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InvisibleMateja
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LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth * 6
    #27007467 - 10/28/20 12:35 AM (3 years, 2 months ago)

This experiment will demonstrate that bacteria will cause turbidity in a LC and that turbidity is easily recognized.


5 nutrient rich liquid mediums were used for this test. The nutrient density is purposefully kept to a minimum so that the liquid medium will be crystal clear and able to effectively contrast turbidity. Three of these broths contain 0.05% malt extract by weight and the other two were made from diluted rye grain water which was then further diluted by mixing with 19 parts water.


Bacteria and mold colonies used for this test were gathered from these plates




1 ME broth was inoculated with a grey/opaque bacterial colony, 1 ME broth was inoculated with a yellow bacterial colony and 1 ME broth was inoculated with a mold colony. 1 GW broth was inoculated with a wedge containing both cube myc and the grey/opaque bacteria and the other GW broth was inoculated with a wedge containing both mold and cube myc. These broths were incubated at 25C (77F) and swirled once a day.



The 5 broths shortly after inoculation:



To inspect for turbidity an object should be placed right behind the LC jar along with a back light that will illuminate both broth and the object. I chose to use a small spore solution vial but any kind of object can be used as long as it has distinct patterns/lines that can be easily observed through the crystal clear LC jar.

Example:




After only 8h of incubation we can already observe noticeable turbidity in LC's that were inoculated with bacteria and the spore vial can not be seen through all the light diffusion inside the liquid media. The light scatters in all directions as it reflects off of countless bacterial cells. LC's containing only mold and/or cube myc are still transparent and the spore vial can be observed through the broth.

Results:





The LC's in those pics above were inoculated with bacterial agar wedges and regardless how tiny the bacterial colonies were they still contained a huge number of cells which contributed to a very fast colonization of the broth. For the next test I will use just a fraction of that and let's see what will happen. These next jars were both first inoculated with 10ml of mold liquid culture each using a syringe and needle. One of these mold LC's was then inoculated with one drop of bacterial solution and the other mold LC was inoculated by gently touching the broth surface with the needle tip of that same needle.


The broth that was inoculated with 1 drop of bacterial solution went turbid within a few hours and the mold colony appeared to stop growing right away. The other (needle tip) broth offered the mold colony a chance to recover briefly but within 24h it also went turbid and no more growth could be observed in the mold colony.

Results:




In the pics below I didn't even use a background object, only light, and the bacterial LC can easily be differentiated from the clean LC and from the control jar which hadn't been inoculated.

Results:




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Edited by Mateja (03/10/22 01:00 AM)


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InvisibleBaba Yaga
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27007489 - 10/28/20 01:00 AM (3 years, 2 months ago)

Thanks for doing this.

:threadmonitor:


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InvisibleMateja
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Baba Yaga] * 1
    #27007624 - 10/28/20 04:38 AM (3 years, 2 months ago)

And it seems that bacteria will fuck up a liquid broth in a matter of hours :shrug:

I placed a spore solution vial inbetween the light and the LC jar as to be able to gauge the anticipated turbidity.
But I guess I should have takes pics every 30min or so lol these photos were taken 8h efter inoculation and that's all folks for this experiment :rofl:





Soooo... If your LC hasn't gone turbid in the last few hours I'd say you can be fairly sure there's not bacterial activity in there. :bongload:


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Cakes inside Water Tub


Edited by Mateja (10/28/20 04:39 AM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja] * 3
    #27007627 - 10/28/20 04:42 AM (3 years, 2 months ago)

Some bacteria will cause turbidity some wont. Some bacteria form biofilms because of their secretions. Those make a broth way more turbid than say most species of lactobacillus would and some lacto make no turbidity


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InvisibleMateja
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: bodhisatta]
    #27007640 - 10/28/20 04:58 AM (3 years, 2 months ago)

I will continue this experiment for the sake of documenting different types of bacteria and mold growth in LC's to learn to decide easier upon future visual inspections. I can understand how some microbes due to secreting make the broth even more turbid. But are you saying that there are types of bacteria that consist of a perfectly transparent cell? And even when those cells are jammed in the broth in astronomical numbers should there really still be no visible turbidity?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27007813 - 10/28/20 08:01 AM (3 years, 2 months ago)

Good stuff. Now who has a spectrophotometer?

Would be interesting to see what happens when you add bacteria, either via agar like you did or a drop of your new bacteria broth, into a clean colonized or colonizing LC.

Inoculation size might matter in some situations- I'm guessing your additions were orders of magnitudes larger than the start of a typical contam. More of an issue in the case where myc might otherwise be able to fight it.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: dfwerydfhg]
    #27008660 - 10/28/20 03:22 PM (3 years, 2 months ago)

Quote:

dfwerydfhg said:
Good stuff. Now who has a spectrophotometer?



You have two of them on your face, kind of :crazy2:

Quote:


Would be interesting to see what happens when you add bacteria, either via agar like you did or a drop of your new bacteria broth, into a clean colonized or colonizing LC.



Yes I will be doing that as well, adding mold and bacterial colonies to clean LC's at various stages to see how growth is affected.


Quote:


Inoculation size might matter in some situations- I'm guessing your additions were orders of magnitudes larger than the start of a typical contam. More of an issue in the case where myc might otherwise be able to fight it.



Yes these bacterial colonies were gigantic in terms of numbers even tho they were tiny specks. Next round i will just dip the tip of the needle or gently swipe a bacterial colony on agar and then just gently touch the surface of the broth with the tip of the needle. Now that I think of it I actually know a couple of ways to dilute bacterial colonies so I end up with just a handful of cells, I might do that as well if dipping the tip of the needle proves too crude as well.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27008677 - 10/28/20 03:34 PM (3 years, 2 months ago)

Yeah! Thank you for doing this experiment! this needed to be done!


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Danger-Dave 1]
    #27008740 - 10/28/20 04:10 PM (3 years, 2 months ago)

:havesomescience:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: verum subsequentis]
    #27008754 - 10/28/20 04:21 PM (3 years, 2 months ago)

Oh shit dude, was it my penis that gave you that nasty plate? I think I can even see some AIDS there too. Cool experiment tho.
:threadmonitor:


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InvisibleMateja
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27008813 - 10/28/20 04:55 PM (3 years, 2 months ago)

I've seen much worse :lol:
and if I didn't have faith in your work I'd probably put the swabs to more than 2 plates. :shrug:
I'm pretty sure I found a handful of cube germ spots in this one plate that I transferred, I'll know in a day or two :typing:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27008834 - 10/28/20 05:11 PM (3 years, 2 months ago)

I always assume a swab is dirty though. These fruits were all flopping big dicks bending over with the caps touching the surface, many of them with the gills facing upwards, so some nastiness is to be expected.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27008941 - 10/28/20 06:11 PM (3 years, 2 months ago)

At no point did I ever consider that the swabs you sent me were clean lol I just have faith that you're not sending me a catastrophe worth shit.
I also have faith in my ability to get dirty spores to germinate successfully on a grow medium :bongload:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27015688 - 11/01/20 12:38 PM (3 years, 2 months ago)

This time I inoculated two new broths with 10ml of mold each and then I added
one drop of bacterial solution to one of them and I gently dipped the tip on the bacterial needle into the other broth.
The jar that got a drop of bacterial solution immediately colonized the broth within hours and completely stopped the mold in its tracks. The broth that was dabbed with the bacterial needle tip offered the mold a chance to gain some momentum for probably 24h or so but the day after inoculation I already noticed obvious turbidity. That's also why one of the broths has a larger mold colony than the other, as visible in the pics. When it comes to the bacterial activity in these broths I'd say both reached 'stationary phase' within 36h, the onset of turbidity was gradual but extremely fast and hasn't increased since. So far I'm observing maximum cell mass reached within a couple of days in 200-300ml broth.


Here is the 'drop' and the 'needle tip' inoculated on 31st




And just for reference, here are two homogenized mold LC's with crystal clear broths.
One is MEA 0.05% the other is boiled rye broth diluted with 19 parts water.
Lately I've been placing this Pan Cyan vial inbetween the broths and the light source as a means to detect turbidity. I don't have a spectrophotometer so the text on the spore solution vial is my tool for measurements. On the other hand I don't really have much need for precise measurements or estimations using devices since I only need to detect any turbidity whatsoever or the complete lack thereof.


Mold in MEA and rye water



The Pan Cyan vial is behind the bacterial LC's as well but the light diffusion inside the solution makes is virtually impossible to see, light scatters all over the place trying to shine through a solution crammed with ("invisible") cells.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27015704 - 11/01/20 12:50 PM (3 years, 2 months ago)

That has been my experience, the few times I got bacteria in an LC it would go to shit fast from just a little needle poke. It got turbid very quickly and the cube myc reacted to the bacteria in a pretty obvious way, it would slow down its growth too. I for one am dying to see this sneaky bacteria that won't cause turbidity in the liquid that all people talk about, the one that only shows it's ugly face on agar, or even an LC that only has 'a little of bacteria' after being colonizing for more than one week.  :ninjalike:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27015767 - 11/01/20 01:31 PM (3 years, 2 months ago)

Practically every type of bacteria that's in our home/substrates divide every 20-30min on average in good conditions. They look wimpy as hell on agar but in liquids they talk a whole lot of smack, really fast as well and they back that shit up. Reinforcements are always right around the corner and you're already fucked
:itseveryone:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja] * 1
    #27015804 - 11/01/20 01:54 PM (3 years, 2 months ago)

:takingnotes:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: tripdawg420]
    #27015874 - 11/01/20 02:42 PM (3 years, 2 months ago)

I would like to see you try inoculating a healthy 100% cube LC with bacteria, that should be interesting too. Now that you're all in with this I'm sure you won't mind. :grin:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27015952 - 11/01/20 03:53 PM (3 years, 2 months ago)

Quote:

Josex said:
inoculating a healthy 100% cube LC with bacteria



No sweat man, I was gonna play the villain anyway :bongload:

Was thinking of squirting moderate/excessive amounts of bacterial solution into a fully or mostly colonized myc LC and then immediately use that to inoculate cakes and grain. Been thinking of doing this for a long time cause I'm curious to see how bacterial gangs go about handling their business on the turfs. I've never (I'm assuming no one has) seen bacteria in action on grain with visible visibility from the eyes only. I'd just pour a 50/50 solution myc/bac on top of rye to see if myc colony will be able to implement their policy and ideology or if they'd all be corrupted resulting in bad guys taking over and ruining the society. And then I'd do the same to cakes. This test will probably have varying results tho depending on the water activity of the grain and cake turf it encounters.


Okej kourrwa I have all the passwords and the keys are activate, I just have to mine some more Mycelium to put inside the warhead together with Bacillus so it's will be weaponize properly kourrwa!
:strangelove:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27016056 - 11/01/20 05:00 PM (3 years, 2 months ago)

I think it'd be interesting to test an LC on agar and verify it is clean. Then inoculate something. Then spit in it, put it on a stir plate or swirl it and noc something else. Then noc a new jar every day and track the progress.

But aint nobody got time for that.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27016137 - 11/01/20 05:54 PM (3 years, 2 months ago)

Quote:

Mateah said:
Practically every type of bacteria that's in our home/substrates divide every 20-30min on average in good conditions. They look wimpy as hell on agar but in liquids they talk a whole lot of smack, really fast as well and they back that shit up. Reinforcements are always right around the corner and you're already fucked
:itseveryone:



There's an unfathomable amount of bacteria out there. Even in beer there's a few dozen common contaminants let alone rare ones. And beer is a environment with a pH that's rough, hop acids that are antibacterial to most bacteria, co2, no air, pressure, yeast has some defenses, and low leftover nutrition.
Some bacteria may only grow to a few hundred cells per mL which is hard to detect with all but PCR


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: bodhisatta]
    #27042223 - 11/16/20 01:14 PM (3 years, 2 months ago)

Quote:

bodhisatta said:
There's an unfathomable amount of bacteria out there. ... Some bacteria may only grow to a few hundred cells per mL which is hard to detect with all but PCR




So,... looking for bacteria in the LC with a microscope isn't likely to help?

Would it be a worthwhile "community project" to collect contaminant reports from LC rejects? It would be great if we knew what contaminants really are most common across different locations. For example, I dumped one LC yesterday because a test plate grew trichoderma. It took 3 days to sporulate in the petri, alongside the mycelium.

I've never found bacteria in my LC test plates, but maybe I just don't know how to look for them. When I make a test plate, I try to catch just clumps of growth after spinning the stir bar as fast and hard as I can make it go. Is that the way to get a representative sample? How large a sample do you inject onto the agar? Are there dyes or other ways to make bacteria more visible in the test plate? Maybe I should be testing pH as well?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: sonoramo]
    #27042539 - 11/16/20 04:58 PM (3 years, 2 months ago)

Quote:

sonoramo said:
Would it be a worthwhile "community project" to collect contaminant reports from LC rejects?



That would be a very worthwhile project in my opinion! I encourage everyone to post their LC's in this thread so we have a variety of different LC 'situations' documented to easier map all the visual indications!

Quote:

I've never found bacteria in my LC test plates, but maybe I just don't know how to look for them.



That's because the standard agar recipe of 2% agar isn't very suitable for detecting bacterial growth. The vast majority of bacteria requires a liquid environment to grow and unless the bacteria is very motile they would in addition need moving water to be able to spread their colonies since most baciera are non motile.  That's why microbiologists use mostly liquid and  sometimes semi-solid media's like motility agar for propagation. Even tho bacteria have the ability to deal with differences in osmotic pressure, some microbes are better at this some are do not handle lower osmolarity all that well. All in all bacteria as an organism no matter of strain all require extremely high water activity rates compared to for example myc or mold, so the wetter the environment the more suitable it is for bacterial growth. Hope that helps!


BTW you have any LC projects going on right now?


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Edited by Mateja (11/16/20 05:01 PM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27042575 - 11/16/20 05:21 PM (3 years, 2 months ago)

Quote:

Mateah said:
That's because the standard agar recipe of 2% agar isn't very suitable for detecting bacterial growth. The vast majority of bacteria requires a liquid environment to grow and unless the bacteria is very motile they would in addition need moving water to be able to spread their colonies since most baciera are non motile.  That's why microbiologists use mostly liquid and  sometimes semi-solid media's like motility agar for propagation. Even tho bacteria have the ability to deal with differences in osmotic pressure, some microbes are better at this some are do not handle lower osmolarity all that well. All in all bacteria as an organism no matter of strain all require extremely high water activity rates compared to for example myc or mold, so the wetter the environment the more suitable it is for bacterial growth. Hope that helps!





Thanks. That is helpful. Most of my experience with bacterial colonies is on food in the refrigerator. Eventually they get big enough to see (and smell!) easily. I'll take a "bottom line" that maybe it isn't so bad if the extra liquid from inoculating a test plate softens the agar.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27042587 - 11/16/20 05:30 PM (3 years, 2 months ago)

Quote:


BTW you have any LC projects going on right now?




I tried to answer by PM so the rest of the community doesn't get sidetracked, but your account doesn't accept PMs. So,...

Yes:

  • GT's (now fruiting and so done with it, but saving LC in the refrigerator)
  • Unknown cubes from deep in my spore-print collection but they are old friends :wink:
  • Cambodians, almost ready to inoculate
  • Quilla, very ready, will probably inoculate this week
  • Oyster culture that's previously "worked" from agar and cardboard, now ready with LC
  • Tarragon oyster from commercial syringe, trying to expand before inoculating


In all cases (other than the commercial syringe) it's taken some effort to go from spores to agar to LC, but I seem to get better at it as I go.

My current interest is growing oysters in larger quantities. I just can't consume cubes as fast as I could grow them now that I have a working LC (or four...).

I'm using 16ga dispensing (blunt) needles to sample from agar culture into squat 4oz mason jars. If a culture seems to be clean, then move up to a pint mason jar. Finally, inoculate oats with 20ml of LC, and I've seen full colonization in about 1.5 to 2 weeks. In all cases, lids with SHIP and Tyvek filters. Newer lids I've used two layers of Tyvek in case the broth splashes.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: sonoramo]
    #27042630 - 11/16/20 05:55 PM (3 years, 2 months ago)

My account doesn't accept PM's? You mean the automatic message said to "ask cultivation related questions in the public forum"? I guess it's aight to send me links or pics if you want but in general it's much more productive for everyone if we conduct research here instead of PM's :super:


Do you have pics of any of the LC's you have going right now?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27042744 - 11/16/20 07:05 PM (3 years, 2 months ago)

Quote:

Mateah said:
My account doesn't accept PM's? You mean the automatic message said to "ask cultivation related questions in the public forum"?...





More like a message that said "this user doesn't accept PM's."

Quote:


Do you have pics of any of the LC's you have going right now?




OK, here are a few samples of what's cooking.

Mexican strain from Quilla (thanks, CaptainFuture). This has pretty much filled the LC, and if I don't withdraw some of it soon it will form floating pads on the surface.


A well-used GT culture (from a local friend), used to inoculate 7 jars so far. I'm done with this because it's been so prodigious. It has formed substantial mycelial mats on the surface.


Commerical tarragon oyster culture from a syringe, expanding it to a pint jar. It's only been there for a few days. Much of what you see is sediment from the MEA powder.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: sonoramo]
    #27042807 - 11/16/20 07:49 PM (3 years, 2 months ago)

Following this.

I have had beautiful looking, clear LC's with strong myc growth that when put to agar are contaminated. I have been lucky with LC's with over 95% success rate, but this has happened more than once. My SOP now includes a mandatory agar test before using.

I would love a visual "test", but i am not sure its possible and/or accurate. There are a multitude of bacteria and molds and each would elicit a different visual outcome IMO.

On a side note: I notice nutrient LC's seem to have a higher affinity to contamination. When i use a simple 4% karo solution, I usually have no issues.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: maxmush]
    #27044391 - 11/17/20 06:43 PM (3 years, 2 months ago)

Mostly posting to subscribe, but for some reason my LC broths always come out of the PC as a really nice golden color. Munton's LME like most everyone uses :shrug:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: MLPismyOPSEC]
    #27044514 - 11/17/20 08:00 PM (3 years, 2 months ago)

These are both inner tissue LCs. Left one was made 11/14 the right was 11/13. I think I can tell which will be no good at this point. Also think I'll tone down the sugar content next time. They're 4% honey.



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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lemgrub]
    #27044547 - 11/17/20 08:30 PM (3 years, 2 months ago)


Right is B+ left is APE.
LME heat dissolved into distilled water with a 45min PC cycle.
Some sediment but no other microbes save mycelia in here.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: mushhead]
    #27046036 - 11/18/20 06:02 PM (3 years, 2 months ago)

Following, will post some pics in a bit


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: D3_Myc]
    #27046219 - 11/18/20 07:19 PM (3 years, 2 months ago)

How would you rate these lc's?



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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: BrownBear]
    #27046265 - 11/18/20 07:40 PM (3 years, 2 months ago)

What do you think:



*MS Srynge: GT
*Water/Honey (500ml/4ml PC:30min/15psi)
*Date: 19/10/20


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: enteogenio]
    #27049841 - 11/21/20 03:47 AM (3 years, 2 months ago)



0.4% LME, PC'd for ~20 mins at 15 psi, noc'd with a pan cyan agar wedge.



0.4% LME, PC'd for ~40 mins at 15 psi, noc'd with a pan cyan needle poke biopsy from a pan cyan agar plate.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: coversall]
    #27050583 - 11/21/20 02:59 PM (3 years, 2 months ago)

Sexy

:wellhello:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: verum subsequentis]
    #27051448 - 11/22/20 02:04 AM (3 years, 2 months ago)

Forgive me, but I need to check, is that a sarcastic sexy? Or is the LC looking ok? I gather it's near impossible to tell whether an LC is clean just by looking at it?

I'm going to yolo one of the smaller batches today.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: coversall]
    #27051693 - 11/22/20 08:24 AM (3 years, 2 months ago)

Img dump of the cultures I have going atm. One at rest and one swirled



The tat spun for a few days before I let it settle. The dark spots are the individual tissue pieces


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: D3_Myc]
    #27051725 - 11/22/20 08:36 AM (3 years, 2 months ago)

Are these clean/tested LCs?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: coversall]
    #27051742 - 11/22/20 08:49 AM (3 years, 2 months ago)

No, they are “I don’t even know what I’m looking at cultures and could use some interjections”


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: D3_Myc]
    #27051755 - 11/22/20 08:58 AM (3 years, 2 months ago)

Hah! Cool, we're in the same camp. A couple of yours look similar to mine. One of mine was used today so I'm watching the results with great anticipation.

:creepylurker:

I did put some of it on agar, but then proceeded to drop both the plates once I took them out my SAB, so not sure they will tell me much as the sample went everywhere in the dish.

:notamused:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: coversall]
    #27052207 - 11/22/20 01:10 PM (3 years, 2 months ago)



Just expanded my firsy LC. a little early, but there was plenty of nice looking myc in the transfer.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: D3_Myc]
    #27052239 - 11/22/20 01:21 PM (3 years, 2 months ago)

Quote:

D3monic said:
No, they are “I don’t even know what I’m looking at cultures and could use some interjections”



Hey D3! So The third, fourth, and eighth pic of yours look a bit turbid.
Otherwise all I see is some really nice looking myc.
Your next step is to grab out some of those agar dishes of yours, make a syringe of each LC, and test it on that agar to see if you've grown mushroom myc.
Which I am pretty confident you have.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: mushhead] * 1
    #27052521 - 11/22/20 04:40 PM (3 years, 2 months ago)

Quote:

mushhead said:
Quote:

D3monic said:
No, they are “I don’t even know what I’m looking at cultures and could use some interjections”



Hey D3! So The third, fourth, and eighth pic of yours look a bit turbid.
Otherwise all I see is some really nice looking myc.
Your next step is to grab out some of those agar dishes of yours, make a syringe of each LC, and test it on that agar to see if you've grown mushroom myc.
Which I am pretty confident you have.



I inspected those closely and I couldn't detect turbidity in any of em, they all look clear as day. Tho jars 3,4 and 8 could at a quick glance resemble a turbid broth, upon closer inspection it's quite obvious that the broth is fully colonized thus not leaving out much of the rest of the uncolonized parts to be viewed, but there are still small parts in all of the jars where you can see parts of uncolonized broth being clear and no light diffusion. And on the other hand if there was a bacterial contamination in those jars it would make it very hard to see myc at all and it would be impossible to distinguish sectors of growth or even just different parts of myc. As light gets diffused in all directions it becomes impossible to see details of what's inside the broth. And always swirl a jar before inspection because bacteria in jars can sometimes settle as sediment and the broth can appear to be clear, but after a quick swirl the broth goes instantly back to being turbid. Hope that helps!


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27052559 - 11/22/20 04:59 PM (3 years, 2 months ago)

Critiques? These were shaken about an hour ago, they will eventually settle on the bottom. Also should you decant the visually uncolonized broth before inoculating? I have done so in the past and had good results. Lately i have not been, i would swirl the LC before pouring to stir up the myc, and i've been having bacterial issues. Curious if it's from too much water in my grains or if it's just straight up my cultures. I'm betting the latter, but i can't rule out the former (yet).



In my eyes, these all look good, they look just like all my LCs i've ever made. Haven't tested them yet, will be doing so later this week. None of my LC's ever colonize more than ~50-60% of the broth. Is that normal?


Edited by MLPismyOPSEC (11/22/20 05:04 PM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: D3_Myc] * 1
    #27052570 - 11/22/20 05:03 PM (3 years, 2 months ago)

Quote:

maxmush said:
I would love a visual "test", but i am not sure its possible and/or accurate. There are a multitude of bacteria and molds and each would elicit a different visual outcome IMO.



Per today scientists use spectrophotometers to measure turbidity inside a broth to roughly calculate cell mass inside the medium. So far after reading a bunch of microbiology I haven't come across a bacterial propagation that doesn't cause turbidity, you're welcome to post links to source for that! All I know is what I've seen from tests I've done, and so far what  microbiology says about bacterial propagation in liquid medium + turbidity seems to be true, granted I've only done a dozen or so tests up to this point but let's just say there are strong-overwhelming indications that for all practical purposes in our hobby we are safe to assume that a clear uncolonized parts of a broth means that there is no presence of bacteria.


Quote:


On a side note: I notice nutrient LC's seem to have a higher affinity to contamination. When i use a simple 4% karo solution, I usually have no issues.



I won't name all bunch of reasons for why I use 0.05-0.1% malt LC or heavily diluted grain water but one good reason is for their transparent properties. Makes it a whole lot easier to detect the slightest increase in particles in your broth when you have a broth that's perfectly clear and has no particles in it.


Quote:

D3monic said:
No, they are “I don’t even know what I’m looking at cultures and could use some interjections”



I felt that too at a point and decided to do tests since no one was interjecting whenever I wanted to discuss it :lol:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: MLPismyOPSEC]
    #27052574 - 11/22/20 05:06 PM (3 years, 2 months ago)

Quote:

MLPismyOPSEC said:
Critiques? These were shaken about an hour ago, they will eventually settle on the bottom. Also should you decant the visually uncolonized broth before inoculating? I have done so in the past and had good results. Lately i have not been, i would swirl the LC before pouring to stir up the myc, and i've been having bacterial issues. Curious if it's from too much water in my grains or if it's just straight up my cultures. I'm betting the latter, but i can't rule out the former (yet).





2 broths look mostly clear and 2 look turbid. When did you inoculate?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27052765 - 11/22/20 06:34 PM (3 years, 2 months ago)

Starting a few more cultures three on left are diluted millet grain water, a 50/50 and rest 4g lme to 1000ml water.





Going to do some cross comparisons


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: D3_Myc] * 1
    #27052841 - 11/22/20 07:27 PM (3 years, 2 months ago)

Don't forget to take 'control' pics of freshly inoculated broths so as to know how the broth looks originally when you know it's sterilized, in case you're not working with crystal clear broths, so it's easier to gauge turbidity early on as it makes its presence. I have my LC photo studio on the same spot on top of the SAB and in the same lighting every time so it's real easy to notice the slightest changes going on in the broth :thumbup:


Edited by Mateja (11/22/20 07:30 PM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: D3_Myc]
    #27052855 - 11/22/20 07:42 PM (3 years, 2 months ago)

Quote:

D3monic said:
Img dump of the cultures I have going atm. One at rest and one swirled



The tat spun for a few days before I let it settle. The dark spots are the individual tissue pieces



I love your labels :super: a professional look


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: AtmozFear]
    #27052948 - 11/22/20 08:43 PM (3 years, 2 months ago)

Hope this isn't off topic for this thread but I was wondering what the best way to continuously use an LC for a few months is, if anyone could be so kind as to clear this up for me.

From what I understand, the nutritious broth of the LC allows the myc to grow.

So,

1: Is there any reason I can't make a bigass jar of LC, let it grow out, then continuously take from it to inoculate 100+ jars over a few months?

2: Is there some kind of limit to how much the myc can grow based on the nutrition provided by normal LME LC recipes? Could I not take like 10% of the LC out, let the myc regrow for a week or so, then take more out again after and continue this and have the myc continually grow between uses?

3: If that is possible/practical, what would be the best way to continuously extract LC from the one master jar? I don't have a flowhood and I'm worried that constantly pouring from the same LC in a SAB would eventually let some contaminants in. I would use a syringe and a SHIP but the consensus around here seems to be that SHIPs are a bit risky as well. 


I'm trying to streamline my process and while LI has been great, it'd save me some time to have a single source that I could keep drawing from to inoculate grain.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lenz]
    #27052968 - 11/22/20 08:57 PM (3 years, 2 months ago)

I'd recommend expanding the master into several new LC's which you can then use at will, the new LC's can be refrigerated as well. I don't personally like the idea of opening closing a master many times or sticking a syringe into it multiple times tho it can be done with sterility but Imo why bother risking it 5 times when you can instead make 5 new broths, inspect that they're clean and then use each one of those only once to be sure that you've got a clean inoculate every time.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27053324 - 11/23/20 03:51 AM (3 years, 2 months ago)

Makes sense Mateah, thank you. I was envisioning being lazy and making one giant LC that I could keep using but it seems that won't be happening.

What are your thoughts on using SHIPs and syringes with LC's? Is it really as stupid/risky as people say?

I'm comfortable with pouring in an SAB since I do it with LI but would love to reduce my time in the SAB lol. Could I even get away with inoculating grain jars outside of an SAB using a syringe and SHIP?

Thanks again.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lenz]
    #27053343 - 11/23/20 04:31 AM (3 years, 2 months ago)

To be honest I've never really heard a convincing or even a reasonable argument as to why syringes or ships should in any way be more risky than pouring. Let me motivate this and you be the judge :shrug:


In my case for example if I was to expand LC I'd have a sterilized broth in a jar with a double lid (full inner lid disc with a SHIP and a SFD patch latched onto the jar with the rubber part down sealed against the rim) and on top of this is the full outer lid (not just the ring) and also a SFD patch on this lid as well. Now you can bet that the inner lid and the SHIP on it will be sterile when I open the oter lid right? Of course. And as I break out a new sterile syringe and a new steile needle I side the SAB and attach them everything is still sterile right? Then I remove the cap from the needle, lift the outer lid and stab the needle through the sterile SHIP. Now where is the so called contam vector in this and where would it possibly come from? :shrug:


This was aspirating the LC from the donor jar, and then to inoculate the receiving jar all I'm doing is one again taking the freshly opened needle and syringe from a sterile packaging that I've used seconds ago to aspire the LC and seconds later I'll be lifting the other lid on another newly sterilized jar and then stab the needle once again through a sterile SHIP to inoculate the broth inside. The fact of the matter is there is nothing that makes syringes and SHIPS more prone to contamination than other methods its all about doing sterile work properly imo :thumbup: if anything syringes and ships are safer than pouring because of the minimal surface area that gets exposed to unsterile air.


And yes I'd feel comfortable inoculating with syringes and SHIPS outside of the SAB but I'd never feel comfortable pouring in open air that's for sure :super: (and if not this deductions says it all then Idk what will lol) :lol:


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Edited by Mateja (11/23/20 04:35 AM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27053421 - 11/23/20 06:00 AM (3 years, 2 months ago)

Quote:

Mateah said:
2 broths look mostly clear and 2 look turbid. When did you inoculate?




Which ones? I have a feeling you will say 2 and 4. Inoculated all of them on Nov 7th.

Quote:

Mateah said:
I've used seconds ago to aspire the LC and seconds later




I know English isn't your mother tongue, so very good reasoning here, but "aspire" isn't correct, that should be "aspirate." After looking into it to make sure i was giving you good info, turns out they both come from the same Latin root of aspirare which means "to breathe, to breathe upon," so therefore logically you using "aspire" here (meaning "to aspirate") would normally be correct but for some odd reason, they were split and distinguished from one another. English is so damn weird :lol:


Edit: Updated pictures just for kicks. Broths are in the same order as before.


Edited by MLPismyOPSEC (11/23/20 08:56 AM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27053550 - 11/23/20 08:23 AM (3 years, 2 months ago)

Quote:

Mateah said:
...And yes I'd feel comfortable inoculating with syringes and SHIPS outside of the SAB but I'd never feel comfortable pouring in open air that's for sure :super: (and if not this deductions says it all then Idk what will lol) :lol:




:whathesaid:

I believe the main skill is sterilizing the syringes.I start with non-sterile "industrial" syringes and blunt dispensing needles. Disassemble the syringes.  I made sleeves for the syringes by cutting up a Tyvek suit and sewing into a condom shape. Drop sleeves, needles, syringe bodies and plungers into boiling water and let them boil for a few minutes. Then, pull them out with tongs and assemble the syringes and needles. Set the sleeves aside to cool and dry. Pump boiling water through the needles and syringes several times. After pumping the boiling water through it, each assembled syringe goes into a sleeve, needle first.

When it's time to inoculate, I  put an alcohol-soaked paper towel over source and destination SHIPs.  Then, flame the needle, pump air a bit to cool it, give the source SHIP a final wipe with fresh alcohol, and insert  the needle.

I really like 5 inch needles, because I can flame them without worry about melting any plastic. I see that you can get all-metal dispensing needles and that would be nice (mine have plastic Luer locks). Long needles easily reach the bottom of a pint mason jar.

I use a fresh needle and syringe for each LC source. If inoculating multiple destinations, I flame the needle before each insertion.

This may seem elaborate, but it's still less time consuming than cleaning a SAB, loading it and waiting for it to be ready to use. Especially if inoculating a PC-load of grain jars from multiple LCs. I don't worry about shutting my house HVAC down.


Edited by sonoramo (11/23/20 08:25 AM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: sonoramo]
    #27053561 - 11/23/20 08:28 AM (3 years, 2 months ago)

You can sterilize syringes in the pressure cooker, no skill or disassembling needed.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex] * 1
    #27053571 - 11/23/20 08:35 AM (3 years, 2 months ago)

Yea, sorry to break it to you, you've been wasting a lot of time and effort all this time doing that :lol:

On the bright side, now you know how to do it the easy way.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27053713 - 11/23/20 10:33 AM (3 years, 2 months ago)

Agreed, you can sterilize assembled syringes in the pressure cooker. That can work well if you sterilize them while processing grain or LC jars. But you'll have to keep the syringes in some kind of sterile environment while the grain or LC cools. Don't forget that contaminants can enter the back of the syringe if the plunger is "down." Cooking grain can evaporate a thin layer of nutrients all over the inside of your PC, and that gets into the back of the syringe.

It can get a bit tricky to fit assembled syringes into the PC for people like me who like long needles. If you sterilize with the needle down, that tends to force water (plus stray nutrients) into the syringe as pressure returns to atmospheric. If you let the syringes lean against the PC wall, that will melt plastic. Once I made the mistake of letting syringe needles touch the bottom of the PC. The needles got hot enough to melt the plastic.

So there's still a fair amount of trial and error to get it right.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: sonoramo]
    #27053726 - 11/23/20 10:42 AM (3 years, 2 months ago)

I Wrap my syringes and needles separately in aluminum foil and pc them in quart jars.


Edited by BrownBear (11/23/20 10:42 AM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: BrownBear] * 2
    #27053746 - 11/23/20 10:52 AM (3 years, 2 months ago)



60ml syringes wrapped in foil and a pp5 test tube full of 14G needles wrapped in foil, all sterilized inside a gallon jar.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27053765 - 11/23/20 11:04 AM (3 years, 2 months ago)

This is all good to know, currently I’m working with just pouring in front of the flowhood but planned on making a few lids with injection ports and getting some syringes. Is there a favorite brand / size of syringe? (I’ll utilize the search button as well)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: mushhead]
    #27054499 - 11/23/20 07:23 PM (3 years, 2 months ago)

Quote:

mushhead said:

Left is B+ Right is APE.
LME heat dissolved into distilled water with a 45min PC cycle.
Some sediment but no other microbes save mycelia in here.




B+ (Left) APE (Right)

A couple other LC testing nute levels.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: mushhead]
    #27054525 - 11/23/20 07:41 PM (3 years, 2 months ago)

From my limited knowledge about those broths (only knowing these pics and what you've described so far) I'd say B+ looks turbid APE looks clear. But it's worth remembering that discernment comes from having the collective data on everything from time of inoculation, temp, changes in appearance no matter how slight, timing of the changes, many many small factors that I myself know very little about I'm totally a baby just discovering as I go.


I'm just pointing out that the one factor we have that appears to be a very rigid measuring device (the turbidity phenomena itself) nevertheless eyes can also be tricked, I've already been punked by some broths a few times, so observing them regularly in the hours/days after inoculation tends to provide the most valuable data ime.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27054535 - 11/23/20 07:47 PM (3 years, 2 months ago)

I like these, they look ready to rip into some grain.





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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Pastywhyte]
    #27054542 - 11/23/20 07:52 PM (3 years, 2 months ago)

Quote:

mushhead said:
I've been working on my LC nute levels. Trying to get them as clear as everyone's here.
My LCs are clean, but have sediment because I've been experimenting with nute levels.
My first batch is 5g LME to 500ml distilled water. LOTS of sediment. 
Second is 2.5g LME to 500ml distilled water. Medium-minor sediment.
Third is 1g LME to 500ml distilled water. Mostly none. Small amount visible on bottom of jar.
Fourth is .5g LME to 500ml distilled water. Pretty clear.
Pics after I finish a bunch of errands today.




I PC for 45min each batch of two jars I did. The fourth batch is just the one jar.
They're in succession from first batch to fourth which I'm sure you can tell which has the highest nutes.
You see I believe that the B+ jar isn't contaminated at all and the turbidity comes from the amount of nutes used which is like 5~6g because it was my first one after trying with Gulf coast which worked amazingly well for me.
EDIT: These jars were kept at 22c consistently without deviation.


Edited by mushhead (11/23/20 08:15 PM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Pastywhyte]
    #27054558 - 11/23/20 08:02 PM (3 years, 2 months ago)

Quote:

Pastywhyte said:
I like these, they look ready to rip into some grain.







Pasty I aspire to be upon your level immensely.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: mushhead]
    #27054565 - 11/23/20 08:06 PM (3 years, 2 months ago)

Got a APE syringe in today, first time working with it. But it looks like they germinated...



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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lemgrub]
    #27054581 - 11/23/20 08:16 PM (3 years, 2 months ago)

The pic isn't good but Ape spores kinda look like that. If they take time to show growth on agar (and they usually will) you can be sure it's spores.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lemgrub]
    #27054582 - 11/23/20 08:17 PM (3 years, 2 months ago)

Quote:

Lemgrub said:
it looks like they germinated...





I'm afraid "they" could mean 'several types of organisms' germinating. Both fungus myc and bacteria is what the pic says to me. I wonder if it'd be possible to salvage a part of the myc in there tho, maybe to dilute a small part heavily in sterile water and then to extract a samle to put to agar? What do y'all think about this guys? I'm sure someone has tried something similar before, to cleaning out myc from a bacterial broth lol


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27054625 - 11/23/20 08:44 PM (3 years, 2 months ago)

About as good a pic as I can get. looking closer I can tell there are spores, but it looks a bit more viscous than usual. I nocced some agar and brf cakes and a glob came out in one of the cakes. I've looked at plenty of spores under the scope and I've seen a large mass of spores sort of suspended in a matrix of hyphal cells, I don't know if they're just dead in some way or if they were sterile monokaryotic mutant lineages or something like that.




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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lemgrub]
    #27054658 - 11/23/20 09:28 PM (3 years, 2 months ago)

Looks like APE spores to me.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Pastywhyte]
    #27054730 - 11/23/20 10:49 PM (3 years, 2 months ago)

Tight

Here's a tissue needle biopsy LC I started 11/13 from Colombian Rust. The liquid has retained the same clarity throughout. Believe it's growing somewhat slowly due to higher sugar content, 4%



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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lemgrub]
    #27054744 - 11/23/20 10:58 PM (3 years, 2 months ago)

Are you swirling it? That will break up that glob and speed up colonization. It's being terribly slow regardless, I would not trust it but you can always test it.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lemgrub]
    #27054790 - 11/23/20 11:49 PM (3 years, 2 months ago)

Quote:

Lemgrub said:
Tight

Here's a tissue needle biopsy LC I started 11/13 from Colombian Rust. The liquid has retained the same clarity throughout. Believe it's growing somewhat slowly due to higher sugar content, 4%





I've done side by sides with clones before and a varience in nutrient density didn't seem to affect growtht speed much if at all. At least not with the cultures I tested. The myc colony in your LC is far to small for a 10day old LC, it should really be close to being fully colonized at this point. This raises some concerns. Also (and I could be wrong) but from the pic it doesn't look like the light source is very close to the jar so it's hard to determine how clear/turbid that broth is. The brighter the light is shining on a turbid broth the more turbid it will appear and the less light that is being diffused inside the broth the less turbid it will appear so the amount of light shining through will hugely affect how the broth is perceived ime. And lastly I have to say that personally I can not come up with a reason to use stronger broths than 0.1%.

Adding a massive amount of particles to a LC like 2-4% malt will make it that much harder to inspect since you don't have a 'default' like a crystal clear broth for example in which case you're able to notice the slightest increase in particles (since the broth goes from 'clear' to 'not clear') but with a broth with already tons of particles in it then it's simply not reasonable to assume you'd even be able to notice a change in amount of particles (relying on noticing the change from 'turbidity' to 'more turbidity'. Hope that helps. Gl


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Edited by Mateja (11/23/20 11:53 PM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Pastywhyte]
    #27055222 - 11/24/20 09:53 AM (3 years, 2 months ago)

Quote:

Pastywhyte said:
I like these, they look ready to rip into some grain.








Is the brownish stuff on the bottom sediment? If it is, it seems like a lot of sediment. Like, if you swirled the jar it would make everything look cloudy. Was it there from when you prepared it, or did it precipitate out while the culture grew?

Finally, what recipe did you use for the broth?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27055238 - 11/24/20 10:03 AM (3 years, 2 months ago)

I gotcha. The (very bright)lamp was probably an inch from the cup. I've had a few LCs contaminate since making this and the difference is stark. I don't believe this one is contaminated to this point.

And as far as the solution I used, I quickly looked at a tek that recommended 4%, so I went ahead and did it since the PC was almost ready to go. Seemed high and further reading supports that. And honey isn't too bad particle wise.

Josex, yes I've been swirling a couple times a day. Finally a few days ago some myc broke off from the biopsy and there's separate colonies now. I think the colonization will speed up now.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: sonoramo]
    #27055256 - 11/24/20 10:08 AM (3 years, 2 months ago)

Quote:

sonoramo said:
Quote:

Pastywhyte said:
I like these, they look ready to rip into some grain.








Is the brownish stuff on the bottom sediment? If it is, it seems like a lot of sediment. Like, if you swirled the jar it would make everything look cloudy. Was it there from when you prepared it, or did it precipitate out while the culture grew?

Finally, what recipe did you use for the broth?




The brownish stuff on the the bottom is a giant puck of solid agar. The “broth” is simply sterile water added to the vessel once the colony on the agar has grown out and looks healthy. Use whatever recipe you like, this was just undiluted grain soak water and 2% agar for the puck.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Pastywhyte]
    #27055286 - 11/24/20 10:23 AM (3 years, 2 months ago)

Chucked an agar wedgelet into some LME broth today... It floated... I-is that ok?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: coversall]
    #27055318 - 11/24/20 10:47 AM (3 years, 2 months ago)

Started some enigma on LC last night. T1 from a hot pour (it wasn’t needed just wanted to try it) This is one in a series of side by sides I’m going to try with grainwater and lme. My hypothesis is that the enigma will be more aggressive in grainwater based on its growth patterns on agar vs when it goes to grain. I’m looking forward to seeing the results



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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Pastywhyte]
    #27055550 - 11/24/20 01:38 PM (3 years, 2 months ago)

Quote:

Pastywhyte said:
. . .
The brownish stuff on the the bottom is a giant puck of solid agar. The “broth” is simply sterile water added to the vessel once the colony on the agar has grown out and looks healthy. Use whatever recipe you like, this was just undiluted grain soak water and 2% agar for the puck.




I think I read about that approach someplace else. You might even be the person who posted it. I've considered taking that approach to make an LC that could be renewed by withdrawing some mycelium and then adding more water. Presumably, clean water is easier to prepare than clean broth. Are there other benefits to this approach?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: sonoramo]
    #27058009 - 11/26/20 12:32 AM (3 years, 2 months ago)

It's gettin there



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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lemgrub]
    #27060429 - 11/27/20 03:37 PM (3 years, 1 month ago)

Took a sterile syringe and busted up the big clump today, then made a receiving LC, an agar check, and inoculated a qt jar. Pretty sure it's a clean one and just hoping I didn't contaminate anything during the process. 1st pic is the good LC swirled, 2nd is bacterial unswirled and 3rd is bacterial swirled for reference. Notice the lemongrab eyes behind the jar.



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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lemgrub]
    #27060491 - 11/27/20 04:18 PM (3 years, 1 month ago)

Quote:

Lemgrub said:
1st pic is the good LC




Those three broths looks identical to me, Idk how you concluded that one is clean and two are bacterial, what did you base that on? Also why isn't myc visible in any of the three jars like in this jar that yu posted earlier?
Quote:

Lemgrub said:




Please provide us with more details on these broths, recipe, method/date of inoculation so we can try to figure out what happened to them :super: gl


Edited by Mateja (11/27/20 04:24 PM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27060703 - 11/27/20 07:30 PM (3 years, 1 month ago)

I guess I should have left the good one still. 4% honey, which I already determined is too much.

Both cups unshook. The first one hasn't changed in turbidity since I inoculated it. The second was inoculated a week later and began getting turbid within a day. Maybe it doesn't show up well in pics, or the plastic of the container distorts the clarity but I can see my fingerprint from the other side in the good one.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lemgrub]
    #27060790 - 11/27/20 08:44 PM (3 years, 1 month ago)

Quote:

Lemgrub said:
I guess I should have left the good one still.
Both cups unshook



I think you should swirl your LC's daily and definitely before deciding on how clean it is or before taking pics to post :thumbup: I've even had a homogenized, known bacterial broths suddenly appear to be clear one day, then after a quick swirl it became apparent that the previously suspended and evenly distributed particles had eventually sunk to the bottom. 7 believe that the bacterial cells will remain suspended 'mid-air' inside the broth during log phase but in death phase or towards the end of the stationary phase as the cells begin to die it kind of make sense that gravity will pull the lifeless cells and the non motile endospores to the bottom. Don't quote me on this tho even tho I'm fairly sure this is the case I'm nonetheless partly speculating as well.


I will just add that glass jars and crystal clear broths make it very easy to spot changes in the environment inside the broth, 4% no matter what is too much even for agar and not to mention a liquid Media which could probably be as effective even if it was diluted up to 50times. I currently have broths which are made from hay and straw water diluted 40 times and it seems to work fine :thumbup:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja] * 1
    #27060947 - 11/27/20 11:03 PM (3 years, 1 month ago)

It's good to know that growth does fine with so little carbs. And I've since made some receiving cups for LC at much lower levels and like the clarity/color neutrality better.

As for the containers, I kind of like them, they're cheap, and easy to find. I can tell changes in clarity pretty easily with them. I do have to inspect liquids for particles in my job after all.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lemgrub]
    #27061084 - 11/28/20 04:44 AM (3 years, 1 month ago)

Hows my ape culture look, I inoculated the lc on 11/22 from an ape clone plate through a ship. I just got done agitating it to spread it out. I'm pretty new to the lc game but I'm playing around with it for real the first time. What do guys think of my lc.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: MH5109] * 1
    #27061090 - 11/28/20 05:04 AM (3 years, 1 month ago)



LC corner. The three ones that are ready are Pan cyan. The two bigger ones were nocd with the needle poke tek, the smaller on with just a small chunk of agar. The two newer ones are pan cambo. Both nocd with a small agar wedge.

No stirrers, unmodified lids. Being vigoursly swirled at least once a day.

All LME, 1g per 500ml of water.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: MH5109]
    #27061865 - 11/28/20 04:22 PM (3 years, 1 month ago)

Quote:

MH5109 said:
I'm pretty new to the lc game but I'm playing around with it for real the first time. What do guys think of my lc.




I'm also brand new to the LC game, this is the first time I'm using it as inoculate :shrug: tho I've grown it lots of times over the years to observe it inside jars in different settings/environments and take notes :takingnotes:
About your LC, from my limited perspective the broth looks clear but only you can (learn) to be the definitive judge of that. Grow as many LC's as you can, observe them, compare results, and eventually you'll begin to understand it more and more (but this goes for anything of course) you learn little by little each time you do something until you've freed yourself from needing to appeal to 'authority' for "how does this look". To tell you the truth you shouldn't in any way see me as an authority on this, at least to the extent of your confidence about the verogoty of my findings posted anywhere, that's why I've said multiple times no one should take my word for anything I'm just posting MY results from MY experience of observing whatever I'm observing. If some growers can find value in the results of my experiments then I'm happy to have contributed to helping someone, I'm mostly just a curious person and I like to 'fish out' information and showing others how to fish instead of just offering them a speculative fish for the day by which they don't learn anything new nor I.


I fish usually with my 'side by side' rod cause that's the most basic scientific approach to roughly assess/estimate things early on, it's not the most precise science and definitely not the way I'm conducting experiments but it is in my view enough to point in some general directions and of course some findings will be spot on and so on.. The more you fish the more valuable fishing spots you'll be able to find, me personally I like to fish in big lakes but also in small ponds, knowing there's mostly crabfish and minnows lurking and I don't necessarily even need to catch the giant catfish, it can be enough just to find out where his hiding place is and that can be of great value for fishing some other time in other places. Now I'm just too much with these parables I know I want to puke as well, but my old man was a carpenter so I can't help it :shrug:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27062079 - 11/28/20 07:15 PM (3 years, 1 month ago)

She was fully colonized so expanded it



Did a test on agar which looks really good




Here’s how the expanded cultures look. One grain water one lme



With a lil swirl, I think the lme is performing a tiny bit better. Had more sediment to grow and feed on



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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: D3_Myc]
    #27062127 - 11/28/20 08:05 PM (3 years, 1 month ago)

Quote:

coversall said:




Quote:

D3monic said:




The grid patterns behind the jars is genius and really compliments the inspection of the broth
:coolpost:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27062177 - 11/28/20 09:02 PM (3 years, 1 month ago)

The LC looks clear, as in I can see my fingerprints through the liquid on the other side. Im going to give them another week to build up and inject some test grain jars. I love the idea of lc for speed and stretching a culture out, but I'm sure I'll catch some fish I don't want to keep as with all mycology. Really interesting thread to look through, thanks for making it.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27062369 - 11/29/20 02:20 AM (3 years, 1 month ago)

Quote:

Mateah said:
Quote:

coversall said:




Quote:

D3monic said:




The grid patterns behind the jars is genius and really compliments the inspection of the broth
:coolpost:




Holy shit, I hadn't even noticed! My pan cambo LC is starting to wake up now. I really need something to pour my pan cyan LC on.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: coversall]
    #27062407 - 11/29/20 03:40 AM (3 years, 1 month ago)

Quote:

coversall said:
Quote:

Mateah said:
Quote:

coversall said:




Quote:

D3monic said:




The grid patterns behind the jars is genius and really compliments the inspection of the broth
:coolpost:




Holy shit, I hadn't even noticed! My pan cambo LC is starting to wake up now. I really need something to pour my pan cyan LC on.




The best way to observe turbidity is by having somwthing to look at through the glass. I have used the grid pattern from my filter on occassion. I have also used a box of gloves and a fan.



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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: BrownBear]
    #27062764 - 11/29/20 10:07 AM (3 years, 1 month ago)


APE LC doing well, no turbidity I can sense (other than high nute level sediment)

RW(left) APE-LC#2(right) both performing as expected, though I did find a rogue body within the APE-LC#2 I am unsure of. All the above were done biopsy method.

B+ LC looking very clean.
This is a three day old plate done with three tiny droplets of LC and spread with my inoculating loop, the liquid had no discernable myc I could make out clearly without aid of a microscope. I have made 10(10cc) syringes of this culture and plan to use it after a few more days of observing the above plate.

I have new equipment on the way!
SH2 Stir plate
Multiple sized magnets.
Proper LC vessels made of Pyrex.
Lab grade Pyrex beakers/flasks.
Glass Syringes(20)
Flow Hood by Phenomenal Fungi (just the cheaper $900 one)
Can't wait to start using this Flow Hood, just getting my lab area prepped so I can fit in upon the desk here.


Edited by mushhead (11/29/20 10:14 AM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: D3_Myc]
    #27063895 - 11/30/20 02:25 AM (3 years, 1 month ago)

Quote:

D3monic said:
Did a test on agar which looks really good







How'd you do the test? Such perfect little growths and no streak marks. Syringe?


Edited by verum subsequentis (11/30/20 02:44 AM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: verum subsequentis]
    #27063896 - 11/30/20 02:28 AM (3 years, 1 month ago)

Yea test plates are always a raging mess for me everytime no matter how careful I want to be. That's if I test at all tho.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27063900 - 11/30/20 02:39 AM (3 years, 1 month ago)

Quote:

Josex said:
Yea test plates are always a raging mess for me everytime no matter how careful I want to be. That's if I test at all tho.




I dropped my last test plates, so most of it ended up on the lid

:singletear:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: coversall]
    #27063905 - 11/30/20 02:44 AM (3 years, 1 month ago)

bet it still grew


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: verum subsequentis]
    #27063908 - 11/30/20 02:46 AM (3 years, 1 month ago)

Lol yeah, and the stuff that did land on the agar grew out ok. Clean enough for me at least.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27063914 - 11/30/20 02:50 AM (3 years, 1 month ago)

Great thread. Thank you to the poster.

Is it 100% mandatory to test LC's before use? Or if its looking good just go for it and knock jars?

Lately i think theres some bacteria hiding in my LC's because ill get 1 flush of small weird fruits then it contams with not trich nessecarily, but a dark squishy patch in the sub, followed by white molds. Also the surface of sub isnt wanting to fill out 100% before it starts knotting and pinning.  I used to push upwards of 7 flushes, i think my plates are fucked and I need to restart from spore. :frown:

But im glad i found this thread and Mads write up on Hidden Contams. So I can start correcting all this. :peace:

https://www.shroomery.org/forums/showflat.php/Number/22020260


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Luminous7]
    #27063918 - 11/30/20 02:52 AM (3 years, 1 month ago)

Quote:

Luminous7 said:


Is it 100% mandatory to test LC's before use? Or if its looking good just go for it and knock jars?






Only if you want to be 100% sure that it's clean. Otherwise just pour that shit and cross your fingers.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: coversall]
    #27063919 - 11/30/20 02:53 AM (3 years, 1 month ago)

I test them on grain jars.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27063923 - 11/30/20 02:58 AM (3 years, 1 month ago)

Quote:

Josex said:
I test them on grain jars.



I do as well sometimes. But i worry about comprimising the LC by testing it.


I do just mason jars with syringe filter disks,  and pour my LC to knock grain.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27063930 - 11/30/20 03:10 AM (3 years, 1 month ago)

Quote:

Josex said:
I test them on grain jars.




:whathesaid:

I test it on potential master grain jars. That way, if it fails there isn't much of a loss.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: BrownBear]
    #27063933 - 11/30/20 03:14 AM (3 years, 1 month ago)

Quote:

Iambrownbear said:
Quote:

Josex said:
I test them on grain jars.




:whathesaid:

I test it on potential master grain jars. That way, if it fails there isn't much of a loss.



well if it fails on a potential GM , then you may have ruined your spawn ratio if you planned your tubs out in relation to your jars.

In the 24qt presto u can fit 10 jars as well as a little wide mouth half pint in the top center. SOmetimes i sterilize a little one of those and test LC on that.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Luminous7]
    #27063937 - 11/30/20 03:19 AM (3 years, 1 month ago)

Quote:

Luminous7 said:
Quote:

Iambrownbear said:
Quote:

Josex said:
I test them on grain jars.




:whathesaid:

I test it on potential master grain jars. That way, if it fails there isn't much of a loss.



well if it fails on a potential GM , then you may have ruined your spawn ratio if you planned your tubs out in relation to your jars.

In the 24qt presto u can fit 10 jars as well as a little wide mouth half pint in the top center. SOmetimes i sterilize a little one of those and test LC on that.




I use 1/4 pint jars of rice for masters and cook them 24+ plus at a time. I use one 1/4 pint to inoculate 3 qt grain bags. So to put it simply, I make more masters than what I need. The ones that don't make the cut get mixed into a shoe box grow or something similar.


Edited by BrownBear (11/30/20 03:46 AM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: BrownBear]
    #27063951 - 11/30/20 03:55 AM (3 years, 1 month ago)

Quote:

Iambrownbear said:
Quote:

Luminous7 said:
Quote:

Iambrownbear said:
Quote:

Josex said:
I test them on grain jars.




:whathesaid:

I test it on potential master grain jars. That way, if it fails there isn't much of a loss.



well if it fails on a potential GM , then you may have ruined your spawn ratio if you planned your tubs out in relation to your jars.

In the 24qt presto u can fit 10 jars as well as a little wide mouth half pint in the top center. SOmetimes i sterilize a little one of those and test LC on that.




I use 1/4 pint jars of rice for masters and cook them 24+ plus at a time. I use one 1/4 pint to inoculate 3 qt grain bags. So to put it simply, I make more masters than what I need. The ones that don't make the cut get mixed into a shoe box grow or something similar.



Interesting , never thought to do something like that. Cool idea!

do you use LC ?

Lately iv been making mass amounts of LC, like a few litres at a time, lets say 4000ml. Ill use quart mason jars and do 500ml per jar.

Im starting to wonder if i should just be doing g2g instead of mass amounts of liquid culture.  LC seems like more potential for error than G2G. I havnt done much G2G work.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Luminous7]
    #27063957 - 11/30/20 04:19 AM (3 years, 1 month ago)

Yes. I use lc. I was growing lc in quart jars but have recently switched to the pint jars. I grow a few lc's of each culture and use the best one out of each culture to inoculate master jars. I use 2.5 cc of lc per 1/4 pint of rice. I can inoculate a little over 5 dozen jars with one pint jar of lc.

I like both lc and g2g. I do not like lc for inoculating large amounts of grain but it is quite useful for small amounts. I like g2g for large amounts of grain because the recovery time is a lot faster, ime, with g2g than it is for lc.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: BrownBear]
    #27063999 - 11/30/20 05:28 AM (3 years, 1 month ago)

My peu and ape cultures are looking clear, here in the next few days they will be going to test jars
[/tex]


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Edited by MH5109 (11/30/20 05:28 AM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: MH5109]
    #27066556 - 12/01/20 03:04 PM (3 years, 1 month ago)



This is an expansion of my first agar-QCd LC.

I used 12cc of the original 8 days ago.

I would appreciate any thoughts or opinions. I am making agar today and will start QC plates tomorrow.

Broth is 1% LME/2% Light Corn Syrup.

I also injected a test run with the source LC that already passed agar QC.



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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: nmd_myco]
    #27066611 - 12/01/20 03:33 PM (3 years, 1 month ago)

That lc looks real nice. How many cc's did you inject into each bag?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: BrownBear]
    #27067844 - 12/02/20 09:26 AM (3 years, 1 month ago)

some of them are in 2 lb bags and some in 3lb bags. They got 6 and 9cc respectively. I was cautioned by a vendor that the biggest issue users ran into was squirting a whole spore syringe in a

This is part of a set of experiments involving sterilized grain from a pool of 7 providers.  I am running the four lowest cost providers against each other first.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: nmd_myco]
    #27067877 - 12/02/20 09:41 AM (3 years, 1 month ago)

B+ LC tested 100% clean, the agar dish is saturated with thick white myc.
Yesterday I expanded the culture into 12 rye jars, two 5LB bags of hpoo, and 7 BRF jars.
All have recovered over night and are moving fast.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: mushhead]
    #27070174 - 12/03/20 02:32 PM (3 years, 1 month ago)

here's the source and QC plate for that last one i posted. its a bit late to see how clean the QC plate is.


source plate


QC plate


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: D3_Myc]
    #27070530 - 12/03/20 06:39 PM (3 years, 1 month ago)

Quote:

D3monic said:

Did a test on agar which looks really good







What method did you use to get it to agar to test?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Sold Out Online]
    #27070544 - 12/03/20 06:48 PM (3 years, 1 month ago)

I just make myself a 10cc syringe of LC and test it, if its good I use it.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: mushhead]
    #27071226 - 12/04/20 05:32 AM (3 years, 1 month ago)

I've got three pan cyan LCs that I made on the 10th of November. They have obviously been ready to pour for while. There was a hold up getting my hpoo, which I am going to finally go and dig up the muck heap on Monday.

Will these LCs be ok to pour? Or should I just start from scratch??


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: coversall] * 1
    #27071671 - 12/04/20 11:40 AM (3 years, 1 month ago)

Colonized LC's can store for a long time in room temp and in the fridge, as long as they've not been compromised. (I don't know this personally but I've seen several growers post about  having good luck with inoculating with LC's stored at room temp for months and months)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja] * 1
    #27071691 - 12/04/20 11:52 AM (3 years, 1 month ago)

I sometimes let them get super thick and just pour globs of mycelium right into the bag. It often recovers in less than a day in those instances.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Pastywhyte]
    #27071802 - 12/04/20 12:45 PM (3 years, 1 month ago)

Ace!


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: coversall]
    #27071813 - 12/04/20 12:52 PM (3 years, 1 month ago)

I just used a month+ old LC that sat at room temp
300 or so ml in a media bottle, poured to 32 quarts around midnight on the 3rd
I hope to see recovery today when I get home, around 5pm will be 41 hours since inoculation


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Pastywhyte]
    #27071828 - 12/04/20 12:57 PM (3 years, 1 month ago)

Quote:

Pastywhyte said:
I sometimes let them get super thick and just pour globs of mycelium right into the bag. It often recovers in less than a day in those instances.



:wellhello:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27075372 - 12/06/20 02:08 PM (3 years, 1 month ago)

it has been almost 3 days since a noced 1g lme 250ml of water. this is my second try at a broth. I just started stirring these 2 days ago.



Edited by kanemush (12/06/20 02:09 PM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Sold Out Online]
    #27075456 - 12/06/20 02:56 PM (3 years, 1 month ago)

Quote:

cdnpsychonaught said:
Quote:

D3monic said:


What method did you use to get it to agar to test?





I just poured a little onto a petri lid and then hit it with the loop

Here’s my expanded cultures, going to send some more to grain and expand again in a day or two.



Looks like lme has been a clear winner in all my tests though I do like the grain water as well.



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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: D3_Myc]
    #27084443 - 12/11/20 05:54 PM (3 years, 1 month ago)

I decided to add a tiny bit of agar to a broth I used for LC to see how it would turn out.

I inoculated this on 12/8 from a tissue biopsy LC that showed to be clean after a agar test a while ago.

The agar is so loose in this LC it looks close to bacterial, but keeps the mycelia suspended for a while after a few swishes which may help it grow:ohwell:

I used about 1ml of the other LC and could barely tell there was any myc in there at first, seems to be growing pretty fast.



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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lemgrub]
    #27084928 - 12/12/20 01:25 AM (3 years, 1 month ago)

So I decided to give LC a go and am pretty pleased with this so far;



It's growing in a uniform ball shape, do I need to try and break this up with a swirl or just let it do it's thing?

You can't see it from this pic but the myc is growing of a dog hair that accidentally got PC'd in the bottle lol.

I know distilled water would give me a clearer medium but I said I'd just give it a go with what's on hand first to see if I like working with it.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: The Dalcassian] * 1
    #27084931 - 12/12/20 01:33 AM (3 years, 1 month ago)

That Jellyfish needs shook! Needed it days ago. I have a hard time watching that thing, my ocd kicks in lol it's so big I feel like reaching through screen and shaking the shit out of it.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27084945 - 12/12/20 01:49 AM (3 years, 1 month ago)

Oh crap lol, ok I'm on it.

You have made me chuckle tho, thanks:laugh2:

Edit; Sorted, although now it floats



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Edited by The Dalcassian (12/12/20 02:00 AM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: The Dalcassian]
    #27084950 - 12/12/20 01:58 AM (3 years, 1 month ago)

It's looks healthy man, that's the important thing. :cheers:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27084955 - 12/12/20 02:06 AM (3 years, 1 month ago)

Thanks dude I look forward to getting to use it.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex] * 1
    #27084959 - 12/12/20 02:12 AM (3 years, 1 month ago)

I also made a APE LC from a agar chunk on a MS plate 12/2. The main chunk stuck to the side of the wall of the cup but it's been seeding off new colonies.



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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lemgrub]
    #27089314 - 12/14/20 06:18 PM (3 years, 1 month ago)

Thoughts on an LC that grows very slowly? I recently discarded an LC that grew but was just very slow. Could this be a sign of contamination or just genetics?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: bongoman]
    #27089384 - 12/14/20 07:05 PM (3 years, 1 month ago)

slow/poor recovery is a bad sign for anything no matter the media.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: mushboy]
    #27092144 - 12/16/20 01:04 PM (3 years, 1 month ago)

Commenting mainly to follow up later...

  • I made two .4% LME (1g to 250ml deionized water) and PC'd for 17 minutes @ 16-17 PSI on 12/13.
  • Inoculated both with Josex poke method on 12/14 using my best (AC) plate.
  • I let them sit for a day before spinning a couple hours each on low yesterday and today.
  • Just seeing cloudy sediment so far and some random detritus that was probably in the medium during PC (hopefully).






Still not sure how to get good pictures of these... maybe bottom lighting instead?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27092160 - 12/16/20 01:09 PM (3 years, 1 month ago)

Running low on plates so I just used some peu lc without testing first. :rockon:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Stipe-n Cap]
    #27092667 - 12/16/20 05:50 PM (3 years, 1 month ago)

Ziploc sandwich bag agar. I had a little bit of agar left after pouring 40 plates. I didn't want to waste the agar so I thought about what I wanted to do with the rest of it. I remembered a friend of mine telling me a few years ago about ziploc bags being packaged sterile and that one could use them for pouring agar. It actually works really well.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: BrownBear] * 1
    #27092911 - 12/16/20 07:53 PM (3 years, 1 month ago)

I use ziploc sandwich bags in lieu of wrapping poured plates.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Pastywhyte]
    #27092932 - 12/16/20 07:59 PM (3 years, 1 month ago)

That sounds about ez, would hate to wrap plates myself, main reason I never got started with petris.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: GrinchGrower]
    #27092938 - 12/16/20 08:01 PM (3 years, 1 month ago)

I believe that with the josex poke, you should give it ample time to recover before spinning.
You see the sample is so microscopic that spinning it too early could possibly kill the culture.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: mushhead]
    #27092952 - 12/16/20 08:07 PM (3 years, 1 month ago)

spinning early makes no sense, it's counterproductive IMO. It's as if you shook a jar when the wedge has barely got time to leap off.

Good all swirling works pretty good tho, can't see no good reason to dick around with stirrers even tho I dick around with stirrers.  :wow:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27092973 - 12/16/20 08:15 PM (3 years, 1 month ago)

Stirred a culture two days after using your method and killed it.
Good news is I got to reuse the LC LOL :shrug:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: mushhead]
    #27093058 - 12/16/20 08:56 PM (3 years, 1 month ago)

I'll let them sit longer next time.

Blue Helix mentioned that spinning early might cause issues, but recommended just letting it sit a day or two.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: GrinchGrower]
    #27093097 - 12/16/20 09:15 PM (3 years, 1 month ago)

Lookin good. 5 day difference. 1ml LC to ~400ml broth 11/9


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lemgrub]
    #27099185 - 12/20/20 11:26 AM (3 years, 1 month ago)


Just wanted to share progress on this lc. I stole pasty's idea and made up a no-pour agar vessel, colonized it with my PESA culture, and added some sterile low nute LME LC.


Edited by mushhead (12/20/20 04:00 PM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: mushhead] * 1
    #27099201 - 12/20/20 11:38 AM (3 years, 1 month ago)

I took a transfer from that liquid culture streak test I posted above... doesn’t get much sexier than this



LC back to agar isnt so bad thats for sure


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: D3_Myc]
    #27099263 - 12/20/20 12:27 PM (3 years, 1 month ago)

:aweyeah::highfive1:
Gorgeous!


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: mushhead]
    #27101307 - 12/21/20 05:25 PM (3 years, 1 month ago)

'


Tex Orange Cap LCs inoculated with Josex poke looking decent. The second one is a bit more difficult to break up just by hand swirling, don't know what I'm gonna do when it's actually time to pour these but oh well lol. Need to test them first anyways. Really enjoying this first try at LC though. :rockon:


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Edited by Lenz (12/21/20 05:25 PM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lenz]
    #27101349 - 12/21/20 06:06 PM (3 years, 1 month ago)

I left my peu lc on the stir plate for what was supposed to be 10min and I fell asleep and then went to work, which turned into almost 20hrs. She was pretty minced up definitely mixed.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: MH5109]
    #27129402 - 01/05/21 10:21 PM (3 years, 22 days ago)

How many quart jars of grain should 500ml of thick LC colonise?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Luminous7]
    #27129410 - 01/05/21 10:29 PM (3 years, 22 days ago)

Maybe you would not want to prep enough grain for that much LC. 5ml per quart jar is already a generous amount of LC, so you could knock up 100 jars with a 500ml LC.
Want to stretch it out a bit? Do 1ml per jar and inoculate the fucking world.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27129425 - 01/05/21 10:38 PM (3 years, 22 days ago)

Quote:

Josex said:
Maybe you would not want to prep enough grain for that much LC. 5ml per quart jar is already a generous amount of LC, so you could knock up 100 jars with a 500ml LC.
Want to stretch it out a bit? Do 1ml per jar and inoculate the fucking world.




At 5ml a jar will it colonise in 10-13 or so days though?

I have been using about 20-25ml per jar, its a bit wet but colonises in 10 days give or take.

Im starting to think this is wasteful. lol


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Luminous7]
    #27129436 - 01/05/21 10:46 PM (3 years, 22 days ago)

It behoves you wait a bit more for full colonization and not throw off the moisture content of your jars imo. 20-25 is definitely a lot for jars, some people do less than that for bags.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27129440 - 01/05/21 10:48 PM (3 years, 22 days ago)

Quote:

Josex said:
It behoves you wait a bit more for full colonization and not throw off the moisture content of your jars imo. 20-25 is definitely a lot for jars, some people do less than that for bags.



i see. Safer on the dryer side eh?

WIll it still colonise in a half decent time that low?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Luminous7]
    #27129456 - 01/05/21 10:55 PM (3 years, 22 days ago)

I'd say 10-13 days is pretty normal from 5ml. In summer I've seen jars done in as little as 8 days from 5ml and it's not rare to see jars done in 6 days . As soon as temps go down colonization can slow down a lot too. Make sure that your grains are correctly hydrated and it'll all colonize faster. I've seen people recommending prepping grains on the dry side just so they could use more LC, this is a terrible thing to do.


Edited by Josex (01/05/21 11:04 PM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Josex]
    #27129507 - 01/05/21 11:37 PM (3 years, 22 days ago)

Quote:

Josex said:
I'd say 10-13 days is pretty normal from 5ml. In summer I've seen jars done in as little as 8 days from 5ml and it's not rare to see jars done in 6 days . As soon as temps go down colonization can slow down a lot too. Make sure that your grains are correctly hydrated and it'll all colonize faster. I've seen people recommending prepping grains on the dry side just so they could use more LC, this is a terrible thing to do.




Word, I think im starting to see where Iv been a bit off myself.

THanks for the insight dude . :peace: :salute:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Luminous7]
    #27151429 - 01/16/21 12:40 PM (3 years, 11 days ago)

Probably a dumb question but does an LC need GE?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: The Dalcassian]
    #27151478 - 01/16/21 01:03 PM (3 years, 11 days ago)

None of my bottles have any GE and seemed to colonize okay.
Just poured some on grain and streaked to agar yesterday, so I guess we will see...


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: GrinchGrower]
    #27151503 - 01/16/21 01:17 PM (3 years, 11 days ago)

Only curious as I've had a pan jar nocc'd with LC doing nothing for nearly two weeks then I shook it. Found there was a tiny bit of colonisation in the center which has since exploded with growth.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: The Dalcassian]
    #27151513 - 01/16/21 01:28 PM (3 years, 11 days ago)

My first go at LC worked out pretty well and they were all in jars with unmodified lids. I did loosen the lids off after swirling.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: The Dalcassian] * 1
    #27151518 - 01/16/21 01:31 PM (3 years, 11 days ago)

LC's do need some "GE" but the air left in the bottle is more than enough. I usually make 200mL of LC in a 250mL media bottle and that has been plenty enough air.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: MLPismyOPSEC] * 1
    #27151522 - 01/16/21 01:34 PM (3 years, 11 days ago)

Nice, I treated myself to a bunch of mini media bottles to make LC in and didn't want to mess up their lids.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: coversall]
    #27151530 - 01/16/21 01:43 PM (3 years, 11 days ago)

I did my first test using a trypsinizing flask w/ side arm and a 250ml media bottle.
The flask makes pouring really easy, but I wish I had a way to flame the neck/rim inside my SAB.


Think that is better suited for flow hood work though unless I want to blow myself up.
At least the caps are all graded for heavy sterilization in the PC...


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: GrinchGrower]
    #27159394 - 01/20/21 11:49 AM (3 years, 7 days ago)

Need some advice...
I poured a splash of this to grain and a streak to agar five days ago.



I am not seeing any growth on either medium, and this flask did not show visible colonization until AFTER my pour/streak when I gave it a good shake and placed it back on the shelf.

Yesterday I noticed small roundish globules of mycelium floating around, so I put it on the mag plate for just a second to break it all up.

Today it looks like they consolidated into more of what I am used to seeing in other LCs here....

Is it possible to tell if the the mycelium looks healthy now, or if I picked up something nasty in my SAB during the first test?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: GrinchGrower]
    #27159648 - 01/20/21 01:51 PM (3 years, 7 days ago)

Quote:

GrinchGrower said:
I did my first test using a trypsinizing flask w/ side arm and a 250ml media bottle.
The flask makes pouring really easy, but I wish I had a way to flame the neck/rim inside my SAB.


Think that is better suited for flow hood work though unless I want to blow myself up.
At least the caps are all graded for heavy sterilization in the PC...




I like this.
:takingnotes: Added to the list of items needed for LC.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: GrinchGrower]
    #27160191 - 01/20/21 07:13 PM (3 years, 7 days ago)

Quote:

GrinchGrower said:
Need some advice...
I poured a splash of this to grain and a streak to agar five days ago.



I am not seeing any growth on either medium, and this flask did not show visible colonization until AFTER my pour/streak when I gave it a good shake and placed it back on the shelf.

Yesterday I noticed small roundish globules of mycelium floating around, so I put it on the mag plate for just a second to break it all up.

Today it looks like they consolidated into more of what I am used to seeing in other LCs here....

Is it possible to tell if the the mycelium looks healthy now, or if I picked up something nasty in my SAB during the first test?




Thats basically why I dont bother testing mine. Just opens up another Vector of contamination.

I take all the precautions possible while making the LC and if it looks clean i use it.
So far this has been working for me.

Best of luck!


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27293817 - 05/04/21 11:17 AM (2 years, 8 months ago)

Here is a mold LC, inoculated from clean GT mycelium via josex poke through a commercially produced SHIP:


After testing on grain:


I honestly didn't expect it to be mold, since it didn't float or sporulate.

In retrospect, I'd say I could see the following differences:

  • The broth had been cloudy and went clear. I thought it was bacterial, but it was also very dense grain water. I'm not sure at this time if it was sediment or bacteria.
  • colonization speed was faster than expected. In 12 hours the blob went from small little ball to 3/4 inch streak.
  • Some mycelium stuck to the glass and won't come off with a shake. I don't think I've ever seen mushroom mycelium do that.



I believe this failed LC is due to lid failure.

If you use plastic lids, always use gaskets, and don't buy the cheap thin red ones. Get the thicker clear silicone ones with a groove in them, from Amazon. The red gaskets are too thin and  difficult to position properly, and then they fall off when you screw the lid. After contaminating your jar several times to remove and replace lid to adjust the red gaskets, they then get brittle and fail in the pc. This is even worse if you are making your LC by opening the jar and transferring as opposed to using a SHIP. I wouldn't recommend opening an LC jar that used a thin red gasket at all. They are too hard to reseat in the sab.


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Edited by karri0n (05/04/21 11:18 AM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: karri0n]
    #27294702 - 05/04/21 09:25 PM (2 years, 8 months ago)

The whole point of this method is to use as crystal clear broth as possible so that you can easily determine when turbidity is occurring (Turbidity=bacterial colony present). So with that being said, to use a cloudy broth would totally defeat the purpose of this type of inspection.



I suggest using a 'control jar' which makes inspection much easier. A control jar is just a broth which hasn't been inoculated. Notice how easy it is to spot which LC's have gone bacterial!




Also for this specific method of inspection I'd advise inoculating with an agar wedge and what this does is allows you inspect the Cubensis culture as it's growing out from the agar wedge and this will give you an idea of how the cube culture looks and acts in the broth and this will enable you to easier spot potential mold colonies growing. And you have several types of white mold which will not sporulate visibly on neither plates nor in broths so don't bother looking for green sporulation as a means of detecting mold. In fact molds will always be able to blend in with healthy Cubensis mycelium and especially if you have a pretty far grown LC that only recently has been infected with mold. Gl


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27608651 - 01/06/22 04:59 AM (2 years, 21 days ago)

This thread is a really good reference point, both visual and contextual. Thanks for starting it and keeping it updated. :sogood:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: JohnS0N]
    #27689841 - 03/10/22 02:20 PM (1 year, 10 months ago)

Just wanted to bump this, and I've updated the OP as well.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja] * 1
    #27689848 - 03/10/22 02:30 PM (1 year, 10 months ago)

Quote:

Mateja said:
The whole point of this method is to use as crystal clear broth as possible so that you can easily determine when turbidity is occurring (Turbidity=bacterial colony present). So with that being said, to use a cloudy broth would totally defeat the purpose of this type of inspection.



I suggest using a 'control jar' which makes inspection much easier. A control jar is just a broth which hasn't been inoculated. Notice how easy it is to spot which LC's have gone bacterial!




Also for this specific method of inspection I'd advise inoculating with an agar wedge and what this does is allows you inspect the Cubensis culture as it's growing out from the agar wedge and this will give you an idea of how the cube culture looks and acts in the broth and this will enable you to easier spot potential mold colonies growing. And you have several types of white mold which will not sporulate visibly on neither plates nor in broths so don't bother looking for green sporulation as a means of detecting mold. In fact molds will always be able to blend in with healthy Cubensis mycelium and especially if you have a pretty far grown LC that only recently has been infected with mold. Gl



Wise idea.

I also like to keep my freshly knocked LC's unagitated for the first week or so , you can easily see the inoculation growing , and see clean broth all around it.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Luminous7] * 1
    #27690511 - 03/11/22 03:43 AM (1 year, 10 months ago)

Quote:

Luminous7 said:
Quote:

GrinchGrower said:
Need some advice...
I poured a splash of this to grain and a streak to agar five days ago.



I am not seeing any growth on either medium, and this flask did not show visible colonization until AFTER my pour/streak when I gave it a good shake and placed it back on the shelf.

Yesterday I noticed small roundish globules of mycelium floating around, so I put it on the mag plate for just a second to break it all up.

Today it looks like they consolidated into more of what I am used to seeing in other LCs here....

Is it possible to tell if the the mycelium looks healthy now, or if I picked up something nasty in my SAB during the first test?




Thats basically why I dont bother testing mine. Just opens up another Vector of contamination.

I take all the precautions possible while making the LC and if it looks clean i use it.
So far this has been working for me.

Best of luck!




I'm in this camp too. I 'test' my LC on cakes. haha. If it works then aweomse. If it was dirty then boo I wasted some BRF. But it wasn't much more work than pouring plates and if it's clean then I am one step ahead.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja] * 1
    #27690990 - 03/11/22 12:06 PM (1 year, 10 months ago)

Quote:

Mateja said:
I suggest using a 'control jar' which makes inspection much easier. A control jar is just a broth which hasn't been inoculated. Notice how easy it is to spot which LC's have gone bacterial!






Nice work.

This is one of the benefits of low nutes for sure, clarity of broth for visual inspection. I ran a test once to see how quickly I could visually ID bacteria with 1% versus .1%, i believe it was 4 days with the .1% whereas the 1% was too cloudy  to begin with.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Stipe-n Cap]
    #27691019 - 03/11/22 12:19 PM (1 year, 10 months ago)

good thread everyone that works with LC should check it out.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: sandman420]
    #27691858 - 03/11/22 10:55 PM (1 year, 10 months ago)

So LC broth only needs .5% lme and it will fully colonize?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: san pedro guy]
    #27691874 - 03/11/22 11:14 PM (1 year, 10 months ago)

You mean 0.05%? Yes I've been using this very low ratio a bunch of times and it never seemed to hinder any of my LC's from colonizing normally :thumbup:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27691877 - 03/11/22 11:23 PM (1 year, 10 months ago)

uhhh yea that’s exactly what i meant haha 😅

Lc seems so much faster than a wedge into grain jars…

If you drop a wedge into grain and another into lc at the same time. Then wait for the lc to colonized to use in a grain jar which one will finish first? I’m talking only a small amount of lc, to colonize a single jar….


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Offlinesonoramo
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: san pedro guy]
    #27692234 - 03/12/22 09:10 AM (1 year, 10 months ago)

Quote:

san pedro guy said:
uhhh yea that’s exactly what i meant haha 😅

Lc seems so much faster than a wedge into grain jars…

If you drop a wedge into grain and another into lc at the same time. Then wait for the lc to colonized to use in a grain jar which one will finish first? I’m talking only a small amount of lc, to colonize a single jar….




I'm not clear whether this was intended as a question. A grain jar inoculated with 20ml of LC will colonize really fast, and will definitely catch up to the agar wedge jar if you don't wait for the agar test. Even more so if you don't shake your grains.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: sonoramo]
    #27692245 - 03/12/22 09:23 AM (1 year, 10 months ago)

Quote:

sonoramo said:
Quote:

san pedro guy said:
uhhh yea that’s exactly what i meant haha 😅

Lc seems so much faster than a wedge into grain jars…

If you drop a wedge into grain and another into lc at the same time. Then wait for the lc to colonized to use in a grain jar which one will finish first? I’m talking only a small amount of lc, to colonize a single jar….




I'm not clear whether this was intended as a question. A grain jar inoculated with 20ml of LC will colonize really fast, and will definitely catch up to the agar wedge jar if you don't wait for the agar test. Even more so if you don't shake your grains.




Yea, it was meant as a question. That’s kind of what I thought. LC colonizes really fast and it colonizes the grains super fast, I don’t get why anyone would do wedges unless they have some contam issues maybe….?

Think I’ll make a bunch of tiny lc jars today and save one control, i like that idea.


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InvisibleLegionOfShroom
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27696120 - 03/15/22 09:47 AM (1 year, 10 months ago)

Thanks to everybody that has contributed to this thread, especially Mateja! This has been a great resource for me to learn from.

I made a 0.15% LME LC and inoculated with an agar wedge on 12/14. I stirred it a few times then left it sitting at room temp until 3/11.

On 3/11 I made another 0.15% LME and inoculated it with 6ml of LC from the jar that was made on 12/14. I’ve stirred the jar that was inoculated 3/11 once per day with a magnetic stir bar.

I’d like to use the 3/11 jar to inoculate 10+ grain bags, it would be my first time inoculating grain with an LC. I’ve had a great success rate inoculating bags with agar recently, but I’m interested in LC for faster colonization times.

I’m looking for feedback on the pictures that I’ve included at the bottom of this post. Also opinions on if it’s ok to move forward with this culture that sat on the shelf at room temp for about 90 days

I understand that it’s best to store your LC in the fridge once it’s colonized, I just forgot about it and made a mistake this time

Inoculated 12/14 (pint jar)
   


Inoculated 3/11 (quart jar)
 

I don’t have a control jar, after reviewing this thread I’ll probably start keeping one around

(note: the surface of the pint jar is more uneven than the quart jar, this distorts the light more and makes it harder to see objects through the liquid clearly during a turbidity test)


2% agar 2% LME test plate inoculated 3/11. This test plate was made immediately after inoculating the quart jar

   

The recovery in the petri dish seems a bit slow to me, but it looks like mycelium is starting to grow and I don’t see anything that looks like bacteria

All pictures in this post were taken today 3/15


Edited by LegionOfShroom (03/15/22 12:16 PM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: LegionOfShroom]
    #27696503 - 03/15/22 03:35 PM (1 year, 10 months ago)

:splooge:


That was a clean ass post in all regards, from the way you take pics to the LC recovering on plates, not to leantion your clean looking work space :thumbup:



Also you hardly even need control jars IMO when you know what to look for. An LC won't ever be 'slightly bacterial' it will either be completely turbid and you won't be able to see through the jar or it will remain crystal clear. Altho I'd advice everyone trying this out for the first time to use a control jar, especially those with a very limited experience and equipment unlike yourself :super:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27696837 - 03/15/22 08:31 PM (1 year, 10 months ago)

Thanks so much, I’m very fortunate to have a dedicated space and good equipment to work with. I put a lot of care into my posts because I try to get the most out of these interactions. After posting I had second thoughts about if this was even the right place to post all of that stuff

This community has helped guide me through every significant milestone in my journey since I started my first PF Tek inside a SAB with some spore syringes back in 2017

I’m not always active here on a regular basis, but whenever I return it never fails to impress me how much knowledge is exchanged and how many talented individuals participate here

Thanks again Mateja and shoutout to everybody that helped me along the way


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: LegionOfShroom]
    #27696846 - 03/15/22 08:35 PM (1 year, 10 months ago)

And posters like yourself inspire me to put in more work into my own posts and to lvl up my equipment game for sure :thumbup:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27697118 - 03/16/22 12:10 AM (1 year, 10 months ago)

Quote:

justanotherhuman said:
Short version: does this rhizzy AF plate look bacterial to you? The culture is a clone of the pin in the center btw





You know what to do to test if it's bacterial or not :popcorn:


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OfflineSpectreOfCommunism
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27699411 - 03/17/22 08:31 PM (1 year, 10 months ago)

Hey Mateja, is it really 0.05% ME by weight? That seems extremely low to me — for example 0.05% of 250ml water would be just 125mg


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: SpectreOfCommunism]
    #27699490 - 03/17/22 09:25 PM (1 year, 10 months ago)

Weighing out 0.05% of LME is tricky enough just aim for inbetween 0.1 and 0.05 and you'll be good :super: idk about y'all's LME but mine comes out pretty dark even at 0.1 that's why I do 0.05% :shrug:


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Edited by Mateja (03/17/22 09:27 PM)


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja] * 1
    #27699765 - 03/18/22 05:34 AM (1 year, 10 months ago)

I do 10 times as much at 0.5% 18 grams to a gallon. Looks like this


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: sandman420] * 1
    #27699807 - 03/18/22 06:41 AM (1 year, 10 months ago)

I normally go 0.2%. This is 0.6g in 300ml



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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: SwabMarley]
    #27699814 - 03/18/22 06:48 AM (1 year, 10 months ago)

I feel like dabbling in the arts of LC but don't have lme at hand, what is a good ratio for a honey or corn syrup recipe? 4%?


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: dyel]
    #27700180 - 03/18/22 01:05 PM (1 year, 10 months ago)

Idk why honey or corn syrup are supposed to be any different from LME? I'll be doing some 0.1% honey and 0.1% white sugar LC next time just to see what's up :takingnotes: I don't think anything unusual gonna be 'up' suga=suga


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27936122 - 09/05/22 08:31 PM (1 year, 4 months ago)

Should an agar wedge float or sink in an LC or does it matter?

I thought I read somewhere that floating was a bad sign for some reason but I can't seem to find much searching for LC and wedges floating vs sinking so I'm not sure where I read it or if I'm misrembering whatever I read


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: wonderousovoid]
    #27937061 - 09/06/22 11:21 AM (1 year, 4 months ago)

large pieces of agar will float, and small pieces will sink. if left undisturbed the myc will colonize the surface and start pinning.


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: SwabMarley]
    #27947614 - 09/12/22 10:49 PM (1 year, 4 months ago)

Quote:

Myc Hunt said:
I normally go 0.2%. This is 0.6g in 300ml






Would it be essntial to add higher ratio of sugar if you want the LC jar to chill for a while? or is it possible the culture could stall out and die from lack of food?

I Have some LC's that are over a year old, stored at room temp and they looks alive and well. Ive had success with them to.

I was adding like 1-2 g per 500ml broth for the longer term ones, its a nice way to save cultures i beleive. They last incredibly long and you can just forget about them and have good LC at any time. I could imagine the shelf life would be alot longer if stored in the fridge. :peace:


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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Luminous7]
    #27947674 - 09/13/22 01:07 AM (1 year, 4 months ago)

I've been rocking LC since my first successful grow. That was over 10 years ago now...crazy, time flies. After a couple failed attempts at PF tek I actually had spores germinate in a vendor syringe, and that ended up going into a 4% corn syrup LC I made. Put it to wheat and spawned into a compost mix I used to use and got my first fruits. I actually had pretty good results going spore syringe to LC back then, but also a few failures here and there. I use less sugar and prints/swabs and agar now, but...

I've definitely noticed that bacterial LC gets cloudy really fast too. I've found that the green spore molds typically float to the top in a couple weeks or less and sporulate on the surface. I've been tricked by a black mold a couple times, even though something didn't seem quite right with the mycelium (extra whispy with odd clumps, and myc that stuck to the glass). Those cases, the myc grew way too fast on grains and turned black pretty quick.

I used to use metal lids with a hole sealed with red RTV silicone, but use plastic lids with a injection port and syringe filter and clear silicone ring seal now. I've found colonization speed is similar, but the cultures last longer without the filter. I had some LC still do well after 3 years or so at room temp back when I was using the metal lids with no filter. I'm trying out long term culture storage with .1% malt LC in capped syringes in the fridge now, I think it should work pretty well.

Ok, I'm done grandpa rambling now...cool thread, I love LC!


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