus Type I.
I think (non-chemist, so there might another reason) the reason you don't see any growth in 25% solution is the same you don't see any growth on molasses, fruit preserves and other foods preserved by high sugar is the osmotic pressure augmented by the sugar. When you dilute your SDS solution your lower the osmotic pressure and bacteria (SDS is bacteristatic) will grow.
Thanks! As an epilogue to this story, my cells actually did not become contaminated (at least not over the 72 hour time course between seeding them and performing the experiment). So if the SDS solution contained live bacteria, perhaps they were not suited for robust growth in DMEM? Maybe I just got lucky? At any rate, I will always filter-sterilize my solutions in the future!
- Badges
- Science method