Available chlorine in the dissinfection

[coline]

Hi, as now I'm getting close to start my first home tissue culturing of carnivorous plants, orchids and other plants, I have been reading again about what concentrations are used for this step of a dissinfection, and I have seen reccomended percentages that range from 5% to even 30%, with different times. But, what I am startled with is, how much of that is the available chlorine in the solution?
My most common household bleach in this country comes with a 5,25% v/v available chlorine (NaClO), so, in this case, how can you make those dilutions so you do not burn all the material?

[adamcross]

Our TC team uses a standard 4% NaClO solution for general sterilisation of vegetative material, and I've used between 2 and 5% with good results for TC and agar work with Aldrovanda (seed and vegetative material). I'd suggest starting with a low concentration (say 2% w/v) and see how it works on material you can afford to practice on, and figure out what works for each species using a stepwise increase until you're pleased with the results.

One thing to bear in mind in addition to the concentration of sterilising agent is the time spent in the solution- immersion a 2% solution for 6 hours can kill tissues just as readily as in 50% for a minute. I'd suggest a 15-30 m immersion at low concentration to start with, and see how you go.

[coline]

Ok, so as my comercial bleach solution has a 5,25% v/v and you suggest me to make it 2%, I need then 38ml of bleach and 42ml of distilled water to make a 100ml 2% solution, and with that one I start trying with different dissinfection times ranging from the 15 to 30 minutes?

[adamcross]

That would be my suggestion; alternatively, try with one or two times and increasing concentrations. It really depends on the time and resources you have available!

[coline]

Ok thanks a lot, I'll try it and post pictures of the results, as soon as the new packet of medium arrives and I can work!

[Medina]

Hi Coline, I used comercial bleach solution, it has a 5,25% v/v, diluted 1:10 (10mL of bleach and 90 mL water it is 0.52% v/v) for 10-15 minutes for seeds. For sterilisation of vegetative material I used bleach diluted 1:3 (10 mL bleach and 20mL water it is 1.75%) o bleach diluted 1:2.5(10 mL bleach and 15 mL water it is 2.1%) for 10-15 minutes with good results.

[coline]

Thanks a lot, very well explained! and even for the picture, it made me ask to you, is that really a piece of stem that you tissue cultured, I say, a piece that does not have the apical meristem? And did you use that dilution of 1:3 or 1:2.5 for that explant?

[Medina]

Its a explant with apical meristem and i use comercial bleach dilution of 1:2.5 and in effect, this explant was cultured in vitro after many tries.

[coline]

Great job then. Are you going to split it for it to make a better callus and multiply?