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thisisfielding
the dude

Registered: 01/30/15
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First time trying out Agar this weekend - A couple general questions
#21305264 - 02/20/15 06:11 PM (9 years, 10 months ago) |
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So heres what I'm working with supplies wise (also have MS syringes, All American 921 and a SAB not pictured). The small container with powder in it is "Gentamycin Sulfate Antibiotic Powder". I've never done agar before and since most of the teks I've read are using household items and I've tried to go the scientific materials route I have some questions. The 1000 mL Sterile Polycarbonate Erlenmeyer Flask I have here is "auto clavable" and looks to have a synthetic filter in its top. Should I still put aluminum foil on the top of the flask? I have an All American 921 PC, how long should I PC the mixture? Should I just only do water + one of the premixed agar mixes or would I benefit of mixing 50/50 of the potato and the malt? As far as the the parafilm goes, do you guys just pre-cut sections of it before using? I'm waiting on some grain jars to finish colonizing so I plan on using multispore on this agar. Would you suggest trying inoculate all of the dishes I make or just a few, then use the others to keep isolating? How many dishes will 1000 ml of agar make? My general understanding after searching the forums and reading posts is as follows (please correct me). 1. Mix water with premixed agar mix into flask 2. Add .10 of a gram of the antibiotic to flask per 1000ml 3. PC flask (for undermined amount of time) 4. After place petri dishes, flask, parafilm in SAB 5. Pour Agar substance from flask into dishes 6. Use parafilm to seal each individual dish 7. Let cool to room temperature 8. After agar is set, then cut through parafilm and add a drop of MS solution to dish 9. Seal with parafilm 10. Wait for results.
Sorry for the wall of text,.. I just kind of wanted to throw it all out there in hopes someone will see the big picture and see if I have it wrong somewhere, or if theres a better way to do this using the supplies I have. Thanks ahead of time for any help!
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cronicr



Registered: 08/07/11
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Re: First time trying out Agar this weekend - A couple general questions [Re: thisisfielding]
#21306659 - 02/21/15 01:02 AM (9 years, 10 months ago) |
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i shall be back....
-------------------- It doesn't matter what i think of you...all that matters is clean spawn
I'm tired do me a favor
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Nos-hroom
Oneironaut's Apprentice



Registered: 08/13/14
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Re: First time trying out Agar this weekend - A couple general questions [Re: cronicr]
#21306670 - 02/21/15 01:08 AM (9 years, 10 months ago) |
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Be careful about heating your antibiotic material. Some of the substances degrade drastically when exposed to heat, even levels well below the ~250F of a PC cycle.
The amount per plate will depend slightly on both the depth desired, and the diameter of the dish itself.
The Erlenmeyer you have appears to be designed for autoclaving (and rather well, at that). Still, if the principles are the same, wrapping your lid in foil would help to prevent any water from dripping back down onto the filter from the PC lid itself.
I'm slightly tempted to answer more of your questions and go into more depth with what I have to offer. Then on the other hand, I feel like Cron is about to blow it out of the water with well-sourced answers and trustworthy advice, so I'd check back when he puts in his answers/info.
Edited by Nos-hroom (02/21/15 01:10 AM)
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Mushroom_J
Hard to the Coir !



Registered: 02/17/11
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Re: First time trying out Agar this weekend - A couple general questions [Re: Nos-hroom]
#21306676 - 02/21/15 01:16 AM (9 years, 10 months ago) |
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Cron is getting high lol He'll be back.
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Mandarinfish

Registered: 01/27/15
Posts: 1,365
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Re: First time trying out Agar this weekend - A couple general questions [Re: Mushroom_J]
#21306697 - 02/21/15 01:49 AM (9 years, 10 months ago) |
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.
Edited by Mandarinfish (07/15/20 02:16 AM)
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cronicr



Registered: 08/07/11
Posts: 62,905
Loc: Van Isle
Last seen: 3 minutes, 2 seconds
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Re: First time trying out Agar this weekend - A couple general questions [Re: Mushroom_J]
#21306719 - 02/21/15 02:06 AM (9 years, 10 months ago) |
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Quote:
Mushroom_J said: Cron is getting high lol He'll be back.
yep....and i'm back! but honestly not much to add, i myself would skip the anri biotics and just use the premixed stuff only doing a few plates, you are gonna need to transfer so save some dishes. i also only wrap my dishes when they get knocked up.
-------------------- It doesn't matter what i think of you...all that matters is clean spawn
I'm tired do me a favor
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thisisfielding
the dude

Registered: 01/30/15
Posts: 178
Last seen: 8 years, 7 months
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Re: First time trying out Agar this weekend - A couple general questions [Re: cronicr]
#21310664 - 02/21/15 08:42 PM (9 years, 10 months ago) |
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Okay awesome - so don't worry about wrapping them initially? How long do you usually wait until you inoculate them? I'm only working with a SAB so trying to figure out the best way to be sterile. What I've gathered is 25ml = 1 dish pretty much,.. I have 100 dishes currently (10 x 10 sleeves). I basically want to play around with this / learn it while I wait for my jars to colonize fully.
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,916
Loc: Milky way
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Re: First time trying out Agar this weekend - A couple general questions [Re: thisisfielding]
#21310839 - 02/21/15 09:17 PM (9 years, 10 months ago) |
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the sleeve they come in is sterile. I try to pour a whole stack. I use some the same day I pour them. Try to pour your agar close to 47-50C. agar starts to gel up at around 45C. pouring at 47C helps eliminate condensation and your dishes solidify in a minute or two. use some dishes for whatever work you need to do and then wrap those ones. store them in new ziplocs or another petri sleeve. the leftover dishes from pouring the whole sleeve, go back into the sleeve they came from. there's now some excess which you can fold over and tape down, now your excess dishes are stored in a sterile pouch for later use. and keeping them together while they cool to room temp also helps to combat condensation.
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,916
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Re: First time trying out Agar this weekend - A couple general questions [Re: bodhisatta]
#21310872 - 02/21/15 09:21 PM (9 years, 10 months ago) |
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don't worry about being a noob at agar.

 almost two years ago, contamination on some dishes, wrapped like a noob. still doing better than pushing spore syringes.

get a bit better at wrapping
 soon you'll have nicely wrapped dishes. these are all wrapped with saran wrap. I couldn't afford parafilm. parafilm is very good stuff but saran works period.
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thisisfielding
the dude

Registered: 01/30/15
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Re: First time trying out Agar this weekend - A couple general questions [Re: bodhisatta]
#21311036 - 02/21/15 09:36 PM (9 years, 10 months ago) |
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Thanks so much for clearing up stuff! Those are great motivational pictures! So 250ml mix = 10 dishes,.. I'll try inoculating 2 dishes with a drop or two of MS syringe,.. and then wrap in parafilm. Leave the other 8 untouched back in sleeve after poured. Once the 2 start to show growth then transfer them to another dish until I finally have a dish that looks white and clean. Do you store the dishes with the MS solution at room temperature? Am I right by saying even if the dish is contaminated I can cut off a piece thats white to transfer to a new dish to salvage it? I guess I am a little OCD so instead of just going head and doing it / trial and error I want to make sure I have all that I need, all the info,..etc. Also would either of those mixes make a decent LC mix? I've made a ton of SHIP / SFD lids that I can use for a LC.
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mushpunx
Fungus Punk



Registered: 04/20/14
Posts: 13,394
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Re: First time trying out Agar this weekend - A couple general questions [Re: bodhisatta]
#21311050 - 02/21/15 09:39 PM (9 years, 10 months ago) |
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I like to wrap what I dont use and then store them in the sleeve. Maybe over kill 
Any who, Bod said it.
Take a roll of cling wrap and with a sharp knife cut an inch or so off the roll. It makes it much easier to wrap.
Yea you can take a clean piece from a dish with contamination. If I were you, since you are working with agar quit the LC
--------------------

Amateur Mycologists United
AMU Q&A
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,916
Loc: Milky way
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Re: First time trying out Agar this weekend - A couple general questions [Re: thisisfielding]
#21311102 - 02/21/15 09:57 PM (9 years, 10 months ago) |
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Quote:
thisisfielding said: Thanks so much for clearing up stuff! Those are great motivational pictures! So 250ml mix = 10 dishes,.. I'll try inoculating 2 dishes with a drop or two of MS syringe,.. and then wrap in parafilm. Leave the other 8 untouched back in sleeve after poured. Once the 2 start to show growth then transfer them to another dish until I finally have a dish that looks white and clean. Do you store the dishes with the MS solution at room temperature? Am I right by saying even if the dish is contaminated I can cut off a piece thats white to transfer to a new dish to salvage it? I guess I am a little OCD so instead of just going head and doing it / trial and error I want to make sure I have all that I need, all the info,..etc. Also would either of those mixes make a decent LC mix? I've made a ton of SHIP / SFD lids that I can use for a LC.
room temp is fine. http://www.shroomery.org/forums/showflat.php/Number/18430998/fpart/all/vc/1
that link should also help some
try out some different things with lc as long as you have agar to fall back on
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Val
Were just Marbles in a Cave...

Registered: 01/31/15
Posts: 120
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Re: First time trying out Agar this weekend - A couple general questions [Re: thisisfielding]
#21311126 - 02/21/15 10:03 PM (9 years, 10 months ago) |
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Quote:
thisisfielding said: 3. PC flask (for undermined amount of time)
In the PC 15 psi for 15-20 mins asssuming your at sea level.
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,916
Loc: Milky way
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Re: First time trying out Agar this weekend - A couple general questions [Re: Val]
#21311137 - 02/21/15 10:06 PM (9 years, 10 months ago) |
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Quote:
Val said:
Quote:
thisisfielding said: 3. PC flask (for undermined amount of time)
In the PC 15 psi for 15-20 mins asssuming your at sea level.
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thisisfielding
the dude

Registered: 01/30/15
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Re: First time trying out Agar this weekend - A couple general questions [Re: bodhisatta]
#21315600 - 02/22/15 11:26 PM (9 years, 10 months ago) |
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So PC mixture > Let Cool > Pour Agar - How long does it take for the agar to set? Should I be able to inoculate shortly after pouring or should I just pour them all, put them back into sleeve, close sleeve up and put in fridge. My plan is to make 500ml of mixture, 2 x 10 dish sleeves,.. use the first 2 in each sleeve (four total) to drop a couple drops of MS syringe solution on. Parafilm them all then see what happens in the next couple days. Should I store them all in the lunch box fridge method or does that completely stall growth on the dishes? Where do you guys usually place the sleeves as they are cooling then after they've been inoculated?
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Nos-hroom
Oneironaut's Apprentice



Registered: 08/13/14
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Re: First time trying out Agar this weekend - A couple general questions [Re: thisisfielding]
#21315646 - 02/22/15 11:43 PM (9 years, 10 months ago) |
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I would not put inoculated dishes in the fridge, unless the intent was to stall the growth.
Agar can set on you shockingly quickly or take ~10-20 minutes, with a high water:agar ratio. Glass dishes on a metal work surface in winter here start to gel up in mere moments, but plastic dishes on a wooden work table will take a the normal 5 or so.
Within less than an hour, you could begin inoculation, if you were eager to get a jump on things.
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thisisfielding
the dude

Registered: 01/30/15
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Re: First time trying out Agar this weekend - A couple general questions [Re: Nos-hroom]
#21315663 - 02/22/15 11:48 PM (9 years, 10 months ago) |
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Ok so PC at 20mins at 15 psi - Do I need to wait till the PC is cool to the touch? I'm worried about trying to take the weight off before the pressure has gone down. What is a decent time table from in PC for the 20 mins at 15psi to inoculation? I literally have 10 mins left in the PC cooker.
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Nos-hroom
Oneironaut's Apprentice



Registered: 08/13/14
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Re: First time trying out Agar this weekend - A couple general questions [Re: thisisfielding]
#21315795 - 02/23/15 12:54 AM (9 years, 10 months ago) |
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Okay, so a good handful of us like to let the PC cool on the burner (prevents boil-over) and even just leave it overnight to allow the pressure to come down slowly and just put the flask in a pot with some simmering water; agar that has set will melt once more when introduced to heat, and (thanks to the filter) will remain sterile, so no worries there.
So that's what I'd do, unless you are super busy during the week: just leave the PC to cool nice and slow, then bring it back to its liquid form (swivel it lots at this point to remix the agar which settles during the PCing) and then pour those puppies in front of the flowhood, being sure to keep surfaces sanitary, air well treated (I use oust + flow hood) and your technique brief and sterile.
Edited by Nos-hroom (02/23/15 12:55 AM)
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twistedty
Forcefully Retired



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Re: First time trying out Agar this weekend - A couple general questions [Re: Nos-hroom]
#21315800 - 02/23/15 12:57 AM (9 years, 10 months ago) |
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i just waited until i knew there was no more pressure in pc, grab the bottle with an oven mitt and place in sab. let cool down until i could handle it with my hands and pour my dishes.
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Val
Were just Marbles in a Cave...

Registered: 01/31/15
Posts: 120
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Re: First time trying out Agar this weekend - A couple general questions [Re: Nos-hroom]
#21323993 - 02/24/15 05:47 PM (9 years, 10 months ago) |
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Quote:
Nos-hroom said:
.....So that's what I'd do, unless you are super busy during the week: just leave the PC to cool nice and slow, then bring it back to its liquid form (swivel it lots at this point to remix the agar which settles during the PCing) and then pour those puppies in front of the flowhood, being sure to keep surfaces sanitary, air well treated (I use oust + flow hood) and your technique brief and sterile.
Im sure your method works but...if you have a hood why would you bother with the additional step of reheating. Once your PC has cooled to zero pressure you should be in the clear of boil-overs (but YMMV).
I just place it in front of the flowhood (PC and all) before it reaches zero PSI. Once it reaches zero PSI (or 15-20 mins later before agar would set up after reaching zero PSI) I remove flask of agar until it cools to about 120F (IR therm), and pour.
Edited by Val (02/24/15 06:08 PM)
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