After washing the soil off the roots you can dip them Tween 20 if the soil does not get washed off, or even place the roots in a sonicator bath for 30 seconds at a time. Then immerse in 70% ethanol for 2-3 minutes and depending on how soft or hard the root is you can vary the hypochlorite concentration from 2% to 6% and vary the time from 1 to 5 minutes.

And then wash off the hypochlorite with a few volumes of sterile water. While we check for surface sterilisation by rubbing the sterile plant on agar or plating out the final water wash, one can never rule out microorganisms that are tightly bound to the outside of the plant as they could survive the sterilisation process. A more appropriate term for microbes that are isolated using these surface sterilisation protocols is "plant associated microorganisms".

I think the method described in the book In Vitro Culture of Mycorrhizas (2005) from the publishers Stéphane Declerck Désiré-Georges Strullu and J.-André Fortin could be a good alternative:

1. Clean the roots thoroughly with sterile (demineralized) water, three times

2. Treat the roots with alcohol (95–98%) for 10 s (pre-treatment 1)

3. Rinse three times with sterile (demineralized) water

4. Treat the roots with the calcium hypochlorite solution for 1–2 min (pre-treatment 2)

5. Rinse three times with sterile (demineralized) water

6. Treat the roots with the solution of chloramine T 2% (20 g / l, with 2–3 drops of Tween® 20) for 10 min

7. Rinse 3 times with sterile demineralized water

8. Treat the roots with the antibiotic solutions for 10 min

9. Remove the antibiotic solutions and transfer the roots to a Petri plate with a fresh antibiotic solution. Keep them in the antibiotic solution until use.

Antibiotics solution: Streptomycin sulphate 0.02% (20mg/100 ml) and gentamicin sulphate 0.01% (10mg/100 ml; 100 ml; solution for step 2).

please read Krimi and al. 2016 there's the procedure for bacterial endophytes and Mohamed Mahmoud et al, 2017 for isolating fungal endophytes

I recommend McKinnon 2016 that addresses this topic very specifically. The paper is named "Plant Tissue Preparation for the Detection of an Endophytic Fungus In Planta". Best wishes

Christopher M M Franco We are also trying similar protocol and we havenot been able to get negative control plates without the growth of bacteria. In this case, how would you define these bacteria in the plates, how much possibility there is that the growing bacteria are endophytes?

Dear Jyotsna,

Please indicate the protocols you use, including the plant and the testing protocol.

If there are bacteria growing when you check your wash solutions or rubbing the sterilised plants then your surface sterilisation has not tremoved all the microbes.

You can increase the concentration of the fresh hypochlorite. Take care that the hypochlorite does not get into the root-you can check the level of bleaching..

As for the question as to whether these bacteria are also endophytes . It is posssible because many endophytes enter the roots from the rhizosphere. However, if the surface sterilisation is done then there should not be a leakage.