Hi,
I have a bacterial infection in my cultures. The cultures consist of primary sourced murine endothelial cells, cultured in endothelial growth medium with antibiotics. The patterns as seen in the first image are patterned on ethanol and UV sterilised PDMS.
In my 6 well plate, 3 wells are infected. A sample of the cells were passaged into a T25 flask and are growing healthy and well.
I am just wondering if anyone can help identify the type of bacteria - I realise the microscope is pretty poor. All my equipment was ethanol sterilised and UV sterilised where appropriate and all solutions were filtered.
The bacteria seems to form a thin film around the cells in the first image and can be seen in clusters in the second.
Thanks
Dear Josh,
According to my opinion you should transfer your supernatant on some basic media such as TSA or Blood agar and then use from some specific and selective agar for final isolation and identification according to your isolated species.
Meanwhile, using of some molecular biology techniques such as PCR or qPCR could be useful for rapid diagnosis affairs.
Good luck
Josh, are you talking about those large lumpy, almost crystal like things in your tiff? Cause those would not be bacteria, bacteria is too small to be seen in your media, you could only tell by the fact that the media is cloudy and you might see brownian motion. Also, you add antibiotics and you still have an infection issue. Its hard to tell from your image but I'm thinking that this might be not be bacteria. Since this is primary tissue have you thought about yeast and other fungal carry over? Do you use an antimycotic in your media. You can buy some and just add it to your media. If you look under the scope and see things that look like little around clusters of grapes those are yeast. If you are certain that this is bacteria though, you can take some of media, spin it down, smear a little on a slide and do a gram stain. That should tell you a bit about morphology, whether its gram positive or negative or even if it is bacteria. You can also take a bit of the spun down material and plate in onto a regular LB plate and see what grows. Good luck!
Thanks for the replies, it was a bacterial infection that couldn't be identified. I killed the wells that were showing infection; 3 of 6 and the rest survived. P/S was not used in the the media to try and reduce the effect on endothelial cell phenotype.
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