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OfflineIndianMycologist
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Registered: 07/19/15
Posts: 5
Last seen: 8 years, 4 months
Chronicles of the First Growing Attempt
    #22031138 - 08/01/15 10:51 PM (8 years, 5 months ago)

Hi,

These are chronicles I'm receiving from SWIM from his first attempt at growing. Will keep updating as more is shared:

Opening Notes
• Project was commenced on 25th July, 2015.
• PF or BRF Tek adopted.
• For round 1, one whole print was used and pulled into 2 syringes at approximately 17ml (3ml was wasted as detailed later).

Glovebox
• Common transparent storage box used- though should have taken a larger one as this was tough to work inside due to small space.
• Holes for hands drilled, and plastic wraps extended from within box as added safety measure.
• Glovebox sterilised by rubbing alcohol and household disinfectant. Box kept closed at all times and sterilised once again just prior to usage.

Syringe Preparation
• RO water boiled for 15 minutes and then brought to a stop.
• Taken into 2 syringes (10ml each) and allowed to cool for 20 minutes.
• Syringe needles flame sterilised between every step of the process.

Print to Syringe
• Activity commenced at 11:30PM, 25th July 2015.
• Work area and glovebox completely sterilised, hands obsessively washed many times over. Body bathed.
• Screwdriver end sterilised to scrape off spores from prints.
• Inside the glovebox, prints scraped off into sterilised shot glass using screwdriver end.
• Pushed water into shot glass via sterilised syringe.
• Shook contents around, spores visible in the liquid medium. Pulled this mixture into syringe.
• Syringe flame sterilised between every step of the process.
• Process repeated twice to get 2 syringes.
• May have fucked up here as process was awkwardly done, especially due to small size of box. Some water (with spores in it) wasted, approximately 3ml.

Substrate Preparation
• Substrate prepared at noon, 26th July 2015.
• Target to make 3 jars. Thus quantities used: 144ml each of water and brown rice flour (BRF), 288ml of vermiculite.
• Water and vermiculite mixed into a moist paste first, BRF added then and mixed into this.
• Added substrate to within 1 cm of all 3 jars. No packing in at all, just let the substrate enter as is, loosely packed.
• Cleaned the top 1 cm inside edge of jar with rubbing alcohol.
• Added dry vermiculite at the top up to the edge to act as filter.
• Closed lid (4 holes in each lid previously poked) and wrapped with tinfoil. Took care that tinfoil is not torn anywhere. During sterilisation stage later, water should not enter the jar at all.

Sterilisation
• Cooking pot sterilised with rubbing alcohol for good measure, though this was not specified in project guide.
• Pot comes with an inside casing explicitly for steaming purposes- solves problem of trying to keep jars a little above water through other ways like lids at bottom or cloth at bottom etc.
• Inside casing installed, water poured into pot till it comes up to just at the inside casing level.
• Four lids placed, tinfoil on top of them, and then the three jars placed above.
• Gas turned on, water brought to a boil and lid closed.
• For 90 minutes, water kept boiling for steam sterilisation and ensured that it does not dry out.
• Boiling started at 12:30PM, and continued till 2:15PM with extra fifteen minutes for good measure and good luck.
• At 2:15PM, shut the gas down and walked away. Pot need not be disturbed for a good 8 hours now to allow the substrate to cool down to room temperature completely.
• No need to rush this step. Substrate must be cooled to the core or else the heat will kill the spores. This takes around 8 hours. Next we will begin at 10:30PM.

Inoculation
• Paranoia. At every step I worry whether I’ve already done something to contaminate the process. The project guide specifies that the tinfoil over the jar lid must not tear or rip. However, during sterilisation the foil over two jars was slightly ripped. Cannot say what that means, yet.
• Inoculation process began at 1PM, 27th July 2015, thus giving the jars close to 11 hours to cool down which is perfect. The glovebox was cleaned with disinfectant and then with rubbing alcohol.
• A mask was also worn so that I don’t breathe wind drafts onto the project area and components.
• Once all apparatus set up, the jars were finally taken out of the pot and placed inside the glovebox. Another paranoia was assuaged here. Throughout the sterilisation process I worried whether the water had all boiled out and the pot was dry. This was now confirmed to be undue worry.
• Syringe 1 was taken out, flame sterilised, and jar 1 was inoculated. I made a mistake though and did not shake the syringe before use. It is to be shaken so that the spores come off the syringe surface and mix well into the solution. But all we need for successful colonisation is two spores to do their thing, so maybe this is paranoia again.
• In either case, I decided to use syringe 2 completely for jar 3.
• Syringe 1 was flame sterilised again before inoculating jar 2.
• Syringe 2 was then flame sterilised, shaken well and used completely for jar 3.
• All 3 jars inoculated, the process was complete and we are now into possibly up to 2 weeks of worry. The forthcoming schedule:
o Colonisation can begin in 2-3 days, but can sometime take up to 2 weeks to commence.
o One should thus not panic before at least 2 weeks.
o Jars are to be examined daily for progress and to spot contamination, which I'm paranoid will set in.
o Jars must not be taken up and placed down for examination. This disturbs the top vermiculite layer and allows possibility of germs getting in.
o Jars must only be rotated around where they sit for examination.

Colonisation
27/7, 1:42AM
• Jars have been colonised, hopefully successfully.
• Observation stage begins.
27/7, 8:20PM
• Nothing much to report, and nothing much to be expected so soon.
31/7, 8PM, 3+ days after inoculation
• Been observing the jars daily since inoculation, today was the first sign of activity.
• Have kept jars covered with a piece of tissue paper. Just in case some bacteria fucks are trying to get in.
• In what will be called Jar 1 from here on, a small patch of fungus spotted growing near the bottom of the dry vermiculite layer at the top. Only on one side, on only one jar so far. But patience is the name of the game.
• As prescribed, the jars are not being picked up and moved about. They are only rotated where they are to observe activity.
• Fucking paranoid that we’ll get infection. Odds say if you inoculate 6 jars you get 2-3 contaminated pretty often. We’ve done 3 jars, in I don’t think completely sterile conditions.
01/8, 1:20PM, 4.5 days after inoculation
• Nice. Jar 1 now shows patches in all 4 inoculation spots. Little white-coloured assurances of immense potential. I have to resist the temptation to have a look at them every once in a while.
• No activity in jar 2.
• A small patch just beginning to form in one spot on jar 3. Good signs all around.
01/8, 11:00PM, nearly 5 days after inoculation
• Jar 1 has seen considerable activity in the past 10 hours, the mycelium patches growing even as I observe.
• Still no activity in jar 2.
• 2 patches of growth in jar 3.
02/8, 10AM
• Jar 1 nicely growing in all 4 spots, very beautiful to observe
• Jar 2 still completely inert.
• Jar 3 now growing in 2 spots, good stuff.


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InvisiblebodhisattaMDiscordReddit
Smurf real estate agent
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Folding@home Statistics
Registered: 04/30/13
Posts: 61,889
Loc: Milky way
Trusted Cultivator
Re: Chronicles of the First Growing Attempt [Re: IndianMycologist]
    #22031228 - 08/01/15 11:11 PM (8 years, 5 months ago)

Someone who is me?


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OfflineSyntheticwords
Victus


Registered: 08/13/11
Posts: 375
Last seen: 6 years, 9 months
Re: Chronicles of the First Growing Attempt [Re: IndianMycologist]
    #22031236 - 08/01/15 11:12 PM (8 years, 5 months ago)

:nothingtoadd:


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InvisibleMunchauzen
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Registered: 06/22/11
Posts: 14,342
Trusted Cultivator
Re: Chronicles of the First Growing Attempt [Re: Syntheticwords]
    #22031961 - 08/02/15 01:59 AM (8 years, 5 months ago)

look at all those bullets! :minigun:


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