I get contamination either fungal or bacterial after a two week successful establishment of Plant callus. Please tell me why it happens? Endophytes are ruled out totally since even after the healthy callus is subcultured this happens after a few days. Kindly help. kindly note: ex-plant ranges from seeds to stem cuttings.
If you are near the plant source should try to use new vegetation collected during growth in a green house and to use vacuum infiltration for cleaning. Recutting under sterilant solution is advised. The time course seems linked to latency of internal contamination but media optimization with less auxin may be easing establishementt. for a review about cleaning http://cdn.intechopen.com/pdfs-wm/26032.pdf
In a micropropagation class I attended, I was taught that 2 weeks is needed for most contamination to show up, but even then, you might still get a few contaminated flasks after that. Bacteria usually shows up after around three days, fungi after 10 days.
Personally I have had explants that are contaminated by fungi on first few days, I think those are endogenous fungi because my explant sources are not very clean. And then the ones contaminated after 10 days are from spores.
So I guess your contaminations come from subculturing.
Also be sure to keep the area as clean as possible, continuously spray down shelves and such. I have had issues lately and I suspect we may have some tiny insects like mites on our shelves because we have people walking in from greenhouses and I have seen damage from time to time on leaves.
Contamination may occur at any time period during tissue culture process, handling with sterilized instruments in sterile environment can help for prevention of contamination. Also, spraying 70% ethanol in culture room can reduce possible risk of contamination.....
It is quite common to get contamination, some endophytes will grow lately from the plant tissue, And make sure, there should be power supply to the tissue culture room alwasy for air conditioning "ON' if there is irregular maininting temperature in the tissure culture room, there is chance for the fungus/endopphyetes to grow (particularly when there is a power cut, that temperature will facilitate the dormant stage fungus/endophytes to grow).
Look back at your subculture techniques and equipment used (e.g. laminar flow cabinet, autoclaves etc). Your contamination may arise from insects (ants or mites) infesting the tissue culture room. Drastic changes in the temperature of your tissue culture room could also cause your cultures to "sweat", facilitating fungal and/or bacterial growth. Is your air-conditioning reliable? Are there too many movements in your working area, some renovations going on near your lab? Do you constantly remove contaminated cultures from your tissue culture room? I have personally experienced each and every factor I listed out, which caused many contamination cycles in cultures that were previously sterile. I hope this helped.
Some of the microbes thrive well in vascular strands (some latent bacteria) that may take some time to grow and appear.
Many of the suggestions mentioned in previous answers are correct. I would like to add a way to possibly reduce or even prevent contamination.
[1] It is important to identify contamination at the earliest stage as possible. To this end use a translucent agar type (like Phytagel) which will help you discover contamination once it appeared in the gel. [2] Inspect your plates frequently (best, daily) so you discover contamination at an earliest point of time. [3] Inspect all the surface of a callus that is in contact with the agar by a stereo-microspcope. In this way you will detect contamination before you are able to detect it by naked eye. [4] Plant in your plate small callus explants, a low number of calli per plate, keep big distances between calli. This will lower the risk of cross contamination between calli. [5] Once you detected a contaminated callus in a plate, transfer uncontaminated calli into a new plate. If you keep these rules and repeat this tactic during frequent and careful subcultures you may eventually succeed to select and grow a contamination-free callus culture.
If you are working on stem cutting you should use anti-fungous like 2% TEBUCONAZOLE. Also, you should know that after producing callus, the cuttings do not need as much water as the first 2 weeks.
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