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Invisibleagar
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:LIQUID CULTURE - THE EASY WAY: * 18
    #4141612 - 05/06/05 09:52 AM (18 years, 8 months ago)



:LIQUID CULTURE - THE EASY WAY:

(how to turn 2ml/cc of spore solution into 20 ??..ten ml/cc syringes - right - cheap & easy.

Assuming you have an empty syringe & another with spore solution.

Go to Kmart, Target or any large discount Mart & buy a 3 pack of EvenFlow plastic baby bottles.

Also buy a small bottle of Karo corn syrup, a tube of silicone sealant & a few glass marbles.

All the baby bottle parts (bottle - screw cap - nipple) will withstand sterilization in a pressure cooker.

Remove the nipple, apply a small amount (1/8th to ? inch) of silicone sealant to the inside (interior) of the nipple, to serve as a self sealing syringe needle injection point. Allow the sealant to cure overnight

Note: the baby bottle has milliliter (ml) graduation marks up to 240 ml (8 fluid ounces).

Add 200 ml of water to the baby bottle (use marks on it - as a measuring point).

Take your empty syringe and aspirate 6 (six) ml of karo syrup into it.

Then inject that into the open baby bottle, resulting in an approximate 3% solution.

Next, add a glass marble to the baby bottle, to serve as an agitator devise - later.

Insert the nipple into the baby bottle (facing down - inside the baby bottle).

Attach the screw ring lid to hold the nipple in place over the mouth of the baby bottle.

:IMPORTANT:
Attach the screw ring loose, to allow steam to escape & pressure equalization during pressure cooking.

Pressure cook the baby bottle (or several) 20 minutes @ 15 psi.

:IMPORTANT:
Allow the pressure cooker to cool to room temperature, otherwise a vacuum can form inside the bottle, if the screw ring is tightened while the bottle is hot..

Open the cooled pressure cooker & immediately tighten the screw rings on the bottle (or bottles).

You now have a sterilized nutrient solution, in which to inject 2 or 3 ml/cc of spore solution into.

Once you inject a spore solution into it, simply incubate it around 78 to 82F in a clean environment.

You should be able to see tiny strands of mycelium (from germinating spores) in the solution within several days. Be patient, as some spores take longer to germinate - than others.

Once you have visible mycelium in the culture, gently agitate in on a daily basis.

Once the mycelium in the culture is fairly thick, it is ready to be aspirated into sterilized syringes, and used an inoculate.

~~~~~~~

FOR THOSE WITHOUT A PRESSURE COOKER:

NOTE: Not as fool/proof - fail/safe as pressure cooking sterilization.

Cut a piece of tyvek in a circle. Use a rubber band to hold it in place over the screw cap of the baby bottle.

Insert the baby bottle into your microwave (be sure the lid is somewhat LOOSE).

Then, microwave heat it until the solution in it - comes to a boil.

Then - stop, wait a moment & do the same thing again. Repeat, several times.

Remove the baby bottle (gently - as the lid is still loose) & allow to cool to room temperature.

Then, tighten lid.

~~~~~~~~~~~~~~


Or, if you want to do gallons, use jars - with filter lids.


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Invisibleagar
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Re: :LIQUID CULTURE - THE EASY WAY: [Re: scatmanrav]
    #4142713 - 05/06/05 02:33 PM (18 years, 8 months ago)

Quote:

scatmanrav said:
How does the baby bottle breathe? Is it unnessesary for gas exchange on LCs?




As I stated, the method is not optimal. The reason why is lack of gas exchange. To cure that with the BB/LC tek, you simply cut a piece of tyvek in a circle & use rubber bands to hold it around the BB botle. That way.... once in awhile.. you can twist the cap a 1/2 turn loose, to allow for some gas exchange.

Also, once you have healthy myc in a BB/LC, place it in the fridge - to stall it.

I don't use the method. I just wrote the BB/LC tek, for the novices to get a start on LC's.

I use larger glass containers (quarts - half gallon & gallons), with rubber tubing through the lid, silicone sealed down & polyfil stuffed, to allow for gas exchange (as in the pic's below).

I also have a magnetic stir plate & place a stir bar in each container. Then, use the stir plate to agitate the culture, as well as shred it into TINY pieces. So, I don't have the problem of getting the filters wet. If need be, I can also clamp them off.

What is GREAT about the rubber tubing is, you remove the polyfil in one, insert a sterile syringe, tilt the jar, asperate culture out, withdraw the syringe & reinsert the polyfil. No drips, no mess.

Also, with two (2) tubes, when you asperate culture out of one, it pulls air in the other filtered tube. So, you don't create a vacuum in the jar.

With this tubing method & without a stir plate, you simply clamp off the tubing, then shake the hell out of the jar. Then, set the jar upright & remove the clamps.





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Edited by agar (05/06/05 02:51 PM)


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Offlinescatmanrav
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Re: :LIQUID CULTURE - THE EASY WAY: [Re: backupwards]
    #4143280 - 05/06/05 04:41 PM (18 years, 8 months ago)

This one works too, I guess I'll throw it in here. Not to draw attention from agars post, just another quick simple dealy...

Grab a Rubbermaid Cool Contors bottle (like the left) and pop off the white thing.


You could stuff polyfil in the hole and go, or you can cut/melt/drill a small whole in the side (like 1/4" or smaller or something) and silicone a peice of tyvek over it. Then throw in a self healing injection port in the top.
https://www.sciplus.com/lw/category.cfm?category=50

You dont need to seal it with anything, it fits in snugly. The whole bottle (including the white top, even though thats the only part thats not PP5) was made with karo, and PCed at 15-17 PSI for 20 minutes. As you can see, it held up fine. Nice Pan Cyan culture growing in there. Thats 600ml of water in it BTW.

I love them self healing ports.


--------------------
"life is like a drop of rain getting closer and closer to falling into a lake, and then when you hit the lake there is no more rain drop, only the lake."

Growing with bags, start to finish (including my new grain and substrate prep)
Anyone looking to start bulk tubs/mono tubs/shotgun hybrids? Good tubs to use..
How I do grain (old still good tips)
Turn your closet into a fruiting chamber
Casing layer colonization and overlay


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Invisibleagar
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Re: :LIQUID CULTURE - THE EASY WAY: [Re: scatmanrav]
    #4143732 - 05/06/05 06:53 PM (18 years, 8 months ago)

Scat....... :thumbup:

You are not drawing attention away from this. Hell, it's all about LC's.

Do you know that life is just SWEET.

Early Friday evening, weather is warm, a big pitcher of Cadillac Margarita?s, a loaf of cheese -garlic French bread, a tossed garden salad, cold slaw, sweet corn on the cob, garlic - butter - shrimp scampi,  meaty pork ribs on the BBQ & a great looking women at your side (wanting you).

It just cannot get better than that. The Club later, some Al Green, Keith Sweat music to dance to??..Time to Party.


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


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InvisibleGnuBobo
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Re: :LIQUID CULTURE - THE EASY WAY: [Re: MrMaddHatter]
    #4144343 - 05/06/05 10:16 PM (18 years, 8 months ago)

Just a question to the more experienced LC people here.  Two products that are available:


The silicone is "self-sealing"?  It's good up to 400 degrees once cured.  So this would be used to put a bead over the injection site?

The Permatex, then, is used for applying things--such as tubing to plastic lids for liquid culture? 

Which is best to use? 

Cheers.  :grin:


--------------------
Jerry Garcia. JERRY GARCIA! JERRY GARCIA!!!!


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OfflineVALIS
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Re: :LIQUID CULTURE - THE EASY WAY: [Re: agar] * 1
    #4146394 - 05/07/05 12:49 PM (18 years, 8 months ago)

When I was searching through liquid culture (LC) techniques a couple months back, I was particularly impressed with SixTango's Easy Liquid Culture Container Tek.

I like the use of baby bottles because of the ease and convenience of the nipple - c'mon, who doesn't love a good nipple?

Anyhow, here's what I did:




Materials - evenflo 4oz glass baby bottle, filter fiber (i.e. polyfill ), glass marble, tubing, scissors.



Snip nipple just below the tip.



Push the tubing through bottom of nipple.



Cut tubing.



Insert filter material into tubing.



Drop the marble in.



Finished product.


Further information for those who don't already know:

Liquid culture is a method where spores, an agar wedge, or mushroom tissue is injected/inserted/placed into a nutritious solution and incubated, for the purpose of inducing mycelium growth, thereby creating a large amount of inoculum that can be used to inoculate cakes or spawn with. The primary benefits of doing this are as follows:

* Lots and lots ( and lots ) of innoculant can be easily created - 1 10cc multi-spore syringe can be turned into 10 jars of LC, and 100mls of LC can create 10 syringes... pretty cool.

* Using LC to inoculate cakes or spawn can vastly speed up colonization - no lag time waiting for spore germination, in addition to the large amount of inoculation points when injected into substrate.

* The LC can be stored over long periods - throw it in your fridge for months.


Following are the beneficial aspects of using a baby bottle as a container for your LC:

* The nipple serves as a self-healing injection point.

* The nipple can be bent over or squeezed just below the filter tube in order to prevent the LC from saturating the filter when shaking or when drawing up solution.

* Easy and quick to prepare - no need to drill or seal.

* Convenient size, and it's nice to have the ml's/oz's right on the bottle.


Other things, not unique to the baby bottle tek, but useful in any LC container:

* The marble is used as means to better stir/mix the LC, and to break up any mycelium clumps that may occur.

* The filter is for gas exchange.


--------------------
Nature is the Technology of the Divine.


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Invisibleagar
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Re: :LIQUID CULTURE - THE EASY WAY: [Re: VALIS]
    #4146557 - 05/07/05 01:13 PM (18 years, 8 months ago)

LOL...... In another life... I posted here as 6T. agar=SixTango.

Yup, you can use a colonized seed/grain to start an LC.

Just make sure the transfer is done in a CLEAN way.... with MINIMAL open air exposure............otherwise - contam farm.


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


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Invisibleagar
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Re: :LIQUID CULTURE - THE EASY WAY: [Re: VALIS]
    #4146838 - 05/07/05 02:00 PM (18 years, 8 months ago)

I did miss it. Excuse = very hung over today.

To much FUN last night


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Invisibleagar
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Re: :LIQUID CULTURE - THE EASY WAY: [Re: Blue Helix]
    #4152603 - 05/09/05 07:01 AM (18 years, 8 months ago)

You dont need to inject 2 or 3 ml of spore solution. Any amount (so long as it has a few viable compatable spores in it) will work. More - simply gets the action going faster, IMHO.

I certainly don't advocate WASTING spore solution. At the same time, I prefer to use the content of a spore laden syringe at one sitting, so I do not have partial syringes laying around.

Once - you master mush/cult & PRINT or SPORE SOLUTION MAKING you generaly have more spores in print or solution form - than a single person (or small group) could EVER USE.

I often use a SPORALATOR to make spore solution:



1/2 or 1 gallon wide mouth jar (Sun Tea type) - with spigot removed & replaced with an injection / asperation port - that reseals, installed in the hole in the jar - where the spigot was.

Lid altered with a gas exchange filter & install a clip of some sort to hold a cap suspended with a stainless steel treble hook through the caps stem area - inside the container.

Fill container with 1000 / 1500 / 3000 ml water (depending on size of container) & PC the whole container with water inside it 20 minutes @ 15 psi &  allow to cool to room temp.

When ready for use, harvest a large cap (veil about ready to tear), inside a G/B or F/H - swab cap top with h202, remove veil, peirce cap stem area with sterile treble hook, open container & attach cap to clip, which hangs over sterile water & will drop spores directly into it (repeat once or twice or more w/same strain cap).

The result is 1000 / 1500 / 3000 ml of spore solution that can be asperated through the injection / asperation port on the side of the jar via a sterile syringe.

Test first syringe on agar/petri/plate to insure spore solution contains no contaminates & (if not) presto - you now have a capacity to fill 100 / 150 / 300  syringes out of a single spore laden solution container - in aseptic conditions.  :thumbup:


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OfflineBlue Helix
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Re: :LIQUID CULTURE - THE EASY WAY: [Re: agar]
    #4154692 - 05/09/05 07:09 PM (18 years, 8 months ago)

Quote:

I certainly don't advocate WASTING spore solution. At the same time, I prefer to use the content of a spore laden syringe at one sitting, so I do not have partial syringes laying around.




Ah, but you, Agar, are the one who has shown us the secret of using vacutainers to store spore solution...



That way you don't leave you spore solution lying around at all. You just poke the needle through the rubber lid thingy, draw out what you need (say 1/4th CC), and the rest is kept sterile and perfect in the vacutainer.


Edited by Blue Helix (05/09/05 07:14 PM)


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Invisibleagar
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Re: :LIQUID CULTURE - THE EASY WAY: [Re: Silly_Cyben]
    #4156485 - 05/10/05 07:33 AM (18 years, 8 months ago)

Quote:

browilliam said: How long can these bottles be stored in the refrigerator? William




Once you have healthy culture growth, it is BEST to stall them, by placing in the fridge - where they can/will last several months.


1 gallon LC on a magnetic stir plate


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