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Offlinesenseit
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Some Basic SAB Sterile Technique Questions
    #23449798 - 07/17/16 12:48 PM (7 years, 6 months ago)

I'm a noob around here. So far I've done two in-vitro grows using Violet's pp5 tek and it's gone pretty well. However, I have some confusion around some parts of the SAB sterile technique that I'm unsure about.

1. Let's say that I have 8 containers waiting to be placed in the SAB. Is it better to wipe down 4 containers, place those in and place another 4 containers in? Or better to have all 8 containers sitting in the open air, then open the SAB once and put all containers in?

2. How often do I need to make a new alochol-soaked paper towel? After every container? After every couple containers? Or do I just need one for all 8 containers? Can I use freshly-laundered rags instead of paper towels to save waste?

3. Once I put the containers in, I usually do a good spray of bleach and oust (actually I'm using ozium and not oust. I think they are essentially the same). Is this overkill? Do I need both bleach AND oust or am I simply trying to kill the contams floating around in the air at this point since I've just sanitized my containers?

4. How often should I re-apply alcohol to my hands and arms? Specifically I wonder about this between agar transfers. I've just done a transfer and I'm re-heating the scapel. Have a bunch of contams just dropped onto my scapel hand? Should I place the scapel in the SAB to cool and then re-apply alcohol to my hands? Do I need to wipe the non-heated part of the scapel down with alcohol?

5. My SAB isn't huge, so let's say I need to do two rounds of containers because everything won't fit. How crazy do I need to get about re-sanitizing my whole area? The combination of bleach, oust, and alochol gets pretty intense smelling.

Thanks for your responses! Any other basics you could pass along re the SAB would be welcomed too!


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InvisibleMushiez
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Re: Some Basic SAB Sterile Technique Questions [Re: senseit]
    #23449812 - 07/17/16 12:53 PM (7 years, 6 months ago)

If you can, I'd recommend a freshly laundered long sleeve shirt, minimizing open skin area to a minimum. Clean long shirt + gloves works well


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Re: Some Basic SAB Sterile Technique Questions [Re: Mushiez]
    #23449896 - 07/17/16 01:26 PM (7 years, 6 months ago)

The SAB needs to be wet, not sanitized. You could spray it with water if you wanted. Wipe down what you want to work with and put it in the box. It's not rocket science lol. Clean rags soaked in iso would be fine instead of paper towels. I usually like a shower before and then do my work while I'm still wet. If everything in the box is wet, there are no contams flying around.

Movements are critical. You could work in a dry box with no gloves if ya had smooth enough movement. Keeping everything wet makes it easier. Never reach over open media. Move smooth and disturb the air as little as possible.


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OfflineMycologist217
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Re: Some Basic SAB Sterile Technique Questions [Re: Pastywhyte]
    #23449951 - 07/17/16 01:43 PM (7 years, 6 months ago)

Just a little more to help with understanding....by creating a wet environment in the SAB with the sprays you are helping to scrub the air...the droplets in the spritz grab contamination out of the air and pull them down out of the air. Which is why water would work as well as multi pupae cleaner with bleach, or oust....

One ISO soaked paper towel for all 8 will be fine,.,,if you don't like that idea....make a jar of ISO soaked cotton balls.....


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Offlinesenseit
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Re: Some Basic SAB Sterile Technique Questions [Re: Mycologist217]
    #23451155 - 07/17/16 08:44 PM (7 years, 6 months ago)

Hey thanks very much for the responses guys. It helps clarify things quite a bit.

Quote:

It's not rocket science lol.




I was actually thinking it was rocket science ;-) I guess I can relax a little. As for the shower, I have been doing that and using a pair of tyvek sleeves on my arms.

Quote:


..by creating a wet environment in the SAB with the sprays you are helping to scrub the air...the droplets in the spritz grab contamination out of the air and pull them down out of the air.




Ah, I didn't quite understand this concept until now. Somehow I thought that the bleach was killing the stuff on the surface and the oust was killing the bacteria, etc flying through the air. I didn't think you were just knocking the contams down to the bottom of the box.

I'm guessing the few contams I have had on agar were from knocking the scapel trying to get the damn wedge off the scapel. I've since read that you can just slice into the agar you're transferring to in order to get the wedge off the scapel.


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Invisiblestareatclouds
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Re: Some Basic SAB Sterile Technique Questions [Re: senseit]
    #23451456 - 07/17/16 10:47 PM (7 years, 6 months ago)

When I first started, I'd use a Tyvek suit or my freshly laundered thin sweatshirt (with the hood on), the facemask, gloves (taped to my sweatshirt), just showered, teeth brushed, etc.

Now I just hop in the shower, come out with facemask and gloves and naked besides the towel (still kinda wet usually) or boxers and go to work. If your technique is solid, you don't need all that extra shit.

It's good to start with because it takes a while to get comfortable making transfers and inoculating shit, but after a while, Tyvek sleeves or taped gloves is unnecessary.

People spray oust and other shit as another measure, which is fine, but a SAB doesn't operate on sterility, it operates on the principle of still air. The soapy water helps contams stick to the walls or drags them to the floor. Just don't have things open too long, move with purpose, don't hover your hand above open stuff, and use 70% iso on everything. Check out Munchauzen's videos.


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InvisibleMunchauzen
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Re: Some Basic SAB Sterile Technique Questions [Re: senseit]
    #23451601 - 07/18/16 12:01 AM (7 years, 6 months ago)

Quote:

senseit said:
I'm a noob around here. So far I've done two in-vitro grows using Violet's pp5 tek and it's gone pretty well. However, I have some confusion around some parts of the SAB sterile technique that I'm unsure about.

1. Let's say that I have 8 containers waiting to be placed in the SAB. Is it better to wipe down 4 containers, place those in and place another 4 containers in? Or better to have all 8 containers sitting in the open air, then open the SAB once and put all containers in?

I load 10 quart jars at a time, or 10 pasty plates at a time.

2. How often do I need to make a new alochol-soaked paper towel? After every container? After every couple containers? Or do I just need one for all 8 containers? Can I use freshly-laundered rags instead of paper towels to save waste?

I usually make a fresh one half way through, so after the fifth lid.

3. Once I put the containers in, I usually do a good spray of bleach and oust (actually I'm using ozium and not oust. I think they are essentially the same). Is this overkill? Do I need both bleach AND oust or am I simply trying to kill the contams floating around in the air at this point since I've just sanitized my containers?

Do not use harsh chemicals in your work space. Tap water or soapy water is fine for SAB work.

4. How often should I re-apply alcohol to my hands and arms? Specifically I wonder about this between agar transfers. I've just done a transfer and I'm re-heating the scapel. Have a bunch of contams just dropped onto my scapel hand? Should I place the scapel in the SAB to cool and then re-apply alcohol to my hands? Do I need to wipe the non-heated part of the scapel down with alcohol?

I remove the foil, wipe down with iso, and loosen the lids on 2 agar dishes at a time, then I ISO my hands and tools before doing those 2 agar dishes. Between these two plates, only one hand has to leave the SAB, which is to flame the blade for my next transfer. After I'm done, I remove the foil on 2 more dishes and repeat the process. When pouring LI, I do it once then do the entire pour without removing my hands once.

5. My SAB isn't huge, so let's say I need to do two rounds of containers because everything won't fit. How crazy do I need to get about re-sanitizing my whole area? The combination of bleach, oust, and alochol gets pretty intense smelling.

No need for all that shit, really. I wipe down my glasstop work surface with iso everytime I load my SAB, then spray the SAB down with more tap water. Forget about the bleach and oust.

Thanks for your responses! Any other basics you could pass along re the SAB would be welcomed too!

watch my vigeos in my signature for SAB techniques. working upside-down with plates is a fave of mine.








Edited by Munchauzen (07/18/16 01:02 AM)


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InvisibleInocuole
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Re: Some Basic SAB Sterile Technique Questions [Re: Munchauzen]
    #23451636 - 07/18/16 12:24 AM (7 years, 6 months ago)

Huffing chemicals is bad.


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Re: Some Basic SAB Sterile Technique Questions [Re: Inocuole]
    #23451650 - 07/18/16 12:37 AM (7 years, 6 months ago)

I have poured dozens of 20 plate 100x15mm plates in a small 66q SAB. Bigger custom made SAB is way more awesome though.

No need for fancy chemicals or even gloves for that matter


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Offlinesenseit
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Re: Some Basic SAB Sterile Technique Questions [Re: Munchauzen]
    #23455675 - 07/19/16 10:20 AM (7 years, 6 months ago)

Munchauzen thanks for the responses. I will check out your videos.

stareatclouds thanks for the info, I'm going to try with just water and stop huffing the chemicals :-)


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OfflineLa Flama Blanca
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Re: Some Basic SAB Sterile Technique Questions [Re: senseit]
    #23457611 - 07/19/16 09:03 PM (7 years, 6 months ago)

So you want the things in the SAB wet too? I could have swore it said in pastywhytes tek that he lets the plates dry for a few hours in the SAB if they seem wet....seems contradictory to me


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Re: Some Basic SAB Sterile Technique Questions [Re: La Flama Blanca]
    #23457640 - 07/19/16 09:15 PM (7 years, 6 months ago)

Nobody said that.. you just want the SAB wet... Everything else should be reasonably dry, or wiped down with iso recently so... dry.


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Re: Some Basic SAB Sterile Technique Questions [Re: La Flama Blanca]
    #23457666 - 07/19/16 09:21 PM (7 years, 6 months ago)

Quote:

cj711 said:
So you want the things in the SAB wet too? I could have swore it said in pastywhytes tek that he lets the plates dry for a few hours in the SAB if they seem wet....seems contradictory to me




He was referring to the condensation inside the plates though...


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OfflineLa Flama Blanca
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Re: Some Basic SAB Sterile Technique Questions [Re: dankington]
    #23457752 - 07/19/16 09:43 PM (7 years, 6 months ago)

Gotcha. Thanks for the clarification bros


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