We are inducing callus from wheat embryos (media-MS+2,4-D), getting lot of contamination (both bacterial and fungal). Which is seems to be arising from seeds not the media, as its appearing around the callus. We tried follwoing changes in our protocol.
1. Taking atmost care in sterilization of seeds and all the tools.
2. We use 70% alcohol and 50% bleach for seed sterilization - here we increased and decreased the alcohol and bleach concentration but no use.
3. Taking care during working in hood to avoide external contamination.
4. Tried adding ppm (plant preservative mixture) to callus induction media. Which is broad spectrum biostatic and biocidal.
5. Subculturing non-contaminated callus as well did not help.
Despite the most stringent use of sterile techniques and aseotic conditions, the contamination remain persistant problem.
Your suggestions are very wel come and much appreciated.
Thanks in advance
Uday
If you work with sterilised seeds, media and tools then perhaps you have some problems with the hood. Have you checked if HEPA filters in the hood are ok? Do you use disposable pipette tips and other plastics? How do you sterilise tools which are used more than once? I would suggest autoclaving if they are ressistant to heat, but you should also check if you autoclave works fine (using sporal). Other idea is that you have contamination inside your incubator.
70% alcohol doest not sterilise surfaces - it only disinfecs, so it leaves fungal spores and bacterial endospores intact.
Sir, try these methods
1.Wash ur seeds first in running tapwater
2.put around 50 seeds and a drop of liquid washing detergent in a closed container with lid containing autoclaved water and shake it well, from this step use autoclaved vessel and water.
3.Give 30 to 1hrs agitation with mg/lml of any fungicide with bactericide
commercial ones include BAVISTIN and streptocyclin, then wash
4.Take it into laf, do surface sterilization with a drop of tweentwenty in0.%HgCl2 solution shake it well in a closed vessel for 2-5 mins, then wash
5.give nice shaking with 70% ethanol, and three water wash,
6.Double sterilize ur equipments, blotting paper,petriplates, use two seperate forceps, flame and use, alternately
Try these, it will definitely reduce, if it persists again , attach the pic of the seeds getting contaminated, we can identify the nature of contamination.
Depending on that I can suggest some more ideas.
In culture media add 200 mg/l cefotaxime (bactericide), and for fungi control. Proper uv treatment in laminar airflow & proper wash of material with Hgcl2 (0.2 % ).
You said that your contaminations were only observed around the callus, not the medium. Judged from that, this contamination can be due to the 'seed' itself or the tools used for dissecting and moving the tissues.
For these, you should make sure that the seeds are properly sterilized, and also, the tools should be properly sterilized before use.
(1) Had you added 'surfactant' in the bleach while you used it for seed sterilization? After you sterilized seeds with ethanol and bleach, was the sterilized (autoclaved) water you used to rinse them ok? To ensure water quality, I always use/open a new bottle of (autoclaved) water for rinsing the seeds during seed sterilization.
(2) Were the tools properly sterilized and kept sterilized in the hood? We autoclaved our tools first. Then, inside the hood, we also briefly flame the tools before every action (cutting the tissue or moving the tissues).
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