Post by ischan on Mar 23, 2008 7:20:49 GMT
Hello everybody I need to ask for some help with tissue culture. I bought Dr. Carol's tissue culture kit, and after 4 L of media and probably a few hundred cuttings, I still have not been successful at sterilizing leaves. I'm only working with drosera. I think it's time I asked for some help.
Here is what I have done previously and the mistakes I've committed and learned since then. I still don't know how to move forward so let me just tell you what I've done and see if you can tell me what I can do to put me on the right path.
All media was made with distilled water, and all sterile water was made with distilled water, and 1ml PPM
Feb 23, Media -- 50% MS, 2 Liters, 0.5 ml PPM per liter (I know this is wrong, I corrected it in the late a recipe.) 2 table spoons of sugar, pH paper to get 5.5 pH. 8 g of agar per liter (I know this is too much, and I reduced it in my next recipe)
Feb 24, cuttings and seeds. Cuttings from filiformis, falconeri, paradoxa, petiolaris, spathulata, burmanii, leaft cuttings. Seeds from nepenthes Viking.
Sterilization technique:
bLeach 5%, 10 minutes, rinse, five minutes, done on leaves
bleach 20%, 10 minutes, rinse, five minutes, done on seeds
49 ml MS Media 50%, 1ml PPM, for hour soak both leaves and seeds
Results:
seeds turned black, still retaining.
Leaves in bleach and leaves in ppm, 100% contamination after the first week.
I used the same media the following weekend March 2 for this batch of leaves:
bleach 10%, 10 minutes, rinse, detergent one drop in the bleach, two rinses, five minutes each
Leach, 7.5%, 10 minutes, rinse, detergent one drop in the bleach, two rinses, five minutes each
Condemnation:
bleach, 7.5%, six out of 12
bleach, 10%, eight out of 13
After a week, all remaining leaves turned black as well. I opened up the containers last weekend. It was very smelly inside, and some of the leaves have begun to leak, it was a white cloudy liquid came out. Others began to decompose and there were fungal attacks.
I made a new batch of media, on March 8. 50% MS Media, 1 ml PPM per liter, two tablespoons of sugar, two different types of agar --- 8 g per liter, and 4 g per liter. I also put BAP, and one liter of media and kept it out of the other liter.
On the ninth of March, I used bleach at 10%, for 10 to 15 minutes, and rinsed the leaves.
Results:
zero contamination for everything. All leaves turned black within a week. Everything was discarded on the 21st of March without any growth. The same leaking of cloudy liquid was observed yesterday during the cleaning process.
I made a new batch on the 16th of March:
bleach 5%, rigorous shaking for 10 minutes, rinse for 1 1/2 hours
bleach 3%, rigorous shaking for 20 minutes, rinse for one and half hours
the same symptoms were observed, last week, all remaining leaves turned black, they start turning back at the tips and then slowly the whole leaf turns black. By now I had known what this symptom meant so I discarded everything again yesterday, some of the leaves were turning black at the tips, and also fungal in the centers of the leaves.
Yesterday I tried another batch, this time I used 1% bleach, five minutes, and two five-minute rinses
I also tried 0.5% in bleach for seven minutes followed by 2, 5-minute rinses
The bleach is becoming so little at 1% and 0.5 so I'm quite sure that it's not powerful enough. So what I tried is to lower the pH to 5.5. I got it down to around 5.4 and then I stopped.
My question is how I can go about sterilizing leaves without everything turning black after a week, but at the same time be able to get rid of all the infection. I see all the recipes online calling for 10% bleach and I have no clue how anything can live after observing what I've been doing.
Only now I read that you're supposed to do this in small amounts. Each time I make, I probably have at least 40 cups. So I've probably killed more than 200 cuttings by now probably not the best way to do things.
I would prefer to stay away from chemicals like nadcc, calcium hypochlorite, and other pool cleaning agents which I don't know how to get my hands on easily. I hear that hydrogen peroxide might work although nobody seems to be willing to share with me a suitable concentration and duration of sterilization time.
I read on this forum that you can get away with bleach. I'm prepared to try bleach with other tricks, or, hydrogen peroxide.
If there's really no choice but I will try other chemicals if you can tell me where to get them. I live in Singapore. Please don't tell me to go to Ralph's or Lowe's, we don't have those over here.
Any help is greatly appreciated, I think after so many badges and so much time and effort, I would really like some guidance now because I have not made any success on my own other than to tissue culture wild growing colonies of fungus and mold.
Here is what I have done previously and the mistakes I've committed and learned since then. I still don't know how to move forward so let me just tell you what I've done and see if you can tell me what I can do to put me on the right path.
All media was made with distilled water, and all sterile water was made with distilled water, and 1ml PPM
Feb 23, Media -- 50% MS, 2 Liters, 0.5 ml PPM per liter (I know this is wrong, I corrected it in the late a recipe.) 2 table spoons of sugar, pH paper to get 5.5 pH. 8 g of agar per liter (I know this is too much, and I reduced it in my next recipe)
Feb 24, cuttings and seeds. Cuttings from filiformis, falconeri, paradoxa, petiolaris, spathulata, burmanii, leaft cuttings. Seeds from nepenthes Viking.
Sterilization technique:
bLeach 5%, 10 minutes, rinse, five minutes, done on leaves
bleach 20%, 10 minutes, rinse, five minutes, done on seeds
49 ml MS Media 50%, 1ml PPM, for hour soak both leaves and seeds
Results:
seeds turned black, still retaining.
Leaves in bleach and leaves in ppm, 100% contamination after the first week.
I used the same media the following weekend March 2 for this batch of leaves:
bleach 10%, 10 minutes, rinse, detergent one drop in the bleach, two rinses, five minutes each
Leach, 7.5%, 10 minutes, rinse, detergent one drop in the bleach, two rinses, five minutes each
Condemnation:
bleach, 7.5%, six out of 12
bleach, 10%, eight out of 13
After a week, all remaining leaves turned black as well. I opened up the containers last weekend. It was very smelly inside, and some of the leaves have begun to leak, it was a white cloudy liquid came out. Others began to decompose and there were fungal attacks.
I made a new batch of media, on March 8. 50% MS Media, 1 ml PPM per liter, two tablespoons of sugar, two different types of agar --- 8 g per liter, and 4 g per liter. I also put BAP, and one liter of media and kept it out of the other liter.
On the ninth of March, I used bleach at 10%, for 10 to 15 minutes, and rinsed the leaves.
Results:
zero contamination for everything. All leaves turned black within a week. Everything was discarded on the 21st of March without any growth. The same leaking of cloudy liquid was observed yesterday during the cleaning process.
I made a new batch on the 16th of March:
bleach 5%, rigorous shaking for 10 minutes, rinse for 1 1/2 hours
bleach 3%, rigorous shaking for 20 minutes, rinse for one and half hours
the same symptoms were observed, last week, all remaining leaves turned black, they start turning back at the tips and then slowly the whole leaf turns black. By now I had known what this symptom meant so I discarded everything again yesterday, some of the leaves were turning black at the tips, and also fungal in the centers of the leaves.
Yesterday I tried another batch, this time I used 1% bleach, five minutes, and two five-minute rinses
I also tried 0.5% in bleach for seven minutes followed by 2, 5-minute rinses
The bleach is becoming so little at 1% and 0.5 so I'm quite sure that it's not powerful enough. So what I tried is to lower the pH to 5.5. I got it down to around 5.4 and then I stopped.
My question is how I can go about sterilizing leaves without everything turning black after a week, but at the same time be able to get rid of all the infection. I see all the recipes online calling for 10% bleach and I have no clue how anything can live after observing what I've been doing.
Only now I read that you're supposed to do this in small amounts. Each time I make, I probably have at least 40 cups. So I've probably killed more than 200 cuttings by now probably not the best way to do things.
I would prefer to stay away from chemicals like nadcc, calcium hypochlorite, and other pool cleaning agents which I don't know how to get my hands on easily. I hear that hydrogen peroxide might work although nobody seems to be willing to share with me a suitable concentration and duration of sterilization time.
I read on this forum that you can get away with bleach. I'm prepared to try bleach with other tricks, or, hydrogen peroxide.
If there's really no choice but I will try other chemicals if you can tell me where to get them. I live in Singapore. Please don't tell me to go to Ralph's or Lowe's, we don't have those over here.
Any help is greatly appreciated, I think after so many badges and so much time and effort, I would really like some guidance now because I have not made any success on my own other than to tissue culture wild growing colonies of fungus and mold.
