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 mikesethnobotany
KCUMB class of 2017
Registered: 07/29/11
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Loc: Missouri, USA
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 Slants 
#15233436 - 10/16/11 02:08 PM (1 year, 7 months ago) |
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I am starting to really wonder what is up with my shiitake mycelium. It is growing extremely slow onto grain, and now I am finding out that my slants are even slow! This is a picture of one slant 14 days after transferring an agar wedge.
What is going on here??
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mikesethnobotany
KCUMB class of 2017
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UPDATE:
So I figured out that my issue was gas exchange. My lid on the vacutainer was completely shut. The lack of CO2 caused the mushrooms to completely stall in growth. I havn't seen many people use vacutainers to make slants, and so it may be beneficial for anyone who has vacutainers and plans on using them for this purpose to know that they will in fact work very well with shiitake. I lifted up the lids so that the slit in the side of the vacutainer's SHIP was revealed to both inside and outside the tube. I then wrapped with 1 layer of parafilm. This has produced at the very minimum, satisfactory results. I would call this great results:
3 days after inoculation....
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 mycoelf
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You might consider your agar formula in your observations, FAE should be zero, your nutrient base should be 1/2 what you usually make agar for petris, also your agar concentration should be at 150% for the same formula, you want really firm, low nutrient base. Also RR uses a tooth pick in the slant to promote growth and allow a small amount of wood for the myc to digest while in storage, I soak my tooth picks in a 10% ME solution before pcing and place the tooth pick in right after the subculture. I open slants once a year to give them fresh air, and subculture to make sure the myc has not died. 
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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
    
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mycoelf
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BTW once the myc has leaped off the inoculum, cool them down, the point of a slant is not to promote growth, but to slow growth, they grow even when cold ever so slow,
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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
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mikesethnobotany
KCUMB class of 2017
Registered: 07/29/11
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Quote:
You might consider your agar formula in your observations, FAE should be zero, your nutrient base should be 1/2 what you usually make agar for petris, also your agar concentration should be at 150% for the same formula, you want really firm, low nutrient base.
To tell the truth, my problem was about gas exchange and has nothing to do with my agar formulation. The mycelium was not growing at all after 11 days on agar because there was no gas exchange.The point of my update was to let people who have vacutainers know that they can use those for master slants instead of cultubes, in case anyone wanted to know.
That being said...
I actually used Mephisto's Master Slant Tek
So thus, this was LMEA, approximately 30 grams LME & 15 grams agar to 1Liter of water. The reason I left out the wood is because I don't have any popsicle sticks or toothpicks. Whenever I make more slants in the future I will add those for sure to extend their storage life.
Quote:
BTW once the myc has leaped off the inoculum, cool them down, the point of a slant is not to promote growth, but to slow growth, they grow even when cold ever so slow,
Yes, yes I know that. What I didn't know is that you are supposed to store in the fridge after the mycelium has jumped off inoculum. Seems like somewhere I read that should be done at 60-70% of the culture. I ALSO didn't know you are supposed to have 0 FAE during storage. How might the mycelium survive without any gas exchange though?
thanks for the input mycoelf
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Javadog
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Registered: 05/03/10
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Loc: USA
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FAE vs GE. GE is necessary for storage.
JD
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mikesethnobotany
KCUMB class of 2017
Registered: 07/29/11
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Loc: Missouri, USA
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Alright so GE is necessary, FAE not? Thanks for clearing that up JD.
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total
Post Office Tyvek Advocator
Registered: 10/03/04
Posts: 9,737
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Id suggest you get a proper storage tube...
Your wasting your time trying to STORE mycelium in such a small tube...
Even with the rate at which the mycelium grows under cold storage, that tube still isnt going to last long...
Id suggest some good Kimble tubes that are >20mm wide...
Top them off with a quality Kim-Caps
You could use those tubes to store a fair amount of liquid mycelium under distilled water thou! 
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EvilMushroom666
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Loc: Canada 
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Re: Slants [Re: total] 1
#15252275 - 10/20/11 04:15 PM (1 year, 6 months ago) |
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Having stored mycelium in similar sized vessels (10CC) I can say that they
will easily last a year under refrigeration and will do the trick until
you have the $$ to invest on proper/higher quality slants.
The Kimble Tubes with Kim caps, simport cultubes, or other glass slants
are always a treat, but are obviously a bit more expensive.
Regardless every year or so you want to take your cultures from slant,
and either inoculate new slants, or take them to agar, let them grow out
a few cm's to assure they are clean and vigorous and then re-slant to assure
that your genetics are safe. Done this way you can easily keep your cultures
within a few P values of the original culture for 20+ years.
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Javadog
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Re: Slants [Re: total]
#15252924 - 10/20/11 06:35 PM (1 year, 6 months ago) |
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Thanks for the links Total. I think that these are similar to what Lipa
uses...I should PM him to see where his best price is...
One thing: the site with the caps seems to offer them only in
"Various sizes available for reusable and disposable culture tubes"
Where I will need only the 20 mm size.
EM laid out the long term strategy very well.
JD
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Edited by Javadog (10/20/11 06:43 PM)
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mikesethnobotany
KCUMB class of 2017
Registered: 07/29/11
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Loc: Missouri, USA
Last seen: 12 days, 11 hours
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Quote:
Id suggest some good Kimble tubes that are >20mm wide...
Top them off with a quality Kim-Caps
I will invest in these whenever I have a little more cash. Right now I have 14 shiitake slants in these vacutainers, which should last me a very very long time lol.
Quote:
...let them grow out
a few cm's to assure they are clean and vigorous and then re-slant to assure
that your genetics are safe. Done this way you can easily keep your cultures
within a few P values of the original culture for 20+ years.
So when you re-slant is that done by growing it out in a petri dish then inoculating a new slant, or do you use the slant to reinoculate? And if the latter is true, do you consider the new slant P1 still? Or will that be like P1.5 (lol)
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EvilMushroom666
Heretic
Registered: 11/18/09
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Loc: Canada
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Some growers inoculate right from the first slant into the new ones for
that year and see no need to transfer to agar first. When the culture
starts to grow out onto the new slant it is refrigerated and good to
go. This avoids a lot of work and materials, but if your culture has become
contaminated you are transferring the contaminate along with the mycelium.
Other growers transfer to a fresh plate, let it start to grow and then take
a transfer from the leading edge to a fresh slant for that year. This
allows you to see that your culture is clean, still vigorous and strong, etc.
A P value is the amount of growth across a standard agar plates. P-1 means
the culture has been grown across 1 standard 100mm petri. P-2, 2 standard
100mm petris etc etc.
If after transferring from your slant to agar you only let the culture grow
out 1cm or so you can see that it would take several transfers to get close
to even one step up in P value. Across 20 years you may only increase the
P value of your culture by 1-3 petri dish's depending upon different variables.
Also note that P value while important can be deceptive from species to
species.
Shiitake for example can easily survive up into the 100 P values with no
noticeable drop in productivity, while other cultures, some morels if I
remember correctly will show a sharp decrease in vigor and fruiting ability
after around P 10.
Here are some notes from RR as well in regards to master slants and P
value:
Quote:
1. Yes, I'll use the same slant for several years. Take a sample, and then put it back. Make new ones all the time, and save them too. Always use your oldest productive slant, which is your youngest cell lines.
2. Don't put too much stock in the P value number. There's a huge difference between letting the mycelium grow on a petri dish for 1/2" and then taking a sample back to slant, or letting the dish fully colonize before putting a sample back to slant. However, both would add 1 to the P value.
3. Some of my best strains are approaching P40. When we expand mycelium by a factor of millions when we do grain to grain transfers, getting worried about a P value less than 100 seems silly to me. It's all about how the samples were taken and how many total cell divisions have occurred.
RR
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mycoelf
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Registered: 06/26/09
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It also depends greatly on the origins of the strains, Multispore germination is the strongest, followed by clones, depending on where you got your start, and where your sender got THEIR start, etc, the only place where I take P values seriously is on strains that I originated either by spores or clone from the wild, In those cases they can go into, like 666 said, hundreds. That being said, the morels that I cloned this april lost the willingness to make micro sclerota after p3, I nailed the clone clean on the first try ran to P5 and they lost their vigor, I went back yesterday to pull for fall kits, and the myc had grown out of the slant and produced sclerota on the OUTSIDE of the slant in the baggie, I also had king oysters grow out of the slant and the caps were covered in myc, so there ya go
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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
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mikesethnobotany
KCUMB class of 2017
Registered: 07/29/11
Posts: 1,204
Loc: Missouri, USA
Last seen: 12 days, 11 hours
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Wow lots of great information everyone! I am surprised by all this feedback, thank you all. Would it be alright for me to boil some sawdust and put that into slants? Obviously I have lots of that laying around .... lol
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mycoelf
Agent Of Chaos
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Posts: 550
Loc: hyperspace
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the tooth pick theory is more about having wood to hide cells in, sort of a protective structure against the cold, not a nutritive thing, RR says it increases viability and I beleive him, I did this last year over the winter and the slants seem to have done better then in years previous, same strains, and in theory I like the idea, I could remove the tooth pick and clip the end off onto a dish, then the cells emerge. If you wanted to experiment with wood in a slant might I suggest that you use the water from the boiled sawdust? I tried grinding grains into my slant agar the first years I did slants and I did not like the way the agar tuned into "oatmeal" looking substance at the bottom of the tubes, and then I did not realize that slant agar should be low nutrients to encourage the myc to run, now I know better
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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
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RogerRabbit
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Also, bear in mind you don't need wood from a commercial source for your slants. I was using medical tongue depressors for a long time, but a year or two ago I couldn't find them when I needed to do a bunch of slants. I just went out on the ground around my trees and found some old twigs and branches that had fallen on the ground and used them instead, and they worked out great. It's all I use now.
I just drop them right in the liquid agar mix after pouring the slant but before sterilization.
To Mike or anyone else who hasn't seen it, the Master Culture Slant video clip might be helpful.
RR
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Aleon
666
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Loc: WI, USA
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Quote:
RogerRabbit said:
Also, bear in mind you don't need wood from a commercial source for your slants. I was using medical tongue depressors for a long time, but a year or two ago I couldn't find them when I needed to do a bunch of slants. I just went out on the ground around my trees and found some old twigs and branches that had fallen on the ground and used them instead, and they worked out great. It's all I use now.
I just drop them right in the liquid agar mix after pouring the slant but before sterilization.
To Mike or anyone else who hasn't seen it, the Master Culture Slant video clip might be helpful.
RR
I really like this idea of twigs from the yard lol! Is any extra sterilization time needed because they are from outside, or just 30 or 45 minutes like regular?
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Javadog
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Re: Slants [Re: Aleon]
#15255911 - 10/21/11 10:45 AM (1 year, 6 months ago) |
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Wouldn't that be closer to the range 20-30 minutes?
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mikesethnobotany
KCUMB class of 2017
Registered: 07/29/11
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@RR
I watched the video. What size test tubes were you using in it? They look larger, such as the 20mm ones Total has recommended. Also, where can i find a scalpel like the one you used in the video?
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total
Post Office Tyvek Advocator
Registered: 10/03/04
Posts: 9,737
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Scalpels
http://www.ebay.com/itm/3-Scalpel-Handle-3L-Surgical-ENT-Veterinary-Instrument-/310353093254?pt=LH_DefaultDomain_0&hash=item48427c6286
Blades
I personally like the 18's and 26's...
RR would use a similar "Culture tube" that is >20mm...
With you using vacutainers, i suggest you get a full metal scalpel that can be autoclaved...
Cause flame sterilizing the blade, and then sticking it in those skinny vacutainers to retrieve your cultures, and the unclean handle banging on the insides, is not going to be good for your cultures...
Im not knocking you...
Ive stored a slant or 2 in them myself... 
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