Hi, there,
I am doing primary culture of colorectal cancer. In recent months my cultures are contaminated. I suppose it is some kind of bacterial. It grows slow and almost always appears after 4 days of culture. It doesn't move a lot and forms single clones on the surface of the plate. The culture media turn yellow easily. The micorbie is penicillin-streptomycin resistant. No antifungal drugs have been tried.
Almost everything has been checked to find out and to remove the contamination. The culture media, digestion enzyme and PBS had been cleared. When they were cultured alone, there was no contamination. It is not caused by the tissue, because when I tried to culture cell lines or other materials, the problem persists. It's probably caused by the incubator. I used a Heracell 150i incubator. I changed a new filter and run the autoclave procedure several times, but the problem still exists. I have tried different cultures more than ten times, but the problem always persists.
Anybody have any idea of what could it be and how to solve this problem? Any help will be greatly appreciated.
I think it is about any kind of bacterial microaerofil, or any fungal infection that needs to be tested with broad spectrum antifungals. I think that more can contribute to microbiologists here.
Dear Wei Chen,
It is highly appreciable that you provided supportive evidence (photo) of your problem. Since, you are using Heracell 150i, the shelves are electroplated with stainless steel. Steel accumulates bacteria, dust and fungi. Also, the inline filters if not changed can transmit contaminants. Just by changing HEPA filters do not eradicate contamination. Finally, the purity of CO2 needs to be checked in house. If O2 cylinder is attached, it favours contaminants.
Let me suggest you the way what I follow:
1. Switch off the instrument
2. Disconnect the gas supply
3. Remove sensors ( discard HEPA and inline filters, discard water from tray)
4. Switch on the instrument
5. Run contra con decontamination (90 degree centigrade) - overnight
6. Switch off instrument
7. Replace sensors
8. Fumigate with KmNo4 overnight (along with lab fumigation)
9. Remove fumes periodically (2 days)
10. Wipe the entire incubator, tray and shelves with 70% ethanol
11. Replace new Filters
12. Fill about 1 liter of water in tray and add 2-4 gms of CuSo4
13. Check the sterility of instrument with open blood agar, SDA plate and Thioglycollate broth
14. Wait for one week (but, change water in the tray once or twice per week)
15. When result is negative, use the incubator after wiping with 70% ethanol.
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