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Mateja


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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) |
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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:


-------------------- Cakes inside Water Tub
Edited by Mateja (03/10/22 01:00 AM)
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Mateja


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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) |
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And it seems that bacteria will fuck up a liquid broth in a matter of hours 
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 
 
 
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.
-------------------- Cakes inside Water Tub
Edited by Mateja (10/28/20 04:39 AM)
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Mateja


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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) |
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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.
-------------------- Cakes inside Water Tub
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sonoramo
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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) |
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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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Lemgrub


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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) |
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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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mushhead
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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) |
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   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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BrownBear
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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) |
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How would you rate these lc's?
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enteogenio
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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) |
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What do you think:

*MS Srynge: GT *Water/Honey (500ml/4ml PC:30min/15psi) *Date: 19/10/20
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coversall
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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) |
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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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D3_Myc
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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) |
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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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nmd_myco
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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) |
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Just expanded my firsy LC. a little early, but there was plenty of nice looking myc in the transfer.
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MLPismyOPSEC
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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) |
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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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Mateja


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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) |
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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?
-------------------- Cakes inside Water Tub
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D3_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 06:34 PM (3 years, 2 months ago) |
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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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AtmozFear
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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) |
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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 a professional look
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MLPismyOPSEC
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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) |
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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 
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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Josex
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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) |
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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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mushhead
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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) |
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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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Pastywhyte
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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) |
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I like these, they look ready to rip into some grain.

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mushhead
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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) |
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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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