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Offlinefunguy_ryzing
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Anyone got time for some answers to some long- winded questions?
    #11640927 - 12/13/09 04:37 AM (14 years, 2 months ago)

Hello Shroomerites.

Firstly, I am not a COMPLETE noob, though I still have bunches to learn, as most all of us do. I have created a new account as I have been out of the game for a few years now and cannot remember my old password.

I have been a Shroomery member for 4-5 years now and had a few successful mycological endeavors back in 2004 or '05.

I am currently working with creeper/ brazilian syringes from Ralphster's.

A bit long- winded, I know, but this is all leading up to my question(s)...

So my syringes arrived a couple weeks ago. I wanted to get a jump on things, so right away I prepped two LC jars (baby food jars, tap water, a bit less than 1/4 tsp. "Silverbow" pure clover honey in each; steamed for 30 min [as I don't have a PC this time around]). These were left to cool naturaly in the pot.

Not much $cratch for supplies either, so no nokbox as I had before- Just sprayed (bleach water), no drafts- slow, deliberate movements;flamed and quenched needle  and knocked up one cc of each race in their respective jars, covered and rubber banded w/ 2 new, clean coffee filters each.

Stupidly, I *FAILED* to  record which jar was which race...

Anyway, as I must be covert (roomates) these jars were inc'd under a futon in my room (near a forced- air heater vent ). Thermo shows that temps there range from 70- 80 deg f. There is a 10 or more degree fluctuation. They were placed in a box/ box placed in a bag (bag open, openig facing away from any drafts).

One jar germed, one did not.

So, to make a long story longer... After about 5 days, maybe 6 (okay, shame on funguy_ryzing, perhaps it was 8 days-- my documentation has improved since this step), I finaly had my 12 1/4 pt --yes 4oz (for quick colonization so I can have some headies while I await future casings), PF jars together and "sterilized" (just a tic over 1hr @ a slow rolling boil in a covered pot, left to cool au naturalle).

I decided to inoculate 6 w/ my (questionable?) LC, which has (still) apparently not moved very far beyond germination though it was swirled daily. There are a couple hundred 1mm +/- white specs. They are NOT floating, I think that's good. I dunno. Doesn't matter, these cultures will be tossed tonight. They never made it to the fridge and are now 2+ wks old. Anyway...

Since I had no empty or new syringes I used up my 9 cc of remaining braz on the first 6 jars  to free up the syringe for sterilization. I sprayed the room as before, then one jar at a time, slow, deliberate in my movements, I removed the foil from the jar, swabbed the lid w/ alcohol, punched through  micropore tape @ one innoc. site w/ a flamed and alcohol- quenched brad (to avoid a vac drinking all my spore solution), uncapped, flamed needle, pushed out a drop of solution, swabbed needle, punched through tape and inoculated w/ +/- .33 cc of spore solution per hole (4 per jar).

The punched holes were left in the existing tape and two new and clean coffee filters were placed under the band of each jar.

The empty syringe was then rinsed, filled w/ water the needle removed and wrapped with foil (that was a fuck- up, hope I didn't screw myself there), the shipping cap was placed on the syringe and it was foil wrapped. These were placed in a double- boiler w/ perfed basket, steamed (covered) for 30 minutes, left to cool. The reason I switched the cap for the needle was I feared the heat would expand the water in the syringe, squirting      it out and causing it to draw in unfiltered air as it cooled.

Once the syringe had cooled, (following the same "sans glovebox" sterile procedure as above) I swabbed the coffee filters over the hole in the LC jar lid with alcohol, uncapped and squirted out the water in the syringe, flamed and swabbed needle, swirled the jar, punched through the swabbed coffee filter and foil directly and drew up 10 cc's of solution. I could not SEE any mycs in the syringe, but MUST have gotten SOME (right?) As the particles were very small-- not large clumps.

The remaining 6 jars were knocked- up w/ 1.5 cc each of this solution. Again, the same sterile practices as above were followed for these 6 jars, although, rather than coffee filters, a new piece of micropore was placed over the old one.

Both batches were inoculated on the 9th of this month.
I in no way expect to see growth today (currently the cusp of the 12th/ 13th.) In the jars that were inoculated from spore.

So, finaly, here are my questions:

1) I have never used LC's before- I see no signs of mycs in the 6 PF jars knocked-up w/ the LC's after 3 1/2 to 4 days. Does this mean that my LC's were dead or had they just used up the small bit of honey in the 5 to 8 days pre- knock and gone dormant? I would think I would see something by now.

2) Since both LC's were prepared at the same time and in the same way, would anyone suspect that one of my syringes was not viable (I've only used Ralphster's in the past, and never had a problem w/ any of the syringes I've bought from his site)?

3) In the case of all my previous grows I was lucky enough to have a very nice glove box and a PC. Considering that I don't have these now, does any one see where I can improve my sterile culture technique in the meantime until these things are aquired? Btw, my oven took a shizzle JUST PRIOR to receiving my order from Ralph!

4) Given my temps, along with the fact that I see no evidence of contams, how long should I give the 6 jars inoculated w/ spores before starting them over.

5) What is the risk of my jars drying out before colonizing given the incubation parameters-- indirect forced air heat?

6) How badly might I have screwed myself when I had to take the storage cap off of the freshly sterilized syringe    in the open air to place the freshly sterilized needle on it? Could this have introduced enough contams to sabotage all 6 of my LC quarter pints?

The 9 cc's of creeper spore solution that are left over are now refrigerated. They were unrefrigerated for just over a week. I think thyey'll be okay as they were only uncapped momentarily to inoculate the LC jar and the needle was then flamed before being recapped. If it is thought that my best bet would be to start over with these, please let me know.

Thanks, fellow mycophiles, for taking the time to read this post. I know it's been anything but brief.

Thanks also to each of you for your contributions to the wealth of knowledge put forth on this site. It is great to be back among your ranks.

And thanx in advance fort any feedback.

Also, before anyone says it... I did search for answers to these specific questions before creating this post, so there!!

Happy Shrooming!!

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OfflineZed
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Re: Anyone got time for some answers to some long- winded questions? [Re: funguy_ryzing]
    #11640944 - 12/13/09 04:50 AM (14 years, 2 months ago)

wow....I started reading it then quickly scrolled to the questions which i cannot answer without reading everything you wrote....sadly i dont think i can force myself to read it all, lol. sorry.

But LC's should certainly show signs in 3-4 days if temps are right (70-82).

As I say, I didnt read all your post but in a question you mentioned drying out via forced air heat....this is a very bad idea!, you need a constant ambiant temp to prevent your jars drying out.

Sorry i wasnt more help and was too lazy to read all your post :smile:

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InvisibleLeopardMan
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Re: Anyone got time for some answers to some long- winded questions? [Re: funguy_ryzing]
    #11640960 - 12/13/09 05:01 AM (14 years, 2 months ago)

Man, this is a novel. You should try to keep it short so we can help you. :wink:


--------------------



You have to die a few times before you can really live.

-Charles Bukowski-

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Offlinefunguy_ryzing
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Re: Anyone got time for some answers to some long- winded questions? [Re: Zed]
    #11640985 - 12/13/09 05:21 AM (14 years, 2 months ago)

Ok. Yeah, this is a long post. Sorry for that. As I said the forced air is indirect, but I had feared this might happen. I will move the jars that were injected w/ spore solution pending more f/back as I still see condensation in the jars. Anyone who relly likes to read novels care to take a closer look at my post?? Luckily I used a little extra water in anticipation of this.

Thanx guys, and in the style of drew carey, one zillion and two shrooms to you!! Thanx.

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InvisibleLeopardMan
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Re: Anyone got time for some answers to some long- winded questions? [Re: funguy_ryzing]
    #11641008 - 12/13/09 05:40 AM (14 years, 2 months ago)

I'll try to answer some of your questions:

1)2)4) I would wait more. You got nothing to lose.

3) You can build a glovebox with pratically no money.

Not sure about 5 and 6. GL


--------------------



You have to die a few times before you can really live.

-Charles Bukowski-

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Offlinefunguy_ryzing
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Re: undefined [Re: LeopardMan]
    #11641032 - 12/13/09 05:55 AM (14 years, 2 months ago)

Thanx, leopardman. I will give it more time, which is the only thing I have to lose.

Also, I've built a  glove box for four or five bucks, and I tried again this time, but I kinda half- assed it w/ too small of a tub, for starters, and rather than investing more time and a few more bucks for some collars, hot glue and hose clamps, I tried to *TAPE* the gloves to the cutouts (ricky retardo!)...Needless to say, it didn't work out. I've simmered down now and have a bit more patience a couple weeks post getting my spores, so I'll likely not be so hasty on my next attempt. THX for the f/b.

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InvisibleLeopardMan
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Re: undefined [Re: funguy_ryzing]
    #11641060 - 12/13/09 06:09 AM (14 years, 2 months ago)

Look at veda's glovebox:

http://www.shroomery.org/forums/showflat.php/Number/10549040#10549040

I've spent like 50 cents for my glovebox and it works like a charm.


--------------------



You have to die a few times before you can really live.

-Charles Bukowski-

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InvisibleDoc_T
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Re: undefined [Re: funguy_ryzing]
    #11641067 - 12/13/09 06:12 AM (14 years, 2 months ago)

LC is a gamble, especially starting from spores. In fact, if you only waited 4 days, you may not have grown anything at all in the LC.


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You make it all possible. Doesn't it feel good?

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OfflineSpongiform
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Re: undefined [Re: Doc_T]
    #11641117 - 12/13/09 06:39 AM (14 years, 2 months ago)

Quote:

1) I have never used LC's before- I see no signs of mycs in the 6 PF jars knocked-up w/ the LC's after 3 1/2 to 4 days. Does this mean that my LC's were dead or had they just used up the small bit of honey in the 5 to 8 days pre- knock and gone dormant? I would think I would see something by now.

2) Since both LC's were prepared at the same time and in the same way, would anyone suspect that one of my syringes was not viable (I've only used Ralphster's in the past, and never had a problem w/ any of the syringes I've bought from his site)?




I'd give LC a minimum 7-10 days before using.  There should be large amounts of visible growth.

Here are some LC pics for reference.


Quote:

4) Given my temps, along with the fact that I see no evidence of contams, how long should I give the 6 jars inoculated w/ spores before starting them over.




It can take weeks for spores to germinate and get going sometimes. So be patient.

Quote:

5) What is the risk of my jars drying out before colonizing given the incubation parameters-- indirect forced air heat?




I'd move them away from any direct heat source.  They will colonize just fine at 70f (albeit slightly slower then 75-80).

Quote:


6) How badly might I have screwed myself when I had to take the storage cap off of the freshly sterilized syringe    in the open air to place the freshly sterilized needle on it? Could this have introduced enough contams to sabotage all 6 of my LC quarter pints?




It's possible, but chances are you're fine.

I flame sterilize then wipe with a damp bleach-water cloth.  I also wipe the lids/injection ports down as well.


--------------------
Spongiform's Plastic Tek - An Alternative to Glass
Spongi's PF Block Tek & Automated Shotgun Project.

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Offlineemptyvessal
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Re: undefined [Re: Spongiform]
    #11641391 - 12/13/09 08:38 AM (14 years, 2 months ago)

By the time i read all that my mushrooms will have pinned thanks ! :wink:


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Offlinefunguy_ryzing
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Re: undefined [Re: Spongiform]
    #11641929 - 12/13/09 10:48 AM (14 years, 2 months ago)

Thanks for the input Spongiforum and Doc-T, and again to you Leopardman.

That budget glove box link got my wheels turning again. Gloves and kling wrap are free to me as a cook!

Tried to att'ch a pic of my LC.

Although I plan to  toss it as it has not had th best care.

I guess I cannot attach a .jpg file. Kinda hard to reformat as my pc is a blackberry...Yes, all that typing in the orig. Post was via bb!

Once again, community, thanks for getting me back on track.

It's funny how having a couple successful grows can make one lax and overconfident.

Oh well, live and learn, as they say.

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Offlinefunguy_ryzing
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Re: **UPDATE** [Re: funguy_ryzing]
    #11644145 - 12/13/09 05:36 PM (14 years, 2 months ago)

Hey, just a little update...

I took a look just a minute ago and there IS growth!!!

Unfortunately it is **GREEN** growth!!!

I've never grown out the green meaniez b4, so this is a new experience for me.

Actually, this is quite a relief as now I know exactly what to do!!

I will be starting over with a better sterile technique and what's left of my creeper spores.
Also, will let the jars colonize at room temp, rather than **on the FLOOR in a draft with extreme temperature fluctuations**!

I am building a glovebox later and will take EVERY precaution this time.

I guess that pretty much rounds out this post.

Thanx Shroomery!

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InvisibleLeopardMan
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Re: **UPDATE** [Re: funguy_ryzing]
    #11644849 - 12/13/09 07:27 PM (14 years, 2 months ago)

GL for your next project :thumbup:


--------------------



You have to die a few times before you can really live.

-Charles Bukowski-

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Offlinefunguy_ryzing
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Re: Thanx, LM [Re: LeopardMan]
    #11660342 - 12/16/09 03:29 AM (14 years, 2 months ago)

Thanx, LM. Went ahead and built a glovebox and rigged up an incubator. Just dialing in the temp now.

,Ew jars will be going in 2nite.

Allow me to pay that good luck forward to encompass u'r endeavors as well.

Bye for now!

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