Although, it is very hard to identify organism(s) that are present in the culture plate in images at the resolution at which photographs were taken, it is likely to be a Gram-positive bacteria that can form filaments under certain culture conditions. If you take culture medium and spot on a glass slide and stain it with Gram's stain, it would be a confirmation of bacteria. Alternatively, it could be yeast that can form filaments (smelling the culture medium can give a clue).

If you are sure that the problem cannot arise from your cell culture practice then it is most likely related to something you're using throughout the culture / maintenance process. Could there be a contamination in the stocks you use to supply your cell culture medium? Any of your tools, e.g. pipette tips or serological pipettes, flasks? Did you clean and desinfect your incubators? Not too long ago I heard from a contamination that came from issues with the MilliQ supply (so actually they had contaminated water which the group used for their incubators). There's loads of possibilities.

Hi Nicole - contamination is a bane of our line of work.

It seems that if new vials of cells (that previously had no problems) now have this artefact present then it must be being introduced after defrosting the cells and placing in culture.

I would recommend (apart from UV sterilising the hood, using separate lab coats for cell culture, cleaning and sterilizing water-baths  etc.) try filtering the media you make (with 10% FBS etc.)  through a 0.2 micron, 500 ml bottle filter before use (in the TC hood). This should remove bacteria and fungal spores that have been potentially introduced. This is expensive so is a short term fix - but if the cells remain free of contamination it may indicate one or more media supplements are responsible.

The are a number of sources of possible contamination. If not already done I would recommend that you clean and sterilise the incubators you use. Also make sure the water added to the incubators has been autoclaved and contains the appropriate bacterial/fungal inhibitor in it.

When performing cell culture make sure the nylon gloves you wear are pulled up and cover the cuffs of the lab coat and over any exposed flesh of the wrist.

Also I would check if anything new has been introduced into the lab since the contamination started to appear. Any work to lab ventilation? A new plasticware or reagent supplier being used?

Although hard to tell from the images you supplied the phrase "media is cloudy " implies this is not due to a change in the cells but it is a contamination present.

Sorry I can't be more help,

Gary

Thank you all for your responses. We had already tried some of the suggestions previously (sterilisation mh the incubator, cleaning and sterilising the water bath, UV sterilising the hood after use, etc). We have now changed our antibiotic from Pen/Strep to Gentamycin and we're still making sure our aseptic technique is up to scratch, so far it seems to be working. I will also try some of the suggestions we have yet to try if the issue persists. Thank you very much!

I think it is best to sterilize everything before starting with the cell culture so as to be sure that there is no contamination in the culture. In addition, one can also use 70% IPA (Iso-Propyl Alcohol) to keep the environment clean and sterilized. Rest one has to be very cautious while using the pippets etc.