Surface Sterilization

[Deebs]

Basically the surface sterilization process involves taking the explant, and chemically sterilizing/disinfecting it. This is used to remove the contamination in your cultures. The rinsing process is preferably done in front of a sterile airflow.
Edit: @SCJedi , thanks for the correction.

Once thing I am seeing here is differences in practices, and wonder if they vary depending if its a meristem or nodal explant, or just the plant itself. For instance in some examples I've notice that the explant was soaked in alcohol very quickly, rinsed with distilled before a 10 min soak in sodium hypochlorite. Then another 3-4min wash with distilled. Others alcohol totally excluded. Is that added ISO step a choice or required for a reason? @SCJedi ? Also categorically speaking would incubation, callus formation, and root/shoot formation also belong in the initiation phase?

I think this should go here, but will move the thread if necessary. Just want to document some of the thoughts I am having.

 

[SCJedi]

First, a mild correction. The sterilization does not need to happen in a hood but once you rinse them you need to keep them sterile. The wash can happen in the lab but the rinse should occur in clean airflow.

Surface sterilization varies primarily on the tissue type you are using. You got it right that a meristem, due to its fragility, is a lot easier to desiccate than a woody node. Sterilants are selected accordingly.

There is a lot of research on various chemicals that can be used to sterilize the outside of an explant. As you previously mentioned, some efforts are combined as opposed to used as a stand-alone procedure. The best method to use is the one that works for your particular experiment. For example, I take meristems from sterile cultures and not from unsterile explants. This allows me to dig away sterile primordia without worrying about scraping through a contaminant that may have been hiding in a recess.

There are tips that you learn as you work through trials. Maybe you wash with soapy water and then do your hypo wash but you still seem some exophytes. You try a 30-second dip in 90%+ ISO in lieu of the soapy water wash and it reduces your contamination level down to zero. You know which way to lean for that cultivar/client.

There are other components that should be added to your hypo wash. Polysorbate (Tween) is a surfactant that will help break the surface tension between the aqueous hypochlorite solution and the waxy epicuticle of your explant. This allows that hypo to come in contact with more surface area decreasing the possibility of contaminants.

Also, keep in mind that this is still a living organism even though you have dissected it. Too many washes, or too strong of a wash, and you are left with a lacy skeleton of your target explant.

 

[Deebs]

That's was a great explanation man, I really appreciate your feedback. ?

BTW I just assumed more done in front of the hood the better..lol

 

[Capt. C]

Basically the surface sterilization process involves taking the explant, and chemically sterilizing/disinfecting it. This is used to remove the contamination in your cultures. Process is preferably done in front of a flow hood.

Once thing I am seeing here is differences in practices, and wonder if they vary depending if its a meristem or nodal explant, or just the plant itself. For instance in some examples I've notice that the explant was soaked in alcohol very quickly, rinsed with distilled before a 10 min soak in sodium hypochlorite. Then another 3-4min wash with distilled. Others alcohol totally excluded. Is that added ISO step a choice or required for a reason? @SCJedi ? Also categorically speaking would incubation, callus formation, and root/shoot formation also belong in the initiation phase?

I think this should go here, but will move the thread if necessary. Just want to document some of the thoughts I am having.

First, a mild correction. The sterilization does not need to happen in a hood but once you rinse them you need to keep them sterile. The wash can happen in the lab but the rinse should occur in clean airflow.

Surface sterilization varies primarily on the tissue type you are using. You got it right that a meristem, due to its fragility, is a lot easier to desiccate than a woody node. Sterilants are selected accordingly.

There is a lot of research on various chemicals that can be used to sterilize the outside of an explant. As you previously mentioned, some efforts are combined as opposed to used as a stand-alone procedure. The best method to use is the one that works for your particular experiment. For example, I take meristems from sterile cultures and not from unsterile explants. This allows me to dig away sterile primordia without worrying about scraping through a contaminant that may have been hiding in a recess.

There are tips that you learn as you work through trials. Maybe you wash with soapy water and then do your hypo wash but you still seem some exophytes. You try a 30-second dip in 90%+ ISO in lieu of the soapy water wash and it reduces your contamination level down to zero. You know which way to lean for that cultivar/client.

There are other components that should be added to your hypo wash. Polysorbate (Tween) is a surfactant that will help break the surface tension between the aqueous hypochlorite solution and the waxy epicuticle of your explant. This allows that hypo to come in contact with more surface area decreasing the possibility of contaminants.

Also, keep in mind that this is still a living organism even though you have dissected it. Too many washes, or too strong of a wash, and you are left with a lacy skeleton of your target explant.

The knowledge and willingness to share from our members on some of these really complicated subjects just blows me away sometimes. NOT your average stoners

 

[Deebs]

The knowledge and willingness to share from our members on some of these really complicated subjects just blows me away sometimes. NOT your average stoners

Thanks for the vote of confidence @Capt. C ! I am the most basic of newcomers when it comes to this topic though, i just love learning. I am just glad to be able to share, far from what anyone should really listen to though. I am even more grateful for having people like @SCJedi, who can help clear-up any misconceptions or misunderstandings throughout the journey

Love it

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