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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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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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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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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: 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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