If any one is facing fungus contamination or other due to eviormental conditions then what precautions are necessary and how he can prevent contamination?
Best to get rid of contaminated cultures. Spores from the culture may contaminate your incubator and can contaminate other cultures. Once you remove the contaminated culture, clean inside of your incubator with a cloth soaked in hypochlorite. Then with a cloth soaked in ethanol. Make sure ethanol vapour are gone before putting new cultures in the incubator.
If you need to rescue the contaminated culture replace medium with medium containing antibiotic/antimycotic cocktails and also add gentamycin.
Best to get rid of contaminated cultures. Spores from the culture may contaminate your incubator and can contaminate other cultures. Once you remove the contaminated culture, clean inside of your incubator with a cloth soaked in hypochlorite. Then with a cloth soaked in ethanol. Make sure ethanol vapour are gone before putting new cultures in the incubator.
If you need to rescue the contaminated culture replace medium with medium containing antibiotic/antimycotic cocktails and also add gentamycin.
The explants can be soaked in antibacterial solution composed of Nystatin (120 mg l−1), Kanamycin (60 mg l−1) and Gentamycin (60 mg l−1) with continuous mild agitation for 24 h so as to avoid the fungal contamination in the cultures
Further use Nystatin or cocktail of antibiotics in the medium by trial and error and should ensure that the concentration of antibiotics should not reduce your success rate. All the best.
Please try using appropriate percent of amphotericin into the media during passaging (sub culturing), it will stop fungal growth but I am not sure whether it will destroy the fungal spores.
Remove the contaminated cultures and sterilize the incubator with ethanol. use sterile explant and add appropriate amount of antibiotics according to your need.
As others have advised, dispose of contaminated cells and sterilise not only the incubator but all laboratory equipment. The cells cannot be rescued. Even if you manage to get rid of the contaminant and keep your cells alive, the high levels of antimicrobial agents used to achieve that would undoubtedly disrupt cell cycle.
The water bath is a common source of contamination, and this can get transferred to the fridge, flow hoods, centrifuge, work benches and any surface. You can use Virkon or another antimicrobial detergent to wipe surfaces, followed by ethanol. Some incubators have heat cycles for decontamination.
I'd highly recommend adding a drop of Sigma Water Bath Treatment to the water bath and incubator water pans every time you top up or replace the water, it is highly effective in preventing contamination.
If you have a particularly stubborn contamination that keeps reoccurring and you are confident that your cell source and technique are not causing it, you may wish to consider fumigating the lab. This is a last resort, as it involves employing a professional to fully seal the lab and set off a paraformaldehyde 'bomb' overnight. This requires the lab to have proper ventilation to evacuate any residual gas but is the most effective way of clearing stubborn contamination.
Good luck!
Follow the previous advices. If you need to keep and clean your cells please wash the cells several times with buffer before treatment with antibiotics , it is important to reduce the concentration of contamination
Discard all the contaminated cultures and rinse the incubator with ethanol and also fumigate the incubator. After proper washing and disinfection leave the incubator and cell culture lab empty for 2-3 days and then start cell culture
If you are isolating your cells from fresh tissue I would recommend a quick dip (20 seconds) in 70% ethanol before washing 3 times in medium containing pen/strep and 5ug/ml amphoteric in B (Fungizone). It sounds drastic but it really works.
To prevent fungus contamination one should sterilized distilled water while preparing media. Mix antibiotics like penicillin in tissue culture media and FCS. Micro filter the media.Fungus generally enters through contaminated water.
- Badges
- Science topic