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mogur
regnartS

Registered: 11/15/05
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Liquid culture parameters
#5274821 - 02/07/06 07:27 PM (17 years, 11 months ago) |
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"I know nothing, I see nothing, and I say nothing!" John Banner [Sgt. Schultz, "Hogan's Heroes"]
What a great man, one who lost his entire family to the Nazi regime. A Jew, he finally was able to spoof the third Reich in the American sitcom, Hogan's Heroes. I salute you, Sargent Schultz.
Since I'm a lot like the sarge, I need to experiment to find out stuff, so here goes. What concentration is best for liquid culture? What sugar is best? And finally, what nutrients are ideal?
I know that 3% Karo and potato juice work quite well. So, with that as a control, here's the experiment. Group one- try various ratios of light Karo syrup. Group two- try various nutrient solutions, with 3% light Karo as a carb base. Group three- try different sugars with a potato nutrient broth as a constant.
Group one: five concentrations of light Karo syrup in well water.
Group two: five types of nutrient broth with a 3% Karo solution. Potato broth is two medium russets boiled for a half hour in 1 liter of water. Broth is strained, settled, and diluted to one liter. Soil broth is just a cup of garden soil, fractionally sterilized, strained through coffee filter, and sediments settled, with supernatant extracted. V8 broth, strained and supernatant extracted. Rye broth, fractionally sterilized, strained and extracted. Bullion is a packet of very low sodium Herb Ox sitting in my cupboard, boiled and extracted.
Group three: Five sources of sugar are diluted to 3% concentration. Dark Karo is from a jar that has been in my cupboard for years. Maple syrup from a plastic gallon jug that my neighbor just gave me, probably a few years old, but unopened until now. Honey is a packet that I just got from my local pub/restaurant. It isn't organic or anything, just honey. Sugar is generic table sucrose. Apple juice is Walmart commercial, with added ascorbic acid (Vitamin C). 30 ml figures to be about 3 grams sucrose, 2 grams fructose, a little glucose, and an unknown amount of sorbitol (probably in the range of .3g).
More boring details and pictures tomorrow.
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agar
old hand


Registered: 11/21/04
Posts: 9,056
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Re: Liquid culture parameters [Re: mogur]
#5274856 - 02/07/06 07:38 PM (17 years, 11 months ago) |
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3% solution of 50/50 mix of light dry malt & dry dextrose is my old standby.
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shroomsynergy
Fungi Freak


Registered: 01/17/06
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Re: Liquid culture parameters [Re: agar]
#5274899 - 02/07/06 07:51 PM (17 years, 11 months ago) |
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i like the honey tek because its simple, and quick, yet i did it in the baby bottle like another tek said. i too like to experiment, and for me the honey worked the quikest. (keep in mind that my environment is hobby, and in no way a government science lab.)
....but i got tiny myc in a little over 2 days, and was able to use my LC in just over a week, were as for me the syrup tek took like 3 days longer (for me).
p.s. AGAR, is that a marble that i see in your LC??? i have never used a marble, just shake the shit out of it. do you recomend a marble??? good luck, and keep us posted.
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Snaggletooth
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Re: Liquid culture parameters [Re: mogur]
#5274905 - 02/07/06 07:52 PM (17 years, 11 months ago) |
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Quote:
"I know nothing, I see nothing, and I say nothing!" John Banner [Sgt. Schultz, "Hogan's Heroes"]
What a great man, one who lost his entire family to the Nazi regime. A Jew, he finally was able to spoof the third Reich in the American sitcom, Hogan's Heroes. I salute you, Sargent Schultz.
Weird....I did not know that, wish I did before now it might have gave some insight, but good for him. 
Ok very cool, I have waited for you to step your foot out, cool this should be a very interesting thread. Honestly I have near no clue as to the possible out come, but am very interested.
Thanks for sharing 
I think that is a stir bar
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shroomsynergy
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Re: Liquid culture parameters [Re: Snaggletooth]
#5274941 - 02/07/06 08:01 PM (17 years, 11 months ago) |
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ok, i'm kinda new, so what the hell is a stir bar, and what does one use as a "stirbar"?
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agar
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Registered: 11/21/04
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It is a STIR BAR, used with a stir plate.
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Hoss
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Re: Liquid culture parameters [Re: agar]
#5274981 - 02/07/06 08:12 PM (17 years, 11 months ago) |
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3%? Damn! I thought it was 4%! LOL!
So that's why my LCs have been performing so crappy!
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shroomsynergy
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Quote:
ShrOOmSynEgry said: ok, i'm kinda new, so what the hell is a stir bar, and what does one use as a "stirbar"?
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Snaggletooth
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mogur
regnartS

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Re: Liquid culture parameters [Re: Snaggletooth]
#5279349 - 02/08/06 09:16 PM (17 years, 11 months ago) |
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This jar is just dirt and well water settled. The 'soil' nutrient is just liquid extracted from the clear part and filtered through coffee filters.

On the left is potato water filtered about eight times, in the middle is the filtered soil extract.

On the left is rye grain broth, in the middle v8 juice extract, and on the right is beef bullion.

Took me forever, but here are all fifteen trials ready to be inoculated.
 Will inoc tomorrow evening, I'm pooped. The trial containers are just cheap (but clear) cups, with tyvek siliconed to the rim. I dab a little silicone to cover up any jab holes. I learned that measuring 100ml plus amounts with a 20ml syringe is an exercise in stupidity.
Thanks for the input, guys. Yeah, agar, I know these aren't the ideal ingredients, but I've seen so many different recipes that I thought I would take the time to investigate some common household items. Who knows? My setup is so cheesy, I probably won't get a lot of good info, but gotta try.
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hyphae
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Re: Liquid culture parameters [Re: mogur]
#5279537 - 02/08/06 09:46 PM (17 years, 11 months ago) |
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So are you saying those have all been PCed right? If not your way over your head already bro, but it sounds like one hell of an interesting thread anyway! GL
-------------------- Getting the most out of your casings!, A pinning strategy. Oyster Shell "Flour" $2 for 1lb. a hell of a deal Not what is overlay but rather what overlay is Gas Exchange vs. FAE "We all have priorities. I used a closet once setup a nice little lab trouble was all the shit that was in there ended up in the bedroom that pissed off the GF then I ended up dumping her as she was getting in the way of my sterile culture technique! Ya I got priorities too!!!"
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Snaggletooth
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Re: Liquid culture parameters [Re: mogur]
#5279542 - 02/08/06 09:47 PM (17 years, 11 months ago) |
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Quote:
mogur said: I learned that measuring 100ml plus amounts with a 20ml syringe is an exercise in stupidity.
Ah man I know what you mean..........LOL
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mogur
regnartS

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Re: Liquid culture parameters [Re: Snaggletooth]
#5282918 - 02/09/06 06:43 PM (17 years, 11 months ago) |
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Noc'd up today. South American CFSR print. 1.5ml per, very heavy spore density.

PC? No. Triple fract. on rye, boil 5 minutes on the rest. Aseptic? No.
Here's the final concentrations- Top row: 25% potato nutrient all. Sugars from left to right, dark karo, honey, sucrose, maple, apple juice, 3% all. Middle row: 3% light karo, all. Nutrients from left to right, potato, soil, rye, v8(15%), bullion (all 25%, except v8). Bottom row: No nutrient added, light karo from left to right, .75%, 1.4%, 3%, 6%, 24%.
 Why is the bottom right cup squashed? Cuz I was experimenting with the broth temperatures. Yeah, that's the ticket, experimenting.
Edited by mogur (02/14/06 06:22 PM)
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hyphae
born to grow


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Re: Liquid culture parameters [Re: mogur]
#5283308 - 02/09/06 08:23 PM (17 years, 11 months ago) |
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Triple frac should work but a 5 min. boil especially the soil LC is going to possibly leave you open to contams and you know how difficult they can be to see in LC's. Either way GL have fun we be watching no doubt about that!
-------------------- Getting the most out of your casings!, A pinning strategy. Oyster Shell "Flour" $2 for 1lb. a hell of a deal Not what is overlay but rather what overlay is Gas Exchange vs. FAE "We all have priorities. I used a closet once setup a nice little lab trouble was all the shit that was in there ended up in the bedroom that pissed off the GF then I ended up dumping her as she was getting in the way of my sterile culture technique! Ya I got priorities too!!!"
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mogur
regnartS

Registered: 11/15/05
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Re: Liquid culture parameters [Re: hyphae]
#5283624 - 02/09/06 09:24 PM (17 years, 11 months ago) |
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I knew there were two that I fracted, but couldn't remember the other one. The soil was triple fractionalized, also. Thanks for reminding me. You know way better than I do, Hyphae, it's just that right now I couldn't get my hands on that many PCable jars, so this may well be just a contam race. I learn from my mistakes, since I pay more attention when I screw up. I should be a genius by now.
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mogur
regnartS

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Re: Liquid culture parameters [Re: mogur]
#5300228 - 02/14/06 06:08 PM (17 years, 11 months ago) |
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Here's an update-

Here's a problem- 

Green mold in the apple juice/ potato broth cup. And, I have to confess, that it was the last cup processed, I got lazy and didn't even boil it. I really didn't mean to leave that info out of my earlier posts, I simply forgot about that, and obviously didn't do proper note-taking, either.
Only minor fluffies so far, guess I need to heed hyphae's advice, and improve my sterile technique.
Floggings are on Fridays, so I think I'll go get drunk on grog for now.
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Snaggletooth
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Re: Liquid culture parameters [Re: mogur]
#5300264 - 02/14/06 06:23 PM (17 years, 11 months ago) |
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You have Floggings Fridays.....I liked Caining Saturdays
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mogur
regnartS

Registered: 11/15/05
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Re: Liquid culture parameters [Re: Snaggletooth]
#5313976 - 02/18/06 05:18 PM (17 years, 11 months ago) |
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Ouch!
Well, here's the haps, paps. The first group are various sugars with potato broth added to all of them. We're missing the contaminated apple juice cup. Looks like ring around the bathtub in most of them.

The second group are all light karo with various nutrient broths added. Most look like sawdust has settled out on the bottom of the cups.

The third group are the various concentrations of light karo only, from .75% to 24%, left to right. All are showing good growth, except the 1.5% cup, which is way behind the others. Why, I don't know.

The most interesting one is the 24% concentration cup, which only has a few fluffy flakes, but a half inch jellyfish looking growth. Maybe more like a transparent mushroom shape. This photo isn't very clear, but it's the best I could get of this growth. I'll try again in a few days.
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Snaggletooth
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Re: Liquid culture parameters [Re: mogur]
#5313993 - 02/18/06 05:24 PM (17 years, 11 months ago) |
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Do you have a way that your going to 'test' them
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mogur
regnartS

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Re: Liquid culture parameters [Re: Snaggletooth]
#5314015 - 02/18/06 05:33 PM (17 years, 11 months ago) |
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Rye grain quarts. Gonna give em another week or so. Will only do the cups that look 'right', that is, fluffy stuff. Maybe one jar of the sawdust, just for laughs.
PS [edit]: Oh yeah, I forgot to mention that they are incubated at 72F to 75F, which may explain the slow growth.
Edited by mogur (02/18/06 05:37 PM)
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mogur
regnartS

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Re: Liquid culture parameters [Re: mogur]
#5347224 - 02/27/06 09:18 PM (17 years, 10 months ago) |
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The final result-
Below is a photo of all surviving liquid cultures.

I have learned that nutritional additives need to be watered down to about 20% of the total liquid volume. The potato/dextrose teks I read before this experiment led me astray. Most of them simply said to boil potatoes in a liter of water, filter, and add water back to 1 liter. This is wrong. More detailed broth teks and my own experience dictate a much more dilute solution. Also, short of vacuum filtering through extremely fine meshes, a settling period is required, and the clear supernatant extracted. Otherwise, the solution is so turbid (cloudy) that bacterial contams are almost impossible to identify.
Mycelium growth was observed in almost all cultures tested, but were so obscured by turbidity that hardly any useful data could be gained. The lack of complete sterility in this experiment also contributed to bacterial and/or yeast contaminations that increased the original turbidity. The lowest concentration of karo only culture finally turned turbid just yesterday, indicating that it had finally succumbed to contamination. The higher sugar concentrations were able to fend off contams and remain relatively clear. When I poured out the cultures in my backyard grave site, I mostly smelled whatever nutrient was used (V8, rye, soil, etc.), although two or three smelled yeasty, so I suspect yeast contam in those. None had a sour or acrid smell. (By the way, don't go sniffing nasty cultures, the bacteria can take hold in your lungs and even kill you.)
I look forward to a much improved experiment with better sterility, varied nutrient concentrations, and higher clarity of the broth. I was surprised in this experiment how fast the apple/potato broth took off (although it contam'd very fast, also). I was also surprised that the highest and lowest karo concentration showed good growth, I expected only the concentrations near 4% to do well.
That's it for this experiment, I nocced four quarts of rye with the 24% karo culture, and one quart of rye with the karo/rye culture. If anything unusual about the spawn should happen, I will report back.
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agar
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Re: Liquid culture parameters [Re: mogur]
#5347256 - 02/27/06 09:29 PM (17 years, 10 months ago) |
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20% canned/bottled apple juice to 80% water, has worked well, for me. Numerous times.
 Above is 10% flat beer, 10% apple juice & 80% water. That is one thick mamma jamma LC.
Suggest next time, you use jars, with tubing filter lids.
  
 Beer, apple & cranberry juice LC
Yup, TURBIDITY is a bitch, with any starchy nutrient.
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Snaggletooth
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Re: Liquid culture parameters [Re: agar]
#5347289 - 02/27/06 09:41 PM (17 years, 10 months ago) |
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Yeah it looks like the 24% Karo kick ass in this one.
Have you came across this Vanilla Extract and Vented lid tek for liquid cultures!
Good luck on the rye, what are you spawning to if you haven't said yet.
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mogur
regnartS

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Re: Liquid culture parameters [Re: agar]
#5347309 - 02/27/06 09:49 PM (17 years, 10 months ago) |
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Thanks for the input, agar, much appreciated. Yes, being a cheapskate, I've been keeping an eye out for smallish jars without the 'jelly decorations' imprinted in the glass, without having to order from a scientific supply company at 4 bucks a container. And really looking forward to trying your beer and apple juice combo. Also want to try synthetic nutes, and other peroxide compatible ingredients.
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agar
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Re: Liquid culture parameters [Re: mogur]
#5347358 - 02/27/06 10:01 PM (17 years, 10 months ago) |
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Glass Miricle whip & Mayo quart jars don't have any embossing on them. (frugal agar, LOL)
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mogur
regnartS

Registered: 11/15/05
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Re: Liquid culture parameters [Re: Snaggletooth]
#5347542 - 02/27/06 10:38 PM (17 years, 10 months ago) |
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The vanilla tek, yes, saw that. Agar's lids strike again! Bet the corn syrup in the extract didn't hurt.
Don't know yet what I'll spawn the rye to. Probably poo/verm.
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