Hi Ali,

Depending on the type of tissue you are working with, you can increase the concentration of HgCl2 up to 0.3%. But please try step by step as if it is too much you tissues will not survive. Prior to sterilization with HgCl2 you may try washing the explants in a mixture of 70% ethanol + Chlorhexidine digluconate + benzalkonium chrorure (these two last compounds are commonly used to disinfect small human wounds. It can be easily found in a pharmacy). Then just dip the explants in 70% ethanol for a few seconds and then use HgCl2 for 10-15 mns and rinse several times with DI water. I have a question: are you sure that the contaminants are located on the surface of your explants and not inside the vascular system? This is important to know for adjustment of sterilization procedures.

Good luck and keep me posted on progress.

Pierre

In my opinion, the best sterilizing agent is sodium hypochlorite. Use at concentrations ranging between 0.5 and  1 - 2%,  considering the explant you need to sterilize. Sterilization must be preceded by washing in running water. Sterilization times can vary between 15 to 60 minutes. This is followed by 2-3 washings in deionized sterile water under laminar flow hood.

I agree with Paolamaria. HgCl2 is toxic, while sodium hypochlorite is safe and easily available (commercial bleach usually contains abiout 5% of NaClO). I usually dilute commercial bleach and use it in the range of 1-2 % depending on explant (1% for soft explants like leaves, 2-2.5% for woody explants). Here the steps

1. wash the explants in running tap water

2. pre-sterilization (to low the amount of surface bacteria) in 70% ethanol, max 1 min

3. sterilization with sodium hypochlorite (20 min), under laminar

4. 2 or 3 washing steps of 10 min each with sterile distilled water, under laminar

Good luck,

Sara

Yes, I agree that sodium or calcium hypochlorite is less toxic than mercury chloride but, from our experience, when donor plants are coming from the fields (not from greenhouse)  most of the time these are not efficient enough to get rid of the contaminants so that HgCl2 is preferred. This has to be evaluated case by case, year by year.

Regards,

Pierre

Apart from the suggestions, offered by. Drs. Devaux & Sara, you may consider treatment with Bavistin (1-2%, w/v)  1 hr. prior to HgCL2/NaCLO. After Bavistin treatment wash the explants in running tap water.

Hi all,I would like to suggest you that  use sodium hypochoride( 0.5- 1.5% + tween 20 surfactant ( 0.1%) and washed for 5-10 min ( It depend on tissue)  TWEEN 20 is a nonionic detergent widely used in biochemical applications.it will definitely help you.

Regards

vrushali

The suggestions are excellent. However, the balance between killing microbes and keeping explant viable is often very delicate. Considering all suggestions, possibility of clean yet healthy cultures could be low in some cases, especially if the explants are of underground/nearground  origin. So, keep trying and good luck!

I have found commercial bleach the most useful, if you are having real trouble use 10% bleach (with some tween 20) for 10 minutes with agitation. It is not very pretty but if your plants survive they should be free of fungi. Good Luck

Here in the US we have a product called FungiGone by BioWorld company

Thank you all above for the answer and for sharing precious information.

Dear Pierre Devaux, you have pointed out an important thing about the endophytic contamination. It seems the same in my case because all my explants died may be due to the chemical bite of 0.1 % HgCl2 for 05 minutes but the contamination still remained there.

Paolamaria Chiavazza, Sara Barberini, Vrushali Gawade and Will Pelton all are suggesting the use of NaClO in one way or the other but will it work for the endophytic contamination (contaminants inside the vascular system) as well?

Apart from this what would be your suggestions about the use of Hydrogen peroxide H2O2, which is considered an efficient anti fungal? What concentration should be used and how should it be used?

Nirmal Joshee, How could FungiGone could be used for the control of invitro fungal contamination either by using it as surface sterilizer for explant or by mixing it into the culture media directly or in any other way and how much is it efficient? Kindly guide me.

Dear Iftekhar Alam the same happened to my explants they did not survived at all but contamination was still there.