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InvisibleViolet
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Registered: 12/06/11
Posts: 4,205
Culturing Tech • Agar containers, "Grain Petri" Tek, Invitro growing & Sterile Spore Printing! * 23
    #19035259 - 10/26/13 11:38 AM (10 years, 4 months ago)

HI! :wave:

I'm here with this technique essay to discuss the simple handling and selection of ideal Cubensis cultures, a topic that seems quite under-discussed!

Culturing is the most important avenue of mycology, be it professional or home hobby. It's possible to grow without culturing but it's likened to throwing countless random seeds of a plant into a field, accepting whatever may come. With quite little materials and effort it's easy to select mycelium that is fast, hardy, prolific, and potent.
But of first importance is being able to handle mycelium apart from contaminant organisms!

It should (but won't) go without saying that this is sterile work to be done in a still-air box or in sterile laminar airflow.


*** This was developed as a culturing approach to a grow method that is essentially a scaled version of this but with more substrate in the container, inoculated with the final cultures, fruited in a variety of humidity chambers, and watered for repeated flushes.
That method, just a small later facet of this culturing approach, is found Here  and is thought to be my most significant post here... although it is not - you're in it currently! ***




GRAINWATER AGAR

This liquid left over from brown rice or grass seed preparations is a suspension of the same nutritive goods the grains contain. It makes for no-extra-cost agar and works great!  Why dump this great stuff down the drain? That amounts to boiling out and throwing away a part of one's paid-for value.
Malt extract is a derivative of grain so malt solutions for agar could be considered a grainwater made from purified concentrate!

For firm agar use 9-10g for 500mL, or .5g per 25mL.

Agar from brown rice may look nearly clear when shallow but mycelium are nonetheless able to get more than they need from just the surface alone. Agar from grass seed may be very dark but visibility is great from above.

Spores germinating on ricewater agar:


Transfers quickly recovering & taking off:



Grainwater can be frozen for later thaw and use!


CONTAINER AGAR DISHES
These screw-top containers (I greatly prefer the Ziploc brand) are recycling code 5 which holds up perfectly under hi-pressure temperature (as long as the cooker doesn't run out of water).
Requiring no modification whatsoever, they are sterilized with the lids slightly cracked loose. Do not attempt to sterilize them while sealed!

On the left, a sealed container. Seal them while removing from the sterilizer until and after inoculation.
On the right, a container with the lid very loose but still threaded in to where it does not come off when lifted. This is the loosest it should be for sterilization, but it's more loose than it should be for anything except maybe growing grains invitro.
In the middle, how I suggest keeping lids cracked - for sterilization or when one's project comes to involve a bit of gas exchange.
This is about One to Two of the "notches" around the edge of the lid.


Amongst their many uses, pint (16oz) containers are re-usable sterilizable "petri" dishes, or simply said, agar dishes.
They have a very wide area per dish for colonies to run-out, possibly coming to require fewer transfers and dishes. The photos above of growth on ricewater agar are examples of these in use.

25mL of solution is enough for each dish, so 500mL of prepared agar still makes 20.
Agar can be mixed in a large batch and distributed, or for small or quick batches ~0.5g of agar-agar powder can be put in each container with 25mL.

I have found using these containers to be far favorable to single-use dispose plastic petris for several reasons:
  • Use a container as such 8-12 times and it's paid for itself and used only a fraction of the plastic, with still more uses to come! I feel all plastics should be permanent-application where possible instead of the revolting wastes of plastic today.
  • They can be "pre-pour" sterilized and opened just once for better sterile success chances!
  • Since there's no pour involved, they can be allowed to cool however and used whenever instead of making sure to catch it & pour before solidified.
  • They have a very wide area for mycelium to run out, so likely fewer dishes and transfers will be necessary, and there's more inoculation power from each one.
  • Since they close air-tight no parafilm need be bought or messed with.


I also find them preferable to any other container I have purchased to these ends and others.  Their use for any is ideal, and they also can be used for nearly anything!

The only potential drawback I have experienced is the higher sides. This likely will not bother you depending on how well you can handle agar with a scalpel or other choice tool. I quickly learned how to use them without trouble and now experience only the advantages listed above.

Edited by Violet (07/20/19 01:42 PM)

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InvisibleViolet
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Culturing • ABOUT SELECTING IDEAL CULTURES [Re: Violet] * 2
    #19035260 - 10/26/13 11:38 AM (10 years, 4 months ago)

ABOUT SELECTING IDEAL CULTURES


Ideal cultures are those with as much as possible of these traits combined: Speed and veracity of colonization, quickness until fruiting initiation, where pins prefer to form, abort likelihood, number of pin sites and number of pins at each, size of those fruits which combined totals "yield", and great potency.

Additionally we may observe and select by physical appearance of fruits, physical appearance of mycelium, apparent susceptibility to contamination, sporulation if much or little/none is desired, dense or hollow stems, and one may even go so far as taste although this is mostly left to the realm of culturing edible species.

It is my opinion that those factors be considered in the following order of priority:
1. YIELD.  This is not concerned simply with a large first flush but the total yield per a spent substrate.
2. POTENCY, which truly is yield also. If two cultures yield the same but one is more potent that one has actually yielded more; a hugely-yielding strain with no potency is still useless.  This is equal to raw yield in importance but is the last factor that can be determined.
3. LOCATION OF PIN SITES makes the difference between clean or hasslesome harvests. It can also effect abort rate (side-pins are more likely to abort) and thus yield.
4. SPEED OF FRUITING.  There's no point in waiting around for a culture that hogs fruiting chamber space for extra time without any other advantage. This is especially important when it comes to fruiting directly from whole grains, where most cultures seem to take their sweet time. That factor refers to nutrition tolerance.  Another significant factor is CO2 tolerance... if possible, we want strains to pin fast even within enclosure.
5. NUMBER OF PINS PER PIN SITE / FRUIT SIZE.  Yield comes from the interaction of these two factors. We want as high as possible of a value of both simultaneously.  The most numerous field of pins may abort or grow to be small fruits; the largest of fruits may grow alone. The best is likely to produce a great number of medium-sized fruits, so dense clusters of well-sized mushrooms is ideal.
6. EASE OF RELEASE AT HARVEST.  For me this has become very important.  My favorite champion cultures are those that pluck easily from their substrate with little effort.  They do not call for cutting in order to avoid damaging the substrate which is especially important for keeping casing layers healthy and full.
7. ABORT LIKELIHOOD.  This is a lower priority simply because it is only potentially of significant relevance. If a certain culture forms 3 times as many pins as another, a 50% abort rate is not actually a problem as there are still more pins capable of growing fully.
8. SPEED OF COLONIZATION.  It's very nice to have a quickly-colonizing culture, but for sterile work it comes to make little difference as long as the culture isn't simply slow. Fortunately strong fruiting strains are often plenty quick.  I care about this one a lot solely due to pickiness, and this method allows for speed to be selected for first.
9. SUSCEPTIBILITY TO CONTAMINATION.  Some cultures can be early giving way to Trich & company before the sub's potential is fully used, but this has more to do with the health and nutrition of the cake
10. PHYSICAL APPEARANCE OF FRUITS AND/OR MYCELIUM.  Mushrooms have many different likeable aesthetics, with many novel possibilities.  Some people care for more consistently rhizomorphic mycelium.
11. SPORULATION.  Good cultures should be heavy sporulators to keep the line going, but a strong fruiter that drops few/no spores is very nice and tidy to have also, and should be considered preferable for long-term storage and wide use.


Few strains are prolific pinners. Not all strains fruit sizably. Most strains will pin in side-pin microclimates if they're available, and many will even pin against the sides and create side-pin microclimates anyway.

Of the few top-yielding strains, not all will stubbornly fruit on the intended fruiting surface like we want them to.  Of the strains that will hardily fruit on the intended surface instead of side-pin, not all will give top yield.  The rare strains found to be ideal in both will still need to be compared for potency!

Once such a specimen is found it should be kept carefully for years and years to come!


The younger our cultures, the better. Mycelium's "age" regards how much it has expanded since germination & mating, multiplying towards and through senescence where performance gradually then drastically drops. By minimizing the expansion required to find and isolate the genes we want & keeping un-expanded samples of them on reserve we can use & expand a single strain seemingly endlessly.
Such culture practise done well results in master cultures that can be spread world-wide and be eaten by millions of people, such as the famous Shiitake75 and so many others, shown again and again to colonize most veraciously and yield most prolifically.

Just a little setup, patient observation, careful transfers, and in this case some small-scale testing, makes for unimaginable differences in ease and effectiveness for lifetimes.

__________________________________________________________________


There are many ways to go about finding a strong fruiting strain of Cubensis, but removing factors of randomness and guessing greatly reduces the sterile work, materials, energy, and space involved. "Cloning" mushroom pins/fruits by taking a sample to agar is a great shortcut to reducing mycelium one handles to fruiting strains.
However this has usually involved expanding to colonize a fruiting substrate and expanding as a mushroom, capable of re-expanding but naturally expecting to die and decay, so it's possible that mycelium has expanded very much by the time the genetics are isolated. Further, depending on the fruiting scenario the clone is taken from we may be somewhat blind to the quality of our selections.

We can set-up conditions where the strongest genetics are as much more likely to shine out from the mix as possible.
Cloning from a "bulk" substrate grow may be a great quick & easy shortcut to finding cultures trusted to do well in those scenarios again. However the amount of expansion involved prior is the most extreme, and there are some aspects to it that can bring haze to identifying and selecting ideal genes.
In brief, RATIO OF FRUITING SURFACE AREA TO SUBSTRATE SIZE.
This 'value' greatly effects the concentration of fruits and thus our ability to clearly perceive how densely-fruiting the genetics we're seeing really are.

Consider two divisions of a single substrate, each with the same 9 square inches in fruiting surface area. One is 1 inch deep and the other is 3 inches deep, colonized with the same culture. The 3-inch-deep substrate has 3x the fruiting capacity, but the greater number/size of fruits it will attempt to grow have but the same surface area to fruit from.
Thus a mycelium genetic that will grow sparse single fruits may nonetheless grow side-to-side on the deeper sub in what appears to be clusters and quite a prolific amount, while the more shallow substrate more easily shows in macro what the genetic's solitary fruiting traits really are.

There are more factors as well that are not explained or understood as simply but are no less at work. Large substrates, particularly bulk substrates dense with water mass, tend to grow larger mushrooms. Nobody can complain about a good yield of big guys, and my early clones were almost always such specimens, but I've come to accept that this is a poor way to select cultures. They tend towards a few hard-to-dehydrate giants that would suit 4 people instead of plentiful quick-drying clusters of numerous fruits. (I also believe smaller fruits to be capable of a slightly greater potency.)

As I gradually moved over to my current grow style as shown in the seed & plastic tek I realized that my somewhat-prized cultures, mostly clones from rye/coir sub mixes, were simply not as close to top-notch as I thought. For some of the reasons named above, they appeared about equally prolific on bulk substrate but their differences and limited adequacies were made quite clear from the small & efficient substrates I then used instead.

So I'm here to advise using very small substrates to both find & test the capabilities of our selected genes!

With my grow tek this naturally comes to manifest as what I affectionately call…

Edited by Violet (12/19/17 02:30 PM)

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InvisibleViolet
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Culturing • GRAIN 'PETRI' TEK [Re: Violet] * 4
    #19035262 - 10/26/13 11:38 AM (10 years, 4 months ago)

MAJOR ADDENDUM!
Use grass seed!

Because of the greater volume of the seed, the nutrition is spread-out and thus in a sense "dilute".
Cubensis greatly prefers to fruit from sites of no nutrition, whether it didn't have any or has all been consumed.
For invitro fruiting of (uncased) grains, grass seed is the only substrate of choice.
Had I used grass seed for the culturing and photos taken in this thread I would have had much clearer results with a greater likelihood of top fruiting.

Although all the photos of this tech are taken with brown rice, grass seed is by far the suggested grain-of-choice for these methods.


Here I have written the process by which I use the below method to observe, select, and test for ideal cultures.
It's a thorough step-by-step of my process so I'd rather link to it than insert so much text here.

GRAIN "PETRIS"


Containers are loaded ~1/2 inch deep with prepared brown rice or grass seed.
Brown rice works, as does millet (maybe even a bit better), but grass seed has notably superior results.  The seed is lighter per volume, and fills that volume ideally to make a seamless puck without extra depth, so a pound goes a longer way.
It's an ideal substrate for Cubensis, yet its lightness and volume make it nutritionally more "dilute" in a sense as well.
This makes it the ideal single substrate for growing Cubensis, which will have greater troubles and longer delays finding a suitable pinning location on increasingly nutrition-dense substrates.

The goal is to make them as evenly shallow as one can without having holes in the flat cake-to-be. (Grass seed makes this perfect easy)

Sterilization times are very short, 15PSI 30-35 minutes depending on cooker load and gas/electric. 25 minutes may work for light-loaded small cookers on gas burners, 40 may become necessary for tall cookers loaded high on electric stove eyes.

()
Inoculate just like an agar dish - right in the middle. ("Dig in" the wedge a bit amongst the grains if you like and can do so without risking sterility; not necessary)

My preference is a single transfer directly from a sterile multi-spore dish with many spore flakes, cuts not made adjacent to each other. This keeps expansion to a mere 3 inches before a clone is possible while giving more genetic diversity opportunity to make a show!

Mycelium growing out into the rice:



I love this little trick! These have many advantages.
  • THEY PIN! Mycelium may pin on agar but this often isn't indicative of much more than capability to fruit as they are tiny and usually abort tiny. However these small grain substrates form full pins like any other and are capable of easily growing mature fruits.
  • Spore solutions are finally easily usable for proper culturing from spore. Just a drop or two in the middle is enough! Instead of leaving loose water all over the surface of agar or favoring and spreading bacteria, they germinate and grow outward just like a spore syringe inoc on grain but in "petri" form!, expanding no more than necessary before selections and never destroying/recovering colonies.
  • Transfers can be done easily and quickly with sterile tweezers! Single grains, chunks, or pins can be moved in a second with the easiest of tools to handle.
  • After their use, instead of becoming waste they can be allowed to play out as a mini-grow! With a cracked lid and little bit of bottom-watering they can have up to the grow power of a small or regular PF cake that requires no fruiting chamber.  Even your "lab waste" can yield, making full use of energy and work! (More about this kind of invitro growing is seen in the sections below)

Here is the "first flush" "lab waste" from 6-7 small rice pucks after cloning pins:

Here I "bottom-water" these, allowing them to have the moisture they need for more!



The pins (advantage #1) are my main purpose for using this method.  It's an extremely easy way to "clone", simply transferring the still-sterile pins via tweezer to a new agar dish, which not only allows for absolutely minimal expansion but selecting genes that want to cluster even on tiny substrates
They may pin on their own with time, but when they've colonized over all the grains I crack the lids loose a bit to allow excess CO2 to be pushed out and a very small amount of gas exchanged. This may help prompt a show of pins from genetics responsive to the cultivator's shift from colonizing to fruiting conditions.

This container has had lids loose for several days:

You can see that the condensation near the top of the container has evaporated.  This is a pretty ideal looseness.

This is not, however:

All of the moisture has evaporated. It's a slight difference between the two, a difference exaggerated by the thinness of the substrate within and how easily it can loose its little moisture.

As a rule-of-thumb I generally loosen the lids by turning them a little over an inch counter-clockwise. For Ziploc containers this is the width of the little nodes along the lids.



Some pins that emerged very quickly from the recently colonized outer edge of a rice puck:

There are options to choose from. At the top a bit to the left a single fat pin that may grow tall. At the bottom another sizable pin with just a single small partner at its feet. On the left a nice cluster with some large fruits but that are deformed or mutated. I choose the sizely member of a medium cluster on the right.


Pins make for not only incredibly easy transfers but powerfully reductive selections in a single transfer and dish!


This here is EXACTLY what I was looking for – a quick-fruiting dense cluster of pins at the top edge of the substrate despite a welcoming side-pin environment just half an inch away. I took a few samples from this and labeled them accordingly to paying extra attention.

Same container later:
Absolutely beautiful. I was happy to see this in just one of eight quart invitro containers (you'll read about these below).
Multiple clustering sizeable pins that seem not concerned about hiding in the side-pin microclimates.



Cubensis likes to pin on places with no nutrition, either with none originally or where they've consumed it, so the high nutrition density of brown rice leads them to seek other places to pin from.  This is almost unavoidable, but can give different observational opportunities for selecting cultures.
All-over the sides of the containers are specimens that show to be masters of migration – migrating nutrition and water that is – pinning numerously on bare-naked plastic closest to the edges of the lid where gas is exchanged and moisture lost.  A few samples of pins from dense areas are taken, especially of clusters in densely-pinning areas.


Many of those fruits became full size, so large that they fell and ripped the mycelium network off the container sides!
How did that mushroom get so big?  Mycelium migrated ten whole grams of water to that one single pin, four inches up the side of bare plastic!
That is quite a gene to snag. Not only did it not hide in the sidepin microclimates but went well out of its way to grow on a bare & exposed surface close to the gas exchange where the moisture is lost!, migrating large amounts of moisture quite a distance over just several days.
Mycelium are truly amazing. They have abilities that past techniques don't take full advantage of.


Sweet! Even after taking 2 pins earlier, there are still tons of them growing from this agar wedge.  They fruit from the grainwater agar and migrate water up from below.
I'm particularly interested in the two expressions of genetics shown above, for growing mushrooms on only agar...


Prolific pins are great but I still ignore them if they insist on forcing side-pinning.
The 2nd photo above is an example of one I may be willing to give a second shot to. Perhaps the environment on top of this cake is drier than my fruiting conditions will be, due to a lid screwed too loose.


Pins will take a few days to fuzz-up and start growing across the agar.


It's best to transfer pins as early as possible into maturity.

Some sterile pins transferred quite late:

At a certain point they may even continue to mature and open sporulating caps. We definitely don't want that for a clone dish.

These dishes were all started with small pins:

Growth is much more ideal; these cultures are and will continue to be more healthy and vigorous.

From there it's a snap to isolate the fastest & strongest of the mycelium that grows from the pin and start a small test batch!


* Test batches of cultures should also be on small substrates for the same reasons mentioned in the prior post (clarity due to fruiting area/substrate size ratio etc.) as well as others. Large batches are needlessly large failures if the strain is little good, a problem even more likely when not selecting fruiting strains first! Also, failures of tests due to contams on the likes of monotubs (which happened to me too many times) are incredibly annoying and wasteful speed bumps in determining a culture's ability.


Here I outline exactly how I utilize this culturing technique to select the best cultures from a round, from cloning to testing.

Edited by Violet (03/11/15 10:44 AM)

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InvisibleViolet
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Culturing • INVITRO GROWING [Re: Violet] * 1
    #19035268 - 10/26/13 11:39 AM (10 years, 4 months ago)

CONTAINER INVITRO GROWING
I've really been enjoying this style of invitro. These containers work as perfectly self-contained tunable fruiting chambers, making each one a single instance of the complete growing process.  Success rate is extremely high, yield can be near ideal, and it's ridiculously quick and easy. Combined with bottom-watering it is an ultimate neglect tek.


I've actually given this method its own tek post, as I think it deserves it!  It's an excellent beginners method, or one for culturers as per this tek, or just lazy people, stealth lovers, contam haters, people who just want to try some alternatives and maybe fall in love with the ease and simplicity.


Containers (especially quarts/litres) are loaded just about 1/4 full to leave fruiting space, sterilized, inoculated and colonized.

On the left, colonized with grass seed. On the right, brown rice.
They grow dense as always:




Those containers are slightly larger than the pints, holding about 20oz. They are excellent for small invitro grows especially to test the strength of a culture using small substrates.
Some genetics might do better than others in an invitro circumstance such as these.

For using brown rice or any other grain besides grass seed, an invitro casing layer is likely required for optimal results


Fruiting conditions are initiated by cracking the lid moderately to very loose. It will take some experience to learn intuitively how loose to leave them for full fruiting since maturing fruits greatly increase the amount of mycelium exuding CO2.


A nice first flush for multi-spore on a small substrate, even after taking some of its best first pins for clones.
It's given a tad of water to absorb for the next round!

With good culture, good conditions, and bottom-watering, quart invitro containers are capable of reaching almost the same yields as other typical "violet tek" style cakes in a very discrete and low-energy fashion. Tiny invitros are even capable of PFtek-like yields.


However it's their ability to retain sterility that makes them useful when there's no need for their discretion.
Sterile cloning like from grain petris is of course possible, but for the same reason it's a cinch to get...

Edited by Violet (04/05/17 05:18 PM)

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InvisibleViolet
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Culturing • STERILE SPORE PRINTING [Re: Violet] * 5
    #19035270 - 10/26/13 11:39 AM (10 years, 4 months ago)

ASEPTIC SPORE PRINTING
Considered an oxymoron by most that I've seen on these forums. It's often said that "No spore print is sterile, since fruits do not grow in sterile conditions." These ones do.


Sporulating caps can be held with sterile tweezers:

... while stems are cut as close to the gills as possible by another sterile tool (I use small hygiene scizzors, scalpels should work):

...then transferred into containers sterilized with square rectangles inside them.

Lid the containers before leaving to drop spores.

If the edges of the cap are turned up enough it's pretty easy to see the progress.
This below is after 10 hours.


After enough time has passed to leave a hardy print (8-30 hours depending on sporulation) the cap can be removed again with sterile tweezers or by carefuly poking into (not through) the cap with a pin or something.


Don't worry if the print is smudged in the process of removing the cap. It's all sterile so nothing is compromised.

But, a little trick I have to avoid this from happening:

With the tweezers against one edge of the cap, turn the container on its side in the direction of the tweezers, causing the cap to drop away from the foil onto the tweezers.



Allow the print to dry. In sterile laminar airflow this happens quickly with the container's lid off. In a still-air/glove box the lid should be left loose, even cracked upwards on one side of the top, to allow the print to gradually dry without contaminants falling downwards onto it.


Once the print is ready to go, only alcohol'd gloves need touch the edges to close it up and fold over the edges.

Placed into a fresh baggie (inside is sterile) this print can be opened and used with a 100% chance of sterility! (Given proper sterile tech of course)


I feel that taking one's prints in a fashion that guarantees sterility is a must.  As I say, "start sterile, stay sterile." Once culture sterility is achieved it's easy to keep it that way, even from one spore to the next.
This technique ensures one's prints to be of top quality, a show of courtesy and convenience not only to yourself but anyone who receives your work as a gift or in trade. After all, the point is to share a sample of a single organism, so we've not done it right if there is more than one species in the sample!

__________________________________________________


Thanks for reading! Hope you enjoyed it even a tenth as much as I enjoy using this method!


--------------------
Intentionally or not, here in mushcult we are purveyors of love culture and enlightenment movement. Let's try to act like it!

PODS TEK - Growing Invitro with BRF/verm or Grass Seed containers
The simplest, quickest, safest tek!  For beginners, culturers, lazy people, stealth lovers, contam haters, and alternative seekers!
Violet's Teks and Posts

Edited by Violet (09/30/17 08:31 PM)

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Invisibleanne halonium
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Re: Culturing • STERILE SPORES [Re: Violet]
    #19035380 - 10/26/13 12:06 PM (10 years, 4 months ago)

:thumbup:


--------------------
:aliendance:

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OfflineOregonMushys
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Re: Culturing • STERILE SPORES [Re: anne halonium]
    #19035401 - 10/26/13 12:11 PM (10 years, 4 months ago)

You swooped the 'sterile spore printing' idea from me. I was gonna post that :lol: i been takin 'sterile prints' from invitro fruiting containers for a bit now, just never got around to post it. You should filter the lids of the printing containers to eliminate all that moisture on the foil.

EDIT: by definition thr is no such thing as a sterile spore print, but taking a print from invitro is as clean as it can get.


--------------------
Ps. Cubensis                                        Ps. Cyanescens                                      Ps. Stuntzii  *GrowLog*
   
                                                                                                      :sasquatch::mushroomgrow::leaf::mushroomgrow::leaf::mushroomgrow:
                                                                                                  :cop:

Edited by OregonMushys (10/26/13 02:53 PM)

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OfflineSagescruffy
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Re: Culturing • STERILE SPORES [Re: Violet]
    #19035460 - 10/26/13 12:25 PM (10 years, 4 months ago)

Awesome!


--------------------
Love.

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Invisiblebarong
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Re: Culturing Tech • Agar containers, "Grain Petri" Tek, Invitro growing & Sterile Spores! [Re: Violet]
    #19035582 - 10/26/13 12:52 PM (10 years, 4 months ago)

Quote:

Violet said:
Malt extract is a derivative of grain so malt solutions for agar could be considered a grainwater made from purified concentrate!




Your 'grainwater' shares NO characteristics with malt, and does not contain the sugars present in malt extract. The malting process of grain involves germination until the acrospire grows, modifying the staches in the endosperm. The starches of this malted grain is then converted to 'malt' via a conversion in liquid at appox 68ºc. This is then reduced under a vacuum, and spray-dry techniques are used to make malt extract.

So while your 'grainwater' may work fine for these purposes, people need to be aware that it's not even close to being a substitute for malt extract.

And I have no idea what "made from a purified concentrate" is supposed to mean.

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InvisibleViolet
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Re: Culturing Tech • Agar containers, "Grain Petri" Tek, Invitro growing & Sterile Spores! [Re: barong]
    #19035737 - 10/26/13 01:27 PM (10 years, 4 months ago)

"Not even close to being a substitute for malt extract."  You cannot say it's not even close as a substitute when here it is clearly being a complete substitute.  I haven't used malt extract in over a year.  It may not be due to the switch but I experience the best agar performance I've ever had.

My point is that malt is a single extracted material from grains (which you also pointed out) and that grain prep water contains the same spectrum of materials that the grains do which are clearly far more than enough for what Cubensis needs, as well as many other species (every species I've worked with so far which is Many!)


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Re: Culturing Tech • Agar containers, "Grain Petri" Tek, Invitro growing & Sterile Spores! [Re: Violet] * 1
    #19035774 - 10/26/13 01:35 PM (10 years, 4 months ago)

Quote:

Violet said:that grainwater contains the same spectrum of materials that the grains




But it does not contain the same spectrum of 'materials' that malt extract does. I'm not suggesting it will or won't work, but it's not even close to being malt extract. How that impact mycelium growth remains to be seen . I'm sure it would work, hell dogfood works.

And for all the time and mess, plus timing issues, or storage requirements, for your grainwater against having malt extract quickly and readily on hand, there's really no point, apart from it being another 'novel' idea of yours. There's plenty of people wishing to reinvent the wheel, and good luck to you, if it works.

Cost of 1lb malt extract = $4.50
Cost of 20g malt extract = 20 cents
Cost of malt extract per 20mL solution for plate = 1/2 a cent !

Cost saving by using grainwater = an almost irrelevant arguement

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Re: Culturing Tech • Agar containers, "Grain Petri" Tek, Invitro growing & Sterile Spores! [Re: barong] * 3
    #19035840 - 10/26/13 01:51 PM (10 years, 4 months ago)

"An almost irrelevant argument."  Yet you're the one making it here, yes?

Sure malt is cheap.  However its cheapness is no concern of mine because spending even a dime on it is a waste since I don't need it, I already buy grains.

Thanks for your demeaning posts.


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Re: Culturing Tech • Agar containers, "Grain Petri" Tek, Invitro growing & Sterile Spores! [Re: barong] * 1
    #19035845 - 10/26/13 01:53 PM (10 years, 4 months ago)

some peeps , wanna make every grow a oliver twist contest.

im convinced.
violet writes a great tek,
with great practical value.

and barong, likes malt. alot.


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:aliendance:

Edited by anne halonium (10/26/13 01:54 PM)

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Re: Culturing Tech • Agar containers, "Grain Petri" Tek, Invitro growing & Sterile Spores! [Re: anne halonium]
    #19035862 - 10/26/13 01:58 PM (10 years, 4 months ago)

And thank you (both of you)for spreading poorly researched misinformation.

keep using those chemical fertilisers on your grows !

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Re: Culturing Tech • Agar containers, "Grain Petri" Tek, Invitro growing & Sterile Spores! [Re: anne halonium]
    #19035880 - 10/26/13 02:03 PM (10 years, 4 months ago)

whatever barong......
can of malt on the tracks, wont stop the nitro V train.

i would note, ive never been fond of foil,
as i prefer baby food bottle bags ( sterile), and slides.
sometimes i combine both.



i totally endorse the rest of this thread though.

i wanna see more violet pics of boomers in quart PP5s!


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:aliendance:

Edited by anne halonium (10/26/13 02:17 PM)

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Re: Culturing Tech • Agar containers, "Grain Petri" Tek, Invitro growing & Sterile Spores! [Re: barong]
    #19036014 - 10/26/13 02:44 PM (10 years, 4 months ago)

If spore prints were sterile nothing would grow from them.

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Re: Culturing Tech • Agar containers, "Grain Petri" Tek, Invitro growing & Sterile Spores! [Re: Mush4Brains]
    #19036091 - 10/26/13 03:03 PM (10 years, 4 months ago)

I like Sterile Spores. It's nicely written.

I've considered doing something similar, but with 1/2 pint wide mouths. PP5 containers might actually be better though because of the size.

Couple questions though.

1) How are you introducing sterile FAE for fruiting? Periodically opening in front of flowhood? SAB?

2) Are you bottom watering? Using sterile water?


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Re: Culturing Tech • Agar containers, [Re: Mush4Brains]
    #19036099 - 10/26/13 03:06 PM (10 years, 4 months ago)

so far, a can of malt hurled, and graffiti on the overpass.
nitro V train keeps rollin.

of note,
theres a wide variety of PP5 containers that work with this.



its direct, its easy, it performs.



spits question above seems legit.
FAE isnt an issue when done as shown , its a mater off loose lids.
bottom watering, would be assumed.

lets see what else violet has to share..........

Edited by anne halonium (10/26/13 03:11 PM)

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Re: Culturing Tech • Agar containers, "Grain Petri" Tek, Invitro growing & Sterile Spores! [Re: Mush4Brains]
    #19036103 - 10/26/13 03:07 PM (10 years, 4 months ago)

In this context, "sterile" means does not contain anything but the desired spores.


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Re: Culturing Tech • Agar containers, "Grain Petri" Tek, Invitro growing & Sterile Spores! [Re: SpitballJedi]
    #19036192 - 10/26/13 03:30 PM (10 years, 4 months ago)

Thanks!

1)  Gas exchange is simply from slightly loose lids. The bottom of the lid's threads sits on the top of the container's threads, keeping the seal of the lid just off the edge of the container.  Similar to how still-air boxes work, contaminants do not easily drift in.  During the rounds of pin transfers that the grain petri photos are from I tweezered 20 pins to 20 agar containers. They've all grown out with 0 contams.

2)  After the first flush I do bottom-water as to make the most of everything. Based on potential 200-330% BE, the 22 dry grams of rice in a grain petri could yield 4.5-8 grams. More per larger the invitro.
I use sterile water for this only when I want to retain sterility for the following flushes. Plain water works once fully colonized.

:]


--------------------
Intentionally or not, here in mushcult we are purveyors of love culture and enlightenment movement. Let's try to act like it!

PODS TEK - Growing Invitro with BRF/verm or Grass Seed containers
The simplest, quickest, safest tek!  For beginners, culturers, lazy people, stealth lovers, contam haters, and alternative seekers!
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