I have doubt about our lab south american FBS because it will be expired march 2015. I have a doubt about FBS also. In my A549 culture this one gave rad and spiral shape contamination. So can you give suggestions for my cells safe culturing?
Verify and decontaminate your hardware sometimes simple things as changing the filter on a pipettor can resolve the issue.
First you have to be sure about the origin of your contamination. Besides filter the culture medium you can test separately each component. Use a multiwell plate and add each component to a single well and let incubate during 24h.
Don't forget to check the solutions that you use for trypsinization.
Good Luck
Dear Senthil,
I used to check the FBS by the following protocol,
Use a FBS from different and reliable source as control.
Use FBS from ur lab and other source with the same DMEM/RPMI and pen strep.
also keep a DMEM and pen strep alone just for check.
Culture your cells in these media preparations.
Incubate your cells for 24 hours and then check under microscope.
Analyse the data according to your protocol.
Good luck!
Thank you very much researchers.., already i done above steps you are all suggested but once again my culture contain 10% black color dots in T25 flask.
Federico Tazón Diego : Each and every time i was filtered my medium with 0.22 micron filter paper and then i was added into the flask.
It sounds like you need to change your filtration process. I would test out the base medium alone and the medium plus FBS before doing any cell cultures. If you use disposable paper filters I would switch to using disposable units, (at least a few just to be sure). There are very few organisms that pass through 0.22um and so if that process is working well and you still get contamination in just your FBS media then I would ditch the serum.
Dear Senthil
you can check the sterility of your medium or serum by using Sodium Thiglycolate medium.
from this sterility test you can confirm that which component of your culture system is contaminated. (Serum or media)
FBS is filtered from the company(0.1um) . make two different mixture.
1: filter ur FBS urself and add it to DMEM,Pen/strep,
2: dont filter FBS and add it to DMEM,Pen/strep
culture two wells with 1 and two respectively,
see if both contaminated it is not from ur FBS and etc,
HI
if you confident about filtration process you could use a LB plate to test the contamination. you could decant some FBS to the plate and place it in the incubator for 24 hours, the contamination will be apparent.
Verify and decontaminate your hardware sometimes simple things as changing the filter on a pipettor can resolve the issue.
Test all your media components , media and FBS on individual blood agar plates .
Contaminated FBS quickly turns turbid - leave some FBS at room temperature over night and see if it still clear. Don't sterile filter infected FBS to re-use it since the bacteria often change the pH. Check plastics and other media component (discard and use new). good luck!
Crossing of expiry date does not necessarly mean that the FBS could be contaminated.Contamination or lack of it is related to the way the FBS is being handled.Asceptic handling during media preparation and cell culturing step are important.
You can always monitor the sterility of the FBS/cell growth medium/other reagents, at every step by either plating on suitable plates or by inoculating into liquid media like SCDM/FTM.
You can use universal primers for bacterial and mycoplasma 18S rRNA and PCR.
you can just take a few microlitres of the suspected FBS and spread it on LB-Agar plates. Parallely, you can just take 1 /2 ml and seed in a 6 well plate and incubate for 24 hours.
if there is some contamination. you will see growth.
I have an unopened FBS solution in -80 degree. I thawed it and noticed some white precipitation in my FBS stock solution and some floating substance. Does it mean it is contaminated? Though date is expired, but if is contaminated, is it possible to fix?
Get blood agar plates and streak a drop your FBS and incubate for 48 hrs.
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