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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: 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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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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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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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] * 1
    #27015804 - 11/01/20 01:54 PM (3 years, 2 months ago)

:takingnotes:


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results :thumbup:


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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 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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InvisibleMateja
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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: 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: 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: 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: 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: 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] * 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: 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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InvisibleD3_Myc
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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: 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: 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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