I noticed one of my friends' cells were contaminated with active moving very small rounded black dots and also some white microbes which are a little bigger than those of black dots. These are definitely live bacteria. To check antibiotic sensitivity tests, can I just use the contaminated culture media straight from the cell growing flasks to spread on nutrient agar plates or what are the things to consider for this procedures? Most people suggested to throw away but if we want to salvage those cells, what the best solutions?
Heard of Normocure, Normocin, combination of ciprofloxacin and piperacillin can be used to kills those microbes. Appreciate your experience sharing.
Throw them away. Antibiotic washes are very inconsistent in eliminating bacteria and you are now using cells that have an atypical processing history - hence there is a significant chance that any results you generate in future will be unrepresentative or even unrepeatable using a healthy cell stock.
You our can plate down some of the super arrant in agar if you want to test for a specific strain but this should anxiously take place away from your primary culture environment.
You will not be able to see bacteria using phase-contrast microscopes. It is likely that you are seeing spores or fungus. Does it resemble this
http://www.geocities.ws/micro2052000/images/Spore20forming20bacteria.jpg
There really isn't any point in doing testing if you know your culture is contaminated unless you are having persistant issues and would like to identify where it could be coming from.
As per my experience, bacteria can be seen easily by 20X or 40X under inverted microscope. Pls try to see them.
Those are active moving but not because of the fluid media movement. As per Mark Joseph Mccall, suggestion, I tried to culture them on agar plate but not grow at all. I think his method might not be appropriate to this bacteria.
After thinking, I think I should culture them in broth 1st before culture it on agar. Will try to do such way and see how.
Can Kiessling, after you have watched the files, let me know how do you think of them?
Watched the video, strange apperance.
Jamail, you cannot resolve bacteria without staining (Gram staining and simialr methods) and at 20X and 40X. If you could resolve bacteria, you would be able to see the mitochondria of the cell as well which you cannot see inside the cell with phase-contrast microscopy. You tried to culture them on agar to confirm but this came negative. Doesn't that suggest that it was not bacteria? Inside your cell media, if it was bacteria that colony you saw would be getting bigger. Bacteria divide very quickly. Does your media get cloudy/turbid within a day? Or even hours. That is the hallmark of a bacterial contamination.
http://www.microbehunter.com/bacteria-in-phase-contrast/
Can Kiessling, my media did not turn to cloudy or turbid within a day. I will culture those contaminated culture into broth 1st before plate them on agar and see how if they grow. If not, they are not bacteria already.
It is possible what you are seeing is debris, i've had similar appereances of black spots. Could be FBS or even cell debris (apoptosis?)
http://www.protocol-online.org/biology-forums-2/posts/25256.html
Thanks. But those are not from FBS definitely. Will cells debris will move in active way? I don't think so. We can simply distinguish between debris and microbes.
Can Kiessling and any other researchers, Pls check the attached video clip which was taken during cell counting. Round big ones are cells but we still see some small dots, stained with trypan blue and some were not. However, obviously they were moving, some active swimming like and some were only around its place. What were those tiny moving things? Bacteria? or else?
I would just use fresh frozen cell stocks and have a clean of cell culture equipment (water baths, microscopes), you really don't need to identify a contaminant unless it's persistant. You may be pulling your hair out whilst trying to identify it instead of doing experiments which are more fun :)
If you were able to identify what microorganisms is in your cell culture, what would you do? You would start from frozen stocks :). So just trash it and hope it doesn't happen again. :)
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