Hi,
We've noticed significant contamination on SEM images of gold-coated optical fibers. We've determined the contamination is likely happening during the protein detection phase, as imaging fibers prior to the experiments show very clean surfaces under identical conditions.
I've attached two images (not same magnification) for comparison. Sorry that these don't have scale bars, but the nanoparticles are about 22nm and the bacteria are > 1um.
It's clear to me that this is partially a bacterial contamination, although some "gunky" crap is also in there that does not seem bacterial. This actually could be aggregated proteins from high-concentration experiments, unless you guys have any other ideas?
Anyway, we've been using filtered buffers, run through a 200nm filter. The clean image, f5, has been exposed to several of these buffers and shows no contamination.
We believe we've isolated two sources of contamination. Either a non-filtered ph10 buffer (http://www.coleparmer.com/Product/Thermo_Scientific_Orion_10_01_pH_buffer_solution_475_mL_bottle/EW-55350-52); could bacteria grow in such conditions? Alternatively, it could be our BSA protein solution that is contaminated. The BSA solution is created from dry powder, but the powder is several years old. Could bacterial literally be living on the solid powder? Also, we usually use BSA that is 1-2 weeks old.
We're doing growth tests to confirm the source, but anyone who has experience with bacteria/cleanliness, can you give me some advice? We were thinking of getting dialysis tubes and centrifuging proteins prior to use.
I would say there are bacterial species to grow in (almost) any humid conditions. pH may be a bit high but alkaliphillic bacteria (some Pseudomonas) can develop in there.
In solid powders, it would be harder because water activity is low but bacteria can survive without growing. I suppose you filtered each and every solution you are using through a .22 µm filter with low protein adsorption features?
Of course, during experiments, you should ensure the air in contact with your sample is clean of bacteria (under an appropriate hood for example).
Hope this helps,
I would add to the previuos answer that you glass or plastic matetial should be properly sterilized with bleech or H2O2 diluted solutions and well risen with distilled water after. Also, you may need to consider make new solutions of BSA from.new powder or may be a borrow powder to check if it is just yours tje one that it is cobtaminated. If it is your buffer... you can just autoclave it and reuse it under sterile condition... let s say a biosafety cabinet. In... you should keep aseptic conditions from the first until the last second that you are preparing your samples..meaning preparing you nanoparticles with sterile water for example and mounting your samples in a clean enviroment. Just.one more thing... as a suggestion... clean you fredge or freezer with 10 % bleech solution, the bacteria could be on the humyd air inside or in the surfaces as well.. good luck and let me.know how it goes!
Thank everyone, we will begin to implement these excellent suggestions.
In addition, you should autoclave your distilled water before use. Since protein solutions cannot be autoclaved, the best method is using membrane filtration technique which I belived you have used. So its likely the contamination is from the gadget. Try and determine the HACCP of the entire set up to know the likely source of contamination. The use of hipochlorite solution in rin
I agree with the previous answers; using sterile solutions and labware and aseptic working conditions will prevent the contaminaton problem. However, I do not think that you need such harsh precuations. Bacteria can survive in all aqueuous buffers, and protein solutions. But using freshly prepared solutions will probably solve your problem. You can filter your buffers and protein solutions through 0,2 um filters after preparation and If necessary repeat filtering prior to the experiment. You can use the filter system such as the one the link (http://www.coleparmer.com/Product/Corning_Disposable_Vacuum_Filtration_Systems_0_2um_NYL_250mL_12_Cs/EW-29530-08). You can use the flow through collector to store your solutions. The filters are single use, but I use the filters few times without problem.
Thanks. While we have been filtering out buffers, we haven't been filtering the proteins, so if the growth is occurring after the protein solution is made, obviously we have a problem. We'll start filtering our protein solutions as well!
Sorry, physicists turned biologists, missing probably all of the 101 lab provisions.
You can try UV irradiation. First al equipments like membrane cell and glasswares then the protein solution and any other solution that you use should be irradiated. You can even irradiate the tested membranes and then keep them in sterile package until SEM imaging. I hope it works for you.
Thanks Parisa. Unfortuantly, we don't have the resources to buy any new equipment now.
Our incubation tests came back to show the BSA protein solution was the culprit as we expected. We've switched powders and will start working with frozen samples. We'll retest these samples soon, and if they are still problematic, I think we'll try to filter with centrifuge tubes.
Thanks for all your suggestions, this was a very helpful thread to which I'll surely be returning for future guidance.
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