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OfflineCorrosiveLiquid
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Botany project (germination time linked to colonization time and end product?)
    #15383748 - 11/17/11 11:41 AM (12 years, 4 months ago)


PROJECT HAS CHANGED SOME READ BOTTOM POST OF THIS PAGE!


I will be starting a botany project starting today. This project will include pictures and steps I took to come up with a result.
Send good vibes and thanks to Gumba and Mycelio for helping me design the experiment and their ideas.

I will be soaking rye berries for 0, 12, 24, 36, 48 and 60 hours in 75-80F water soaked paper towels. After the time is up for each batch, I will be cooking the grains in the oven at 250 deg F for 15min. They will then be laid out to dry in front of a fan (12hrs). I will then take the rye berries and grind them into a flour and make PF cakes with them(steam sterilized). All the cakes are inoculated in an identical manner with Pleurotus ostreatus LC from SWorks. The cakes are incubated at 80 degrees F. I will be recording each groups time it takes until birthing them (myc growth).

The cakes will be birthed and each group is soaked overnight for 12 hours. The cakes are then "dunked and rolled" in vermiculite and placed into a fruiting chamber.

The duration of time it takes for pinning to occur is recorded for each cake. The wet weight is recorded for each mushroom.
Using means and standard deviations of the recorded values for the cakes of each group, a decision will be made on which group fruited the best.

"The foundation of this experiment is that seeds are dormant in the absence of water. When hydrated, seeds will produce hormones like gibberellins, auxins, cytokinins. These hormones will induce the production of enzymes like a-Amylase which cause the carbohydrate starch nutrition of the endosperm in the seed to be broken down into more simple sugars, which will be converted into energy that will be devoted to growth.  The hormones of the seeds might affect the growth of the mycelium, the length of time it takes for the mycelium to generate pins, and the morphology of the fruit. The conversion of the carbohydrate starch to more simple sugars also might affect the mushroom's growth. Less time soaking the seeds = more starch and less hormones. More time soaking = more simple sugars and more hormones. The presence of enzymes is irrelevant because they are proteins, and will readily denature with heating like a hard boiled egg. Unfortunately, the hormones will also degrade with heat, but not as readily as the proteins so it is crucial to apply the minimum amount of heat necessary for sterilization." -Gumba

Edited by CorrosiveLiquid (12/01/11 12:22 AM)

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Invisiblemycoelf
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Re: Botany project (soaking time linked to colonization time and end product?) [Re: CorrosiveLiquid]
    #15383971 - 11/17/11 12:36 PM (12 years, 4 months ago)

:goodluck:


--------------------
Mycoelf

Sterility is a process that can be likened unto infinity, which is a long walk, the closer to the end you start before beginning, the more achievable  the goal of infinity becomes.  Remember, cleanliness in next to goddessness

:aliendance::aliendance::wicca::aliendance::aliendance::pipesmoke:

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Re: Botany project (soaking time linked to colonization time and end product?) [Re: mycoelf]
    #15387267 - 11/18/11 05:34 AM (12 years, 4 months ago)

How is that a botany project?  Aren't you pooking around in the wrong kingdom?

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OfflineTerry M
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Re: Botany project (soaking time linked to colonization time and end product?) [Re: johnm214]
    #15387445 - 11/18/11 07:47 AM (12 years, 4 months ago)

Mycology is classified as a branch of botany (perhaps for historical reasons?). I've noticed that in universities, people doing mycology are in the botany department.


--------------------
Liberté, égalité, humidité.

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OfflineCorrosiveLiquid
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Re: Botany project (soaking time linked to colonization time and end product?) [Re: Terry M]
    #15387643 - 11/18/11 09:10 AM (12 years, 4 months ago)

:whathesaid:


--------------------
"In order to be a leader, you must FIRST be a follower!"

"Emancipate yourselves from mental slavery, None but ourselves can free our minds."
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Re: Botany project (soaking time linked to colonization time and end product?) [Re: Terry M]
    #15389266 - 11/18/11 02:48 PM (12 years, 4 months ago)

Really?  Is that pretty widespread?

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OfflineTerry M
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Re: Botany project (soaking time linked to colonization time and end product?) [Re: johnm214]
    #15389294 - 11/18/11 02:55 PM (12 years, 4 months ago)

Quote:

johnm214 said:
Really?  Is that pretty widespread?




I just Googled "Mycology," and got the Wikipedia entry for it:
http://en.wikipedia.org/wiki/Mycology

This says,
Quote:

Historically, mycology was a branch of botany because, although fungi are evolutionarily more closely related to animals than to plants, this was not recognized until a few decades ago.




--------------------
Liberté, égalité, humidité.

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Invisiblejohnm214
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Re: Botany project (soaking time linked to colonization time and end product?) [Re: Terry M]
    #15389818 - 11/18/11 04:46 PM (12 years, 4 months ago)

wow, I can't believe I've been reading this website for so long (and have taken a decent amount of biology dept courses, though mostly molecular bio related) and never knew that.

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OfflineMycelio
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Re: Botany project (soaking time linked to colonization time and end product?) [Re: CorrosiveLiquid]
    #15390294 - 11/18/11 06:15 PM (12 years, 4 months ago)

Interesting experiment!

CorrosiveLiquid, please mention your soaking temperature, as this is an important factor, speeding up or slowing down the process.

Then I must add that something else will be going on in your jars. To observe the effect of germinating kernels you would have to store them in a wide vessel, in a shallow layer with enough fresh air. In tall vessels with a small water surface and the lid on there won't be much germination action from the kernels. Bacteria and yeasts sitting on the surface of the kernels will multiply after a couple of hours, feeding on starch and simple sugars in the liquid,  using up all oxygen quickly and the continuing anaerobically. Without oxygen, the kernels will fall back into a dormant state and then die. After 2 to 3 days (depending on temperature) they are usually dead and a full scale lactic acid fermentation will be going on, including a PH drop. You will be basically starting sourdough with complete kernels. One or two days more and the grain may be too acidic for cube spore germination. With a maximum of 60hrs you should be fine though.

So you may observe different, but still interesting effects. Lactic acid bacteria and yeasts are good sources of amino acids and vitamins. They will definitely produce lots of amylase too.

Do you do any replications for each soaking time?

Cheers, Carsten

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OfflineCorrosiveLiquid
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Re: Botany project (soaking time linked to colonization time and end product?) [Re: Mycelio]
    #15390653 - 11/18/11 07:31 PM (12 years, 4 months ago)

Thank you Mycelio i was really wanting some input of things that needed work. I will be restarting the experiment using your recommendations. :thumbup:

I knew something was not right because they have gotten very smelly very fast.


--------------------
"In order to be a leader, you must FIRST be a follower!"

"Emancipate yourselves from mental slavery, None but ourselves can free our minds."
"Don't gain the world and lose your soul, Wisdom is better than silver and gold"

Edited by CorrosiveLiquid (11/18/11 07:36 PM)

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OfflineMycelio
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Re: Botany project (soaking time linked to colonization time and end product?) [Re: CorrosiveLiquid]
    #15392314 - 11/19/11 07:25 AM (12 years, 4 months ago)

You're welcome, I'm glad if I can help.

Whenever I germinate grain, I submerge the kernels for a few hours, then I pour off most of the water and spread them on a flat plate. Wrapping things up in wet paper towels should work fine too.

Regarding measuring results, the harvested weight should be most important. For oysters, cap size and stem length can vary a lot due to light and air exchange.

Cheers, Carsten

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Re: Botany project (soaking time linked to colonization time and end product?) [Re: Mycelio]
    #15404535 - 11/21/11 06:39 PM (12 years, 4 months ago)

Are you calling your soak time of 0 your control?

How many trials are you doing of each soak time? You'll want a good number of data points for each before you can even think about implying correlation.

Also are you comparing the amount of time spent in the fruiting chamber? A good way to measure things when all is said and done might be:

(grams mushrooms, dry weight)/(time from inoculation to end of first flush)

This will give you a rate to work with for each, which will speak to the efficiency of each different soak time as a substrate, which may be the most useful way to view things. You can then later extend this to carry through to the final flush but the first one would have the highest number of controls in place and most likely to show correlation.

Also if they are fruiting at different times you will want to make sure you're misting at equal intervals throughout the entire process or your misting variations could lead to experimental error.

And finally you might want to consider how you're going to ensure each jar gets exactly the same amount of spore solution. Not too hard but something to make sure you think about rather than eyeballing.

Just my two cents, not trying to tell you how to run your experiment, just give you a few ideas to make it more scientific.

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OfflineCorrosiveLiquid
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Re: Botany project (soaking time linked to colonization time and end product?) [Re: Rammy]
    #15404963 - 11/21/11 08:05 PM (12 years, 4 months ago)

Quote:

Rammy said:
Are you calling your soak time of 0 your control?

How many trials are you doing of each soak time? You'll want a good number of data points for each before you can even think about implying correlation.

Also are you comparing the amount of time spent in the fruiting chamber? A good way to measure things when all is said and done might be:

(grams mushrooms, dry weight)/(time from inoculation to end of first flush)

This will give you a rate to work with for each, which will speak to the efficiency of each different soak time as a substrate, which may be the most useful way to view things. You can then later extend this to carry through to the final flush but the first one would have the highest number of controls in place and most likely to show correlation.

Also if they are fruiting at different times you will want to make sure you're misting at equal intervals throughout the entire process or your misting variations could lead to experimental error.

And finally you might want to consider how you're going to ensure each jar gets exactly the same amount of spore solution. Not too hard but something to make sure you think about rather than eyeballing.

Just my two cents, not trying to tell you how to run your experiment, just give you a few ideas to make it more scientific.




love the input dude... the more the better. I will be using a nearly identical amount/strain of oyster LC that i got from a sponsor and made a bulk sterile media with it. I dont have the time to do more than one run at this for my botany project but i could do this 3-4 times with the amount of LC i have now and post results on shroomery. I plan on recording colonization times, fruit weight, and fruiting length. I also will be misting a the same time of day with the "same" amount of FAE as i possibly can.


--------------------
"In order to be a leader, you must FIRST be a follower!"

"Emancipate yourselves from mental slavery, None but ourselves can free our minds."
"Don't gain the world and lose your soul, Wisdom is better than silver and gold"

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Offlinedkmonk
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Re: Botany project (soaking time linked to colonization time and end product?) [Re: CorrosiveLiquid]
    #15405134 - 11/21/11 08:41 PM (12 years, 4 months ago)

Wouldn't you need an isolate of that strain to get consistent results since multispore is a crap shoot?

I just skimmed over, and disregard if this doesn't make sense.


--------------------
First Grow Golden Teacher Koh Samui - in progress
http://www.shroomery.org/forums/showflat.php/Number/15330714

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OfflineCorrosiveLiquid
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Re: Botany project (soaking time linked to colonization time and end product?) [Re: dkmonk]
    #15405217 - 11/21/11 08:59 PM (12 years, 4 months ago)

Quote:

dkmonk said:
Wouldn't you need an isolate of that strain to get consistent results since multispore is a crap shoot?

I just skimmed over, and disregard if this doesn't make sense.




I believe sponsors use isolated strains in their LC's I could be wrong tho.


--------------------
"In order to be a leader, you must FIRST be a follower!"

"Emancipate yourselves from mental slavery, None but ourselves can free our minds."
"Don't gain the world and lose your soul, Wisdom is better than silver and gold"

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OfflineCorrosiveLiquid
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Re: Botany project (soaking time linked to colonization time and end product?) [Re: CorrosiveLiquid]
    #15447988 - 12/01/11 12:21 AM (12 years, 3 months ago)

The project changed some due to time restraints but will be ongoing.

FIRST LEGIT LAB REPORT... any pointers? there are standard deviations and graphs but dont want to do the work to get them on here.

The effect of seed germination time on the growth rate of Pleurotus ostreatus mycelium.


By: Joe Momma







Abstract:
The main idea of this experiment is to find out if germination time of rye seed has any effect on Pleurotus ostreatus mycelium. The results were: the longer the germination time, the less growth. This could be due to more endospores being germinated and hindering growth rates. This could be that more PGR’s hinder growth of mycelium.  More testing needs to be done to rule out the endospores and the hormones. That will prove it has to do with starch being converted into simple sugars.
0hr Group: Jar1 - 9.5cm, Jar2 – 9.5cm, Jar3 – 9cm, Jar4 – 9cm. (90-95% colonized in 10 days)
12hr Group: Jar1 - 2cm, Jar2 – 2cm, Jar3 – 1.5cm, Jar4 – 1cm. (10-20% colonized in 10 days)
24hr Group: Jar1 – 9cm, Jar2 – 9cm, Jar3 – 8.5cm, Jar4 – 8cm. (90-80% colonized in 10 days)
36hr Group: Jar1 - 8cm, Jar2 – 7.5cm, Jar3 – 7.5cm, Jar4 – 7cm. (80-70% colonized in 10 days)
48hr Group: Jar1 – 7.5cm, Jar2 – 7cm, Jar3 – 6.5cm, Jar4 – 6.5cm. (75-65% colonized in 10 days)

I am interested in mushroom production because it is a hobby of mine that I started a few years ago. This experiment will possibly help me speed up the time it takes from inoculation to fruit. My hypothesis is that the group that soaks the longest will perform the best. The foundation of this experiment is that seeds are dormant in the absence of water. When hydrated, seeds will produce hormones like gibberellins, auxins, cytokinins. These hormones will induce the production of enzymes like a-Amylase which cause the carbohydrate starch nutrition of the endosperm in the seed to be broken down into more simple sugars, which will be converted into energy that will be devoted to growth.  The hormones of the seeds might affect the vigor of the mycelium. The conversion of the carbohydrate starch to more simple sugars also might affect the vigor of the mycelium. Less time soaking the seeds = more starch and less hormones. More time soaking = more simple sugars and more hormones. The presence of enzymes is irrelevant because they are proteins, and will readily denature with heating. Unfortunately, the hormones will also degrade with heat, but not as readily as the proteins so it was crucial to apply the minimum amount of heat necessary for sterilization. The main purpose of this experiment was to see if I could find the optimal germination time of rye seed for Pleurotus ostreatus mycelium growth rates.

Methods & Materials
• 1/2pint mason jars with lids
• Hammer/nail
• 5 plastic “shoe” box’s
• Strainer/bowl/pot with lid/baking pan
• Paper towels/foil
• Measuring cups
• Rye grains
• Oven/stove
• Fan
• Blender
• Vermiculite
• Gypsum (drywall)
• Liquid culture (Pleurotus ostreatus)
• Permanent marker
• 91% rubbing alcohol
• Candle
• Glove box (still air box)
• Incubation chamber
• 10-14 days

Read completely before starting.

STEP ONE – Germination
I germinated rye seed for 0, 12, 24, 36, and 48 hours in water soaked paper towels at 75F. The rye grains were measured out one cup per group. Each group needs rinsed in a strainer for 5 minutes to remove any dirt then drained for 5 minutes to remove excess water. The paper towel was laid in the bottom of a plastic “shoe box” container. The grains were dumped on top of the paper towel then spread out to a depth of 1cm. The rest of the paper towel was laid on top of the grains and the water (2/3 cup) was added, then the lid was placed on the container. The lid should not be air tight because the lack of oxygen will cause the grains to become anaerobic and possibly die.  Group 0 hours grains were placed immediately into the room temperature baking pan that was then placed in an already pre-heated 210F oven for 20 minutes. This is done to stop enzyme activity and halt germination.  After 20 minutes the grains were placed in front of a fan for 12 hours to cool and remove any moisture in the grains or on the grains. When the 0 hour group is done under the fan they were taken to the blender and set on grind for 5 minutes. The 12 hour group will now be ready to go in your already 210F pre-heated oven for 20 minutes. After 20 minutes, place the grains in front of the same fan on the same speed setting for 12 hours.  Keep doing this process/cycle until you have baked all 5 groups and all 5 groups have been dried by the fan then ground. Remember to keep each group labeled so you don’t get the germination times mixed up. You will also need to be preparing “cakes” immediately after their done in the blender.

STEP TWO - Cakes
You will need to prepare your 0 hour group immediately after they are done in the blender. You do this by mixing together 2 cups of vermiculite and one teaspoon of gypsum in a mixing bowl. This is done to keep the gypsum from clumping together and not mixing properly. Add 1 cup of water and mix the vermiculite/water/gypsum together. After mixing thoroughly you need to add 1 cup of your ground up rye grains. Mix the rye “flour” for about a minute into your vermiculite/water/gypsum. This is done last to keep the rye flour from clumping together. Spoon the mixture into 1/2pint canning jars. Leave about ½ inch of the jar empty at the top, you will be putting straight vermiculite in this top ½ inch to act as a contamination barrier. Make sure to wipe any of the mixture off of the glass if there is any before adding your dry vermiculite, this can lead to contamination if it is left there. Place the lid on the jar and label it 0hr: jar 1. Do this 4 more times so you have 4 labeled jars with the mixture in them and dry vermiculite on top of the mixture. You want to take a hammer and a nail (bigger than needle) and puncture 4 holes into your lid very close to the outside edge. These will be used later to inoculate the cakes. Place foil on the lid so it covers the lid completely leaving the bottom half of the jar uncovered. This is to keep moisture from getting in your jars when you steam sterilize them. Repeat for other groups.

STEP THREE – Steam Sterilization
Take a pot big enough for all 4 jars with a lid and place a few paper towels in the bottom of the pot. Put your jars in the pot on top of the paper towels and add room temperature water so it comes up 1/2 of the jars height. Turn the stove on high and place the lid on the pot. Once the water starts to boil, turn the stove down to medium and start a timer. You want the water to continue boiling but not a “rolling boil”.  Keep this boil for 90 minutes. Remember the placement of your boil setting, you will need to use the same setting for the other groups. After the 90 minutes is over place the pot by a fan to aid in cooling the jars.  Repeat for other groups.

STEP FOUR – Inoculation and incubation
I used an isolated strain of liquid culture to inoculate my jars. I used a still air box that was cleaned with 91% rubbing alcohol. Each jar was wiped down with alcohol then placed in the still air box. Shake your syringe to evenly distribute the liquid culture. The needle was flame sterilized then wiped with an alcohol soaked pad, do this between each jar. Inoculate each hole in each jar with 1cc of liquid culture for a total of 4cc of liquid culture per jar. Try to keep the needle under the dry vermiculite layer and close to the glass when you inject the culture. Place the jars in an area that will maintain a constant temperature. This temperature can be 75-80F but should not fluxuate. This area should not receive any light to remove the variable of light intensity and light hours effecting the growth of the mycelium. After 10 days of incubation you are going to want to record the amount of growth. I picked 10 days because I did not want them all to be 100% colonized.  Jars are 10cm tall. Measure growth per 0.5cm.








PROCESS OF START/FINISH (11am – 11pm)
Day 1
0hr group bake > fan (12hrs)
Night 1
0hr group boil > fan (12hrs), 12hr group bake > fan (12hrs)
Day 2
0hr group inoculated, 12hr group boil >fan (12hrs), 24hr group bake > fan (12hrs)
Night 2
12hr group inoculated, 24hr group boiled > fan (12hrs), 36hr group baked > fan (12hrs)
Day 3
24hr group inoculated,  36hr group boiled > fan (12hrs), 48hr group baked > fan (12hrs)
Night 3
36hr group inoculated, 48hr group boiled > fan (12hrs)
Day 4
48hr group inoculated
Day 12
0hr group growth measured
Night 12
12hr group measured
Day 13
24hr group measured
Night 13
36hr group growth measured
Day 14
24hr group measured


Results
0hr Group: Jar1 - 9.5cm, Jar2 – 9.5cm, Jar3 – 9cm, Jar4 – 9cm.
12hr Group: Jar1 - 2cm, Jar2 – 2cm, Jar3 – 1.5cm, Jar4 – 1cm. (CONTAMINATED)
24hr Group: Jar1 – 9cm, Jar2 – 9cm, Jar3 – 8.5cm, Jar4 – 8cm.
36hr Group: Jar1 - 8cm, Jar2 – 7.5cm, Jar3 – 7.5cm, Jar4 – 7cm.
48hr Group: Jar1 – 7.5cm, Jar2 – 7cm, Jar3 – 6.5cm, Jar4 – 6.5cm. (Possible contaminates)


I am positive the 12 hour group contaminated. I do not know why it contaminated but I believe it has something to do with germinating the endospores. The 48hr group also had a funky smell coming from the jars but the mycelium looks very healthy. I need to run more experiments under these same conditions and see if the same things happen to 12 and 48. The results were the total opposite of my hypothesis. I didn’t think any of the 0hr group had a snowballs chance in hell. Mainly because in my mind not soaking the grains would not germinate any endospores in the grains. I thought this would be the first to contaminate but it looks like, not giving the endospores any germination time when steam sterilizing is a benefit. I also want to see how each group performs under fruiting conditions. This will be my next experiment with these cakes. I will compare fruit weight of each group. I also want to determine if the hormones have any effect on the growth by pressure cooking the grains instead of steam sterilizing them. The differences in growth may or may not have anything to do with PGRs. Pressure cooking the grains at a high temperature should destroy or alter most of the hormones.

References: Gumba, Mycelio, and Rammy of Shroomery.com for helping me design the experiment and giving me ideas.

http://www.mathsisfun.com/data/standard-deviation-calculator.html

www.shroomery.com


--------------------
"In order to be a leader, you must FIRST be a follower!"

"Emancipate yourselves from mental slavery, None but ourselves can free our minds."
"Don't gain the world and lose your soul, Wisdom is better than silver and gold"

Edited by CorrosiveLiquid (12/01/11 12:36 AM)

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InvisibleJavadog
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Re: Botany project (soaking time linked to colonization time and end product?) [Re: CorrosiveLiquid]
    #15448704 - 12/01/11 08:46 AM (12 years, 3 months ago)

Well, I have more reading to do! 

....but 24 hours seems to have held up which is good.

Thank you for sharing!

JD


--------------------
Boyd Rice told my brother that life is a corny pack of freesakes

Myco-tek.org

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