Re-Sterilizing Tissue Culture Explants!

Are you throwing away your contaminated explants? Don't do that! Because you can re-sterilize them and use them again that will save your time and money put in the culturring processes. So, in this video we are going to reuse one of our contaminated culture of Mostera deliciosa and show you the re-sterilization process by using its roots.
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[@dracolusus]

Coming from a fungi culturing background, is it not preferable to open open that contaminated vessel in a still air box? I was taught to clean up contam'd agar dishes SAB, to prevent the contam spores being blown all over you, your workspace, etc by the laminar flow hood. Can do the retrieval and Iso clean in the SAB, then move to the flow hood, just my 2c

[@PlantCellTechnology]

Absolutely right! This was just for video and education purpose.

[@alfredouriarte8078]

what happened at the end of the experiment? does the explant present contamination again in the new media culture?

[@TheDuckofDoom.]

Bleach is shipped with excess NaOH to keep it stable in storage. Adjust the pH of the bleach solution down to 6.5 This is the recommended solution for washing bulk produce and general sanitation with as low as 20ppm active Cl. The disinfection effectiveness of chlorine bleach drops 2-3 orders of magnitude as pH rises from 7.0 to 8.5. HOCl is a far better disinfectant than OCl and -OH. Below pH 4.0 it becomes too unstable and forms chlorine gas.

[@PlantCellTechnology]

Francisco definitely handles all that and takes care of those parameters. Thanks for the concern. :)

[@TheDuckofDoom.]

@@PlantCellTechnology But it was not mentioned or demonstrated in the video, if the adjustment was made there is no evidence. So I put the information here to help others (and maybe help Francisco if it is something he forgot.).

[@PlantCellTechnology]

@@TheDuckofDoom. Sure! We appreciate it. Thanks!

[@deegerhaupt]

Thank you so much.

[@realchristopher4334]

I am confused about the calculation. 1mL bleach 7.5% stock concentration to 100mL water should result in approximately 0.075% bleach solution. Not 1%.

[@alessandramoraes7697]

I have the same doubt

[@TheDuckofDoom.]

He said 1% common bleach, not 1% sodium hypochlorite. Common chlorine bleach is a mixture and typically a combination of 6%-8% sodium hypochlorite with enough excess NaOH from the production reaction for a pH over 12 for good shelf stability. Although the correct chemistry measure for the active component would not be percent NaOCl it would be normality (basically moles of reactive sites or sub-component of interest, OCl- in this case) and that would allow direct conversion to calcium hypochlorite [ Ca(OCl)2 ] or hypochlorous acid and possibly other related chemicals.

[@AquaMayne]

Can you use any dish if you spray it with 70% alcohol or do you need to autoclave the dish too?

[@PlantCellTechnology]

No, spraying a dish with 70% alcohol alone is not sufficient for tissue culture purposes. Autoclaving is the recommended method for sterilizing reusable glassware. Or you can use disposable, Pre-sterilized Dishes. These are the safest and most convenient options. They come pre-sterilized and are readily available, minimizing contamination risk.

[@MrJvartanian]

Great Video.

[@PlantCellTechnology]

Thanks!

[@thembrelectric]

Thank you so much for all your info sir

[@sermsaklimpadapun6054]

Please update the result. I am having problem too.

[@rubiajv7478]

Is a good idea introduce the contaminated explant into de cabinet flow? Thanks for sharing

[@PlantCellTechnology]

It is not ideal, but sometimes you have to do it. Just make sure all your tissue culture flasks are closed while working with contamination. Make sure to clean the working area with 70% isopropyl alcohol or 70% ethanol before and after you are done.

[@ashirana5759]

@@PlantCellTechnology l have asked a question that if a explant like bulb before use get some fungus in store condion in fridge then what should do before use

[@PlantCellTechnology]

@@ashirana5759 Did your cultured explants got contaminated in their storage condition?

[@ashirana5759]

@@PlantCellTechnology yes in test tube even after 5 days fungus appeared......

[@ashirana5759]

@@PlantCellTechnology one thing more my bulb get some fungus in freezer as i store them in aluminium foil..but before use i wash bulb with detergent also use alcohol+ bleech

[@matchmedicines8209]

great advice, thanks

[@PlantCellTechnology]

You're welcome!! Stay tuned to our channel for more videos like this.

[@jinty1232]

What size of culture jars do you use and which company sells them? How much agar media do you add to each jar? Does the lid need to have a filter or can the explants survive happily without it?

[@PlantCellTechnology]

I use jars from plant cell tech. Here's the link: www.plantcelltechnology.com/Lab-Equipment/ I also use some autoclavable plastic containers that I purchase from my local market. You need to add around 20-25 ml media to one container. Lids with filters work better for the growth of plants as they allow easy exchange of gases. However, the plants can also survive without them, but care is needed.

[@jinty1232]

@@PlantCellTechnology Thanks for the information. I'm trying to do some tissue culture just now but on petri dishes at the moment. Jars will come later hopefully!

[@PlantCellTechnology]

@@jinty1232 Amazing. Good luck with your cultures!!! And keep watching PCT videos.

[@davebayles9695]

Hi my friend. Can you make the solution for making the media for stems and leafs and the media for making roots. And the process of moving the cultures from one media to anther.

[@PlantCellTechnology]

Yes, we are going to experiment with plant hormones in future videos!

[@user-xo4tk7jt3w]

is the method applicable for plant contaminated by bacteri? if not, how do we resterilizing the plant?

[@PlantCellTechnology]

surface sterilization effectively removes any microbes on the surface of the explant. However, it would be best if you used other molecular techniques to detect endophytes.

[@cedrickalguzar5654]

Hello. Will this work if I re-sterilize multiple explants simultaneously in one flask? Also, is re-sterilization advisable if your plant species houses endophytic fungi?

[@PlantCellTechnology]

Sterilization is of course essential in this case. And, before culturing you will have to ensure that your explant doesn't have any fungi, for which you will have to use a microscope. It's recommended to use a healthy explant to avoid all rework and save your time and money spent on the procedure.

[@cedrickalguzar5654]

I see. Anyway, can I re-sterilize multiple explants simultaneously in one flask?

[@PlantCellTechnology]

@@cedrickalguzar5654 Yes. You can definitely do that.

[@cedrickalguzar5654]

@@PlantCellTechnology Alright. Will definitely try this. Also, do you have a video on how to treat explants with antioxidant solution to control their browning?

[@shanain6993]

I want to know how to sterilize a rosemary plant

[@PlantCellTechnology]

You can google to find the protocol of your desired plants.

[@ReversedCircle]

Hello, thanks for all advice content in this video! Do You use vinegar just for pH change or is there any other reason for this step?

[@PlantCellTechnology]

No, the vinegar and hydrogen peroxide is also used to sterilize the explant. Combined, they make a very strong oxidizer and a very effective sterilizing solution.

[@mayurshah8319]

@@PlantCellTechnology can Vinegar also encourage root formation? I want to do at home but I am not getting benzylaminopurine and Indole acetic acid here. What are the alternatives?

[@stevenfontana2905]

@@PlantCellTechnology fascinating! no plain water rinsing is necessary?? thank you for making these videos, they are very helpful.

[@PlantCellTechnology]

@@mayurshah8319 No, you need to add auxin to induce rooting in plants.

[@PlantCellTechnology]

@@stevenfontana2905 Do rinse with the distilled water to remove any remains of chemicals. Because you don't want them in your cultures.

[@kaminiveer7942]

how do you sterilize the magnetic stir bar?

[@PlantCellTechnology]

70% isopropyl alcohol.

[@ritambharabhutani2473]

hey. i am doing plant tissue culture of banana in my laboratory, is this method can be helpful for bacterial contamination?

[@Pips.Hunter]

Is Cleaning vinegar same as cooking vinegar ?

[@PlantCellTechnology]

Cleaning vinegar is stronger, but not by much. If you are using cooking vinegar, you may want to increase the time.

[@Pips.Hunter]

Ok, since i cant find cleaning vinegar in my country. Thanks!

[@Darthsura]

Hola Francisco, gracias por tus videos, ¿harás algún video para esterilizar desde 0 las plantas? Saludos 👨🏻‍🔬👨🏻‍🌾👍🏻

[@PlantCellTechnology]

Gracias por tu apreciación. El procedimiento de esterilización difiere según la especie de planta. Entonces, ¿para qué planta quieres tener el video?

[@Darthsura]

Dionaea muscipula y Darlingtonia californica 🙋🏻‍♂️

[@PlantCellTechnology]

@@Darthsura Recientemente publicamos un artículo sobre Darlingtonia californica. Puede que le resulte útil. Aquí está el enlace: www.plantcelltechnology.com/blogintroducing-darlingtonia-californica-to-tissue-culture/

[@shanain6993]

​@@PlantCellTechnologyI want to know how to sterilize a rosemary plant😊😊😊

[@PlantCellTechnology]

We haven't worked on the plant yet. But, you can find a suitable protocol on Google. Many studies must be available on the tissue culture of plants. @@shanain6993

[@Mattytube18]

What brand of wrap do you like to use in the lids?

[@PlantCellTechnology]

We use saran wraps, sold for lab uses. You can buy these from any brand available to your place.

[@burakdurmaz6791]

why didnt you use ppm for contamination ?

[@PlantCellTechnology]

PPM was already in the tissue culture media. However, you can definitely use it for surface sterilization as well, but we need to curate the procedure right for each plant and type and level of infection. So, we are totally working on it.

[@lamontcranston2256]

What is the purpose for wearing a mask but no gloves?

[@PlantCellTechnology]

It is extremely dangerous to wear gloves; if you are exposing yourself to something that can burn your hand, the chemicals on the gloves will further the burn.

[@watercooler94]

im wearing mask and no glove. using glove for me will be danger because i keep burn the scapel into fire

[@kavithaanand5304]

can I re sterile banana also