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Offlineesse_jeremy
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d(r)ied print???
    #23419742 - 07/07/16 10:08 AM (7 years, 6 months ago)

I made several print to agar attempts with this procedure:

-half the agar recipe to a soft surface

-less nutes

-sterelized for 30 min in pc (no pour tek, litle hole with micropore tape as filter)

-cool down and place in a SAB

-use a loop (red hot, cooled in the reciving plates, grab spore, tuch the plates)

-store the innocced plates in a dark and everyday cleaned place


now, before I bought my SAB I got a contam after 3 days I innoc. them (so I assume I sterelized them properly but I fail during innoc)

after several tries, i bought a SAB and inoocc. two plates

the problem is: after a 1.5 week there's nothing growing on the plate.....ok I got no contam so the SAB worked well...but I also have no MYC....nothing.

I'm scared to death that the print is dead or dry...but the agar should reidrate the spore......so why they don't germ??

I did something wrong with my loop? I have to put a drop of sterile water on the print??

I'm afraid...this is my 6th attempt and I never had a myc (also I spend 3 hours a day reading post on the forum to understand the theory and improve but the only thing I saw are people having amazing grow with my same tek....and sometimes also with less gear or with improper sterile tek)


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Invisiblemupetmower
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Re: d(r)ied print??? [Re: esse_jeremy]
    #23419761 - 07/07/16 10:15 AM (7 years, 6 months ago)

It could be the spores. I mean sometimes they can just have a hard time germinating.  Just keep trying. Keep them for at least 3 weeks to make sure it's not jaunt taking a while for them. I've had a couple plates not grow for about 3 weeks and then when I was about to throw them out I noticed they had started germinating finally.

After about a month I would throw them out, though. But just keep trying. You should get some germination as long as the print isn't super old.


--------------------
-The wise man never stops seeking knowledge.


-I wanna feel the change consume me, feel the outside turning in. I wanna feel the metamorphosis and cleansing I've endured within.


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Invisibleweetsie
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Re: d(r)ied print??? [Re: esse_jeremy]
    #23419781 - 07/07/16 10:21 AM (7 years, 6 months ago)

Is there a visible amount of spores on the agar?

A dry print is ok, they still work, just take longer and occasionally stick like a fucker to the foil making it hard to transfer them.

You can try making a spore syringe or digging a little hole in the agar with the loop and putting a drop of water in it with the spores.

Old spores can take a while, 3+ weeks and beyond a certain point it really becomes a numbers game. If you're not noticing contams you can increase your odds by adding spores to multiple sites per plate, I do about 5 or 6 with stubborn spores and even then it sometimes takes 3 plates and a month before you get any that agree to germinate.


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Offlineesse_jeremy
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Re: d(r)ied print??? [Re: weetsie]
    #23419833 - 07/07/16 10:40 AM (7 years, 6 months ago)

I think I can experiment adding spore to one of the plates

Also yes there is a visible ammount (not clearly visible but if u get closer u can see some spore).

And what are pros and cons of making several sites per plates??


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Invisibleimpatientguy
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Re: d(r)ied print??? [Re: esse_jeremy]
    #23419846 - 07/07/16 10:46 AM (7 years, 6 months ago)

You should hydrate the spores IMO

They will germinate much more readily that way.


--------------------
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Offlineesse_jeremy
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Re: d(r)ied print??? [Re: impatientguy]
    #23419918 - 07/07/16 11:18 AM (7 years, 6 months ago)

Quote:

impatientguy said:
You should hydrate the spores IMO

They will germinate much more readily that way.





I'm afraid that if I open them again I will have a contam....I don't know if it's worth 'cause lot of ppl said that this will not speed up germination....I'm so confused


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InvisibleMad Season
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Re: d(r)ied print??? [Re: esse_jeremy] * 1
    #23420040 - 07/07/16 12:05 PM (7 years, 6 months ago)

You can also drop the agar a bit to make it more watery, and up the nutrients. Lower nutrients will be harder to germinate

1.5g of agar and 2g of malt extract would be awesome for germinating.


--------------------
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Invisibledankington
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Re: d(r)ied print??? [Re: Mad Season]
    #23420353 - 07/07/16 02:09 PM (7 years, 6 months ago)

Quote:

Mad Season said:
You can also drop the agar a bit to make it more watery, and up the nutrients. Lower nutrients will be harder to germinate

1.5g of agar and 2g of malt extract would be awesome for germinating.




you might mention the 200ml or so of water. :thumbup:


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InvisibleMad Season
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Re: d(r)ied print??? [Re: dankington]
    #23420382 - 07/07/16 02:19 PM (7 years, 6 months ago)

Lol woops. Actually per 100ml :wink:


--------------------
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Improve your sterile techniques! (A comprehensive guide to agar)
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AMU Q&A
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Invisibledankington
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Re: d(r)ied print??? [Re: Mad Season]
    #23420388 - 07/07/16 02:21 PM (7 years, 6 months ago)

oooh, okay. yeah. it get tricky with agar ratios sometimes. :lol:
I wasn't sure where you were directing him. But yeah, 100-120ml of water for 1.5g agar 2g ME sounds pretty stellar for spore germination.

especially being I'm in the US, so more used to other units of measure. maybe I should move back to canada lol.


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Offlineesse_jeremy
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Re: d(r)ied print??? [Re: dankington]
    #23421252 - 07/07/16 07:27 PM (7 years, 6 months ago)

Quote:

dankington said:
oooh, okay. yeah. it get tricky with agar ratios sometimes. :lol:
I wasn't sure where you were directing him. But yeah, 100-120ml of water for 1.5g agar 2g ME sounds pretty stellar for spore germination.

especially being I'm in the US, so more used to other units of measure. maybe I should move back to canada lol.





is the exact ammount of agar-nutes-water that I used for my plates 1.5% agar 2% nutes


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InvisibleGrey
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Re: d(r)ied print??? [Re: esse_jeremy]
    #23421425 - 07/07/16 08:26 PM (7 years, 6 months ago)

Is there a date on your print? Did you buy it?


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Offlineesse_jeremy
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Re: d(r)ied print??? [Re: Grey]
    #23422753 - 07/08/16 08:34 AM (7 years, 6 months ago)

I bought it but there's no date, the vendor is a sponsor of the forum...and is professional...I don't think they sell me dead print, I hope!!!!

also I storing them upside down after innoc.  can this affect the GE and the grow of myc??


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Re: d(r)ied print??? [Re: esse_jeremy]
    #23422777 - 07/08/16 08:47 AM (7 years, 6 months ago)

Okay. I don't think they would either.

No, storing upside down is fine.  Sometimes they just take some time to germinate.

After you cool the loop and scrape up spores,  do you swipe over the spot you cooled your loop?  It's usually a little watery where you cool the loop/blade and is helpful for Germination.


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Offlineesse_jeremy
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Re: d(r)ied print??? [Re: Grey]
    #23422825 - 07/08/16 09:08 AM (7 years, 6 months ago)

Yep, I've done it, it's possible that the spore remain on the loop and didn't stick to agar??(just my paranoid)


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things does not exist,
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InvisibleGrey
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Re: d(r)ied print??? [Re: esse_jeremy]
    #23422847 - 07/08/16 09:16 AM (7 years, 6 months ago)

Possible,  but the spores are very tiny so you may have still swiped some. 

I think someone suggested it,  but instead of just swiping across the surface try stabbing or "cutting" the agar. Basically drag your loop through the agar. 


If your worried they're old or dry,  maybe check this thread out.

https://www.shroomery.org/forums/showflat.php/Number/15383615


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Offlineesse_jeremy
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Re: d(r)ied print??? [Re: Grey]
    #23423369 - 07/08/16 11:55 AM (7 years, 6 months ago)

Ty for the info man!!!!

Just one more question, where can i retrive the stopper for the syringe??
I never heard about it, is there a way to do that with similar obj??


--------------------
things does not exist,
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InvisibleGrey
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Re: d(r)ied print??? [Re: esse_jeremy]
    #23423533 - 07/08/16 12:55 PM (7 years, 6 months ago)

Just about any of our sponsors would carry them.  Just make sure they're autoclavable. I can pm you a link if you need.

I'm not sure of another way to plug a syringe for that process.


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InvisibleMunchauzen
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Re: d(r)ied print??? [Re: esse_jeremy]
    #23423541 - 07/08/16 12:58 PM (7 years, 6 months ago)

Quote:

esse_jeremy said:
Ty for the info man!!!!

Just one more question, where can i retrive the stopper for the syringe??
I never heard about it, is there a way to do that with similar obj??



No need for a stopper or rubber band. Idk why stro thought that was necessary, because they will hydrate just by being in the syringe.


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InvisibleMad Season
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Re: d(r)ied print??? [Re: Munchauzen]
    #23423553 - 07/08/16 01:02 PM (7 years, 6 months ago)

The prints are how old? I really doubt them being dry is the problem here unless they're at least 2 years old.. 1.5 weeks is a bit long, but not like OMFG these spores are unviable!


--------------------
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How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
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AMU Q&A
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Invisiblemupetmower
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Re: d(r)ied print??? [Re: Mad Season]
    #23423673 - 07/08/16 01:46 PM (7 years, 6 months ago)

Yeah I'm wondering if it really isn't some user error. I doubt a vendor bought syringe/print would be over 2 years old, especially since they go through so many so often. Unless it was some crazy rare variety that they rarely ever sell, which I'm sure it wasn't.

But I guess you never know.


--------------------
-The wise man never stops seeking knowledge.


-I wanna feel the change consume me, feel the outside turning in. I wanna feel the metamorphosis and cleansing I've endured within.


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Offlineesse_jeremy
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Re: d(r)ied print??? [Re: Mad Season]
    #23423676 - 07/08/16 01:48 PM (7 years, 6 months ago)

I hope, I really hope!!

I don't have date of the print, maybe I'm just paranoid....but the are so many factors that I'm going to became crazy!!!

People use spore that are 2 yrs old......the one I bought from sponsor can be no more that 3 month old....I hope


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Offlineesse_jeremy
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Re: d(r)ied print??? [Re: esse_jeremy]
    #23429676 - 07/10/16 03:39 PM (7 years, 6 months ago)

UPDATE
I got cobweb in my older plates, just one day that I was out and I can't clean the place with alcool and I have contam GODDAM

GODDAM GODDAM I'm tired to grow only bacteria and contam since May

I never had a myc!!!! NEVER

I'm a beast, I'm giving up all my activities to focus on this fucking spore and agar that are raping my mind

plz HELP


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Invisibledankington
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Re: d(r)ied print??? [Re: esse_jeremy]
    #23429684 - 07/10/16 03:42 PM (7 years, 6 months ago)

perhaps you're moving too quickly?
make slower, more deliberate movements. move only with purpose. just a though.


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InvisibleSupalemonhaze
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Re: d(r)ied print??? [Re: dankington]
    #23429957 - 07/10/16 05:58 PM (7 years, 6 months ago)

One thing I find helps is to stab the agar with the loop rather than streaking the spores on the agar. Stabbing the agar will get the spores stuck in there, sandwiched, which will hydrate them pretty well with a soft agar recipe.

You could also try a warm pour. PC a batch of agar, soft preferably, and wait for it to cool enough for it to start to thicken. Being careful it's not too hot (needs to be only warm) pour that over your spore inoculated plate. What this will do is sandwich the spores between two soft agar recipes and the warmth of the pour will soften the spore walls, aiding in germination.


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OfflineEywa_devotee
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Re: d(r)ied print??? [Re: Supalemonhaze]
    #23430137 - 07/10/16 07:09 PM (7 years, 6 months ago)

I use sterilized flat tipped wooden toothpicks to scrape spores off foil, the little plastic loops are too bendy and slippery to get a decent scraping easily and the metal ones tend to tear the foil. You can steam sterilize toothpicks by wrapping them in foil and placing them in a steaming basket above the water in a pressure cooker, PC at 15 PSI for 15 minutes to sterilize them and store in a sterilized jar. To get the agar going quickly apply a drop of sterilized water to a portion of your spore print, let it set covered with a empty pitri dish for several hours (8 to 12 seems best), then dip your loop in the wet spores and transfer to your agar plate.

Another method that works well is to get a small glass bottle and a miniature funnel and scrape the spores in sterile water to make your own spore juice. You'll get a lot more failed jars with this method, but the ones that don't Oh boy...


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InvisibleSupalemonhaze
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Re: d(r)ied print??? [Re: Eywa_devotee]
    #23430171 - 07/10/16 07:22 PM (7 years, 6 months ago)

Loops don't tear foil, you're only rubbing it gently, not stabbing the print. Wire loops with metal handles are the best I've ever used. Some vendors send what is called as a lancet with the loop. It's like a very small lance, I found that even better for sandwiching spores between the agar. I've lost the lancet unfortunately and I can't seem to find a new one on ebay.


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InvisibleMad Season
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Re: d(r)ied print??? [Re: Eywa_devotee]
    #23430224 - 07/10/16 07:42 PM (7 years, 6 months ago)

Quote:

Eywa_devotee said:
I use sterilized flat tipped wooden toothpicks to scrape spores off foil, the little plastic loops are too bendy and slippery to get a decent scraping easily and the metal ones tend to tear the foil.



:lolwut: that's literally never happened to me before o.0. You must be using some CHEAP AF tinfoil..

Quote:

You can steam sterilize toothpicks by wrapping them in foil and placing them in a steaming basket above the water in a pressure cooker, PC at 15 PSI for 15 minutes to sterilize them and store in a sterilized jar.



You also should be sterilizing your loops too, not just tooth picks, you probably didn't mean to say that, just came off that way. Also dude swabs/q-tips are 100000x better than toothpicks, and can be PC'd too, though they come out of the package sterile.

Quote:

To get the agar going quickly apply a drop of sterilized water to a portion of your spore print, let it set covered with a empty pitri dish for several hours (8 to 12 seems best), then dip your loop in the wet spores and transfer to your agar plate.



Are you being serious right now? You want the print to have water on it for 8-12 hours before transferring? Just scrape with a loop or use a swab.

Quote:

Another method that works well is to get a small glass bottle and a miniature funnel and scrape the spores in sterile water to make your own spore juice. You'll get a lot more failed jars with this method, but the ones that don't Oh boy...



:facepalm: So your advice is to add tons of other unnecessary contam vectors to the print doing unnecessary stuff? Is this like a joke or something?


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


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InvisibleSupalemonhaze
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Re: d(r)ied print??? [Re: Mad Season]
    #23430248 - 07/10/16 07:49 PM (7 years, 6 months ago)

I wonder what he means by "oh boy". Are these cultures somehow stronger in the presence of god knows how many contams? :lol:

We get some pretty thin foil over here,even the heavy duty catering stuff is pretty weak compared to the foil some vendors use or what you guys in the US have. I still never really tore a print before, and I've used my lancet for most of my inoculations. A lancet is pretty much a needle. If you know how to use your tools, you'll be just fine.

You PC your loops as well mad? I haven't PCed mine for a while, flaming seems to get the job done well. I do the same with my scalpel, flame it half way up the handle and let it cool in front of my hood while I wipe my plates or jars with iso.


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InvisibleMad Season
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Re: d(r)ied print??? [Re: Supalemonhaze]
    #23430260 - 07/10/16 07:51 PM (7 years, 6 months ago)

I PC my loop and scalpel together. It's the only thing going over open media... I don't want to have any satellite contams


--------------------
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How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


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InvisibleSupalemonhaze
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Re: d(r)ied print??? [Re: Mad Season]
    #23430293 - 07/10/16 07:59 PM (7 years, 6 months ago)

I used to do that too, now I just wipe them with iso to get rid of any large dust particles and flame them well to kill everything that's left. My loop's wire is quite long and the handle isn't sturdy, I find that flaming about an inch up the handle is enough. My scalpel can handle more abuse though, I flame that bitch halfway up the handle. I have to let it cool before I start working or it will stick to my hand :lol:

Haven't PCed either of them for a while, I need to buy a good loop handle though, the two I have are made from shitty metal.


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Offlineesse_jeremy
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Re: d(r)ied print??? [Re: Supalemonhaze]
    #23433081 - 07/11/16 08:03 PM (7 years, 6 months ago)

UPDATE 2

ok I dropped a drop of sterile water in the plates (just a drop right??)

Now what I have to expect??

if i look inside the plates I don't see lot of different, it looks as watery as was at the beginning....dunno


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InvisibleSupalemonhaze
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Re: d(r)ied print??? [Re: esse_jeremy]
    #23434049 - 07/12/16 05:04 AM (7 years, 6 months ago)

Spores will take days to germinate. Agar is good, but not that good. :rofl:

Did you squirt a drop or two on the table before inoculating your agar? After flaming the needle, the spores in the first drop would be burnt and dead so the first drop needs to go on the table so you won't have a bunch of useless water sploshing around in your petri. Second drop will have viable spores, that's the one you want to be inoculating with. Just a heads up in case you didn't know.


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InvisibleAdden
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Re: d(r)ied print??? [Re: esse_jeremy]
    #23434143 - 07/12/16 06:27 AM (7 years, 6 months ago)

Use swabs for your spores to agar and skip all this other nonsense. Then you'll really know.. I don't think loops are good for much except transfers. I find them unwieldy for putting spores to agar. Using a softer agar with swabs has always worked fine for me but loops not so much. More for second and third transfers.


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Offlineesse_jeremy
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Re: d(r)ied print??? [Re: Supalemonhaze]
    #23434456 - 07/12/16 09:30 AM (7 years, 6 months ago)

Quote:

Supalemonhaze said:
Spores will take days to germinate. Agar is good, but not that good. :rofl:

Did you squirt a drop or two on the table before inoculating your agar? After flaming the needle, the spores in the first drop would be burnt and dead so the first drop needs to go on the table so you won't have a bunch of useless water sploshing around in your petri. Second drop will have viable spores, that's the one you want to be inoculating with. Just a heads up in case you didn't know.




I drop just sterile water over spore that I inoc. with a loop 7 days ago, so my drop was only water the spore were alredy on agar


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InvisibleSupalemonhaze
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Re: d(r)ied print??? [Re: Adden]
    #23435109 - 07/12/16 02:31 PM (7 years, 6 months ago)

You don't have to drop water on the spores, they will germinate just fine on agar. Especially if it is soft agar.

Quote:

Adden said:
Use swabs for your spores to agar and skip all this other nonsense. Then you'll really know.. I don't think loops are good for much except transfers. I find them unwieldy for putting spores to agar. Using a softer agar with swabs has always worked fine for me but loops not so much. More for second and third transfers.




To each his own I guess. Personally, I hate swabs, I wouldn't use them at all if PE weren't so damn shy about dropping spores. I find loops awesome and easy to use, I've had this thing called a lancet that came with my loop and it was even better for stabbing the agar. I streak mainly due to impulse, stabbing has always germinated faster for me.


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InvisibleMad Season
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Re: d(r)ied print??? [Re: Supalemonhaze]
    #23435126 - 07/12/16 02:42 PM (7 years, 6 months ago)

Lol I don't really have a proper loop. Maybe that's why I have no issues putting spores on agar? I actually went to a craft store and bought clay tools locally.

generic clay tools. Mine have metal handles.


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InvisibleSupalemonhaze
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Re: d(r)ied print??? [Re: Mad Season]
    #23436692 - 07/13/16 12:45 AM (7 years, 6 months ago)

It's not hard to scrape and steak spores from print to agar. I used to use those tools when I had crafts in school :lol: Just took me down memory lane.


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Offlineesse_jeremy
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Re: d(r)ied print??? [Re: Supalemonhaze]
    #23437288 - 07/13/16 08:18 AM (7 years, 6 months ago)

UPDATE 3

I got a contam on the elder plate after I drop the water,
on the 7 days old one I drop the water but nothing change, again stuck in this phase :frown:


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