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Invisiblecoversall
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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 10:58 AM (1 year, 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 03:10 PM (1 year, 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 03:21 PM (1 year, 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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InvisibleMateja
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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 06:40 PM (1 year, 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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OfflineMLPismyOPSEC
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27052559 - 11/22/20 06:59 PM (1 year, 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 07:04 PM)


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InvisibleMateja
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: D3_Myc]
    #27052570 - 11/22/20 07:03 PM (1 year, 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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InvisibleMateja
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: MLPismyOPSEC]
    #27052574 - 11/22/20 07:06 PM (1 year, 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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InvisibleD3_Myc
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27052765 - 11/22/20 08:34 PM (1 year, 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 09:27 PM (1 year, 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 09:30 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 09:42 PM (1 year, 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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InvisibleLenz
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: AtmozFear]
    #27052948 - 11/22/20 10:43 PM (1 year, 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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InvisibleMateja
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lenz]
    #27052968 - 11/22/20 10:57 PM (1 year, 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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InvisibleLenz
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27053324 - 11/23/20 05:51 AM (1 year, 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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InvisibleMateja
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Lenz]
    #27053343 - 11/23/20 06:31 AM (1 year, 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 06:35 AM)


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OfflineMLPismyOPSEC
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Re: LC Training Camp: Interpreting Visual Cues To Predict The Quality Of A Broth [Re: Mateja]
    #27053421 - 11/23/20 08:00 AM (1 year, 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 10:56 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 10:23 AM (1 year, 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 10:25 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 10:28 AM (1 year, 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 10:35 AM (1 year, 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 12:33 PM (1 year, 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 12:42 PM (1 year, 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 12:42 PM)


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