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OfflineMephistophelian
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Mephisto's Master Slant Tek * 24
    #12968316 - 07/28/10 06:26 PM (13 years, 7 months ago)

Its easy...doesn't take special equipment or nonsense and is very straightforward. It also works with any type of slant you may take interest in be it PP Screw Cap, Simport CulTubes, Pyrex Screw Cap, or Vac-Tainers. This works with them all...especially if you don't have a sterilizer setup or a test-tube rack.

Tube Measurements:

  • Vac-Tainers - with a red top are commonly 10ml and 16mm x 100mm
  • CulTubes - depends on your model, I used the largest being 17mm x 100mm (They are also PP)
  • Absolute Smallest Recommended Dimensions is 16mm x 100mm, no less.


So lets begin.

Materials Needed:
  • Wood Sticks - Popsicle, Match Stick, whatever type you find that is flat and suitable for the job
  • Slants/Tubes w/ Caps- Vac-Tainer, CulTubes, Pyrex or PP
  • Agar Mix - I used Lab Standard MEA
  • Measuring Container - Mix agar
  • Syringe - Whatever size & gauge you have laying around.
  • Pint Jars - Quart jars would work as well, although I wouldn't want to personally do it with Quart jars
  • Cooking Pot - Any size is suffice Optional
  • Scale - For measuring agar mix (unless you have premix)






Step 1: Preparation

First thing, measure your sticks to see if they fit sufficiently. I had to break all my sticks in half lengthwise then in half width wise to fit right. Do this before anything else. Pre-soak your woodsticks. I boiled some water in a pot and dropped them in. Took it off the stove, covered with a lid and let soak. You can move onto step two while you wait.




Step 2: Preparation Continued

Fill your slant with water WITH a stick in it, to approximately 7ml. No more then that. As low as 5ml but no more then 7ml WITH the stick in it. Without the stick, it will vary by .5ml. You need this to be no more then 7ml because when you go to lay your slant down, you will lose surface area for your mycelium to grow. That's no good. You'll see when you pour your water in and slant it so the water doesn't go past the edge (remember that the lid will take up a small space inside the tube and you don't want agar building a wall up against it inside).



Now, you want to go into your Lab/Room and inside your GB or in front of your flow hood, have your spot where your slants will sit while they cool before hand. It sucks to do it while your unloading the jars and getting it ready last minute. Not wise. We see here that my little racks sitting on 4 popsicle sticks was suffice to make the perfect slant.



Now that we have that setup, lets go check on our sticks sitting in the hot water.

Step 3: Sticks 'An Tubes



Looks like the sticks are ready and saturated. In a pinch, you can soak these overnight instead of hot soaking, but hey...sometimes you forget or get an urge to do something right-this-second and well hot soak works well. Now what you do is get your tubes ready and lay them out and put your sticks in them. Once you've got your sticks laid out, and sticks in them. We can go to the next step. See how easy this is? You could be drinking a beer while doing this. But make sure your still on your first by this point otherwise your in for a world of trouble later. :wink:


Step 4: Agar Fun

Now lets get some agar mixed up. Some prefer to do it in a pot with it being warmed, some do it cold. Personally I do it with warm water poured into the mix but not over a pot. Had some of my agar go jelly in the pot one time, never bothered with it since. I've got enough temperamental things in my life.

The agar recipe I used was the standard formula of 2% nutrient base 'lab standard' from Fast Freds Cookbook.

10G Malt Extract
15G Agar
1L Water

For myself, I used 300 ml of the agar for this project (the picture it shows just above 500ml with displacement of the powders the rest of the agar was used elsewhere) which makes the recipe cut in half. What you need to do is decide how many your pouring and do the math. 7ml x (how many slants) = total water needed. Adjust nutrients and agar to suit. Simple no? Here is my agar & ME on the scale.



Now stir it all together.



Time for the syringe. Stir up your agar mix to get all the powders that aren't dissolved and suck up into the syringe and start to fill your slants with maximum 7ml a piece. This is simple enough. If you happen to bugger off and come back a few minutes later, make sure to shake your syringe around to get the powders sloshed about to make an even mix for each slant.

Again, STIR BEFORE DRAWING SYRINGE

If you have CulTubes, there are two snaps. First snap is aerobic and lets air through, press harder and its air & water tight. Just leave it loose so air can get in. They'll pop off if you don't. For Vac-Tainers see attached picture. For Pyrex screw tops, just leave it slightly unscrewed to let air through. Don't worry about contams right now, just keep reading the guide, we'll get to how to avoid those.



Get a pint jar to start to lay these in. You'll fit no more then 8 using CulTubes, I think it'll be 8 with Vac-Tainers as well. Grab two lids of whatever type you decided on and lay them down then lay your pint jar down on them so you can slide the tubes in easier.



Repeat this until all tubes are filled up and all your jars are full.


Step 5: Preparing Jars for PC

Now I don't know if this is absolutely necessary but, I like to take precautions and would not want my newly poured slants to get all 'splody in my cooker while they're cooking. So I fill my jars up with some water just about to the same level as the agar in the tubes. I try not to go too high since that water may get up into the slants and throw the content off some. Your welcome to follow suit or do your own thing...don't matter to me. I'm just showing you how I get it done.





Now that all your jars are filled with a bit of water and all your tubes are capped (loosely). Now its time to put on your grain lids that you use in all your grain jars. Tyvek filters, synthetic filters, whatman custom lids, whatever...you just need some filtered G/E like you would with grain for these. This lets air pass through and around it all without needing a sterilizer to vacuum seal the whole ordeal.




Step 6: PC & Cooling

Toss them all into the PC now. I do it for 15-20 minutes at 15 PSI which is suffice for agar. I fill my PC to match the height of the water in the jars to help slow down the cooling in the jars and slants. This helps reduce or eliminate any boil over you might find. Once they are all PC'd and the PC cools enough to open it up, take your jars up to the Lab/Room with your Hood or Glove Box.

Treat these jars like you would with grain jars. No different, keep it all clean and good. Now, open your jars up and when you open up the jars the first thing you do with the slant you pull out is to either snap the lid down tight (CulTube/Vac-Tainer) or screw the lid tight (Pyrex/PP Screw top). This way you stop contams from getting in while they cool. Then lay them down so your wood stick is on the bottom of the slanted agar and let them cool in that angle till your ready to go. They will stay sealed and contam free for a long long time at room temperature. I still have slants that are in VacTainers that I didn't use and are still sitting in my drawer clean and problem free over a year later. On the note of vactainers, when your inoculating them, be sure to keep the vent hole up. Its kind of a pain, but its absolutely necessary (with either glad-wrap or parafilm, both work). But you gotta have the vent up.





And there you have it. Slants to use for your precious cultures or for precious isolates of cultures your studying and working with. You don't even need petri dishes to transfer cultures to these (although it is highly recommended). On a side note, I did have a couple of my slants have half the water/mix in them then they did when it started. It happens every time I work with these and its only 3 or 4 (even within a jar of other full well made ones). So if it happens to you, don't be bummed out. One thing for the nay-sayers out there for the method of the agar, as I finished typing up this guide I went to check my tubes, all jelled and hardened nicely as they always have without heat treatement or 'heavy mixing' like some folks say you have to do.

I hope you enjoyed this and that it will suit you well for many many years. :sun:

Edited by Mephistophelian (02/11/12 03:39 PM)

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InvisibleFooMan
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Re: Mephisto's Master Slant Tek [Re: Mephistophelian] * 1
    #12968513 - 07/28/10 07:05 PM (13 years, 7 months ago)

Nice writeup :thumbup: Bookmarked.


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OfflineBreakfast Crew
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Re: Mephisto's Master Slant Tek [Re: FooMan]
    #12968779 - 07/28/10 07:59 PM (13 years, 7 months ago)

well done  :congrats: .  Your caps changed colors :wink:

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InvisibleBase Icks
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Re: Mephisto's Master Slant Tek [Re: FooMan]
    #12968846 - 07/28/10 08:11 PM (13 years, 7 months ago)

Quote:

FooMan said:
Nice writeup :thumbup: Bookmarked.



Same here. Thank you for your time and energy, you may have jsut opened my mind.

heres a quick one.. what size vacutainers? 10ml?

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OfflineDamion5050
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Re: Mephisto's Master Slant Tek [Re: Breakfast Crew]
    #12968852 - 07/28/10 08:11 PM (13 years, 7 months ago)

Very nice tek...

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OfflineMephistophelian
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Re: Mephisto's Master Slant Tek [Re: Damion5050]
    #12969114 - 07/28/10 08:59 PM (13 years, 7 months ago)

Thanks for the support guys. :grin:

I edited the Tek to answer your question Base Icks but yes, Vac-Tainers are 10ml and 16mm x 100mm where the CulTubes I used are 17mm x 100mm and PP not Pyrex (which drastically changes cost).


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Re: Mephisto's Master Slant Tek [Re: Mephistophelian]
    #12969565 - 07/28/10 10:21 PM (13 years, 7 months ago)

Wish I had read this weeks ago when I first started playing with culture slants. Very nicely done sir, I now know what to do for next time.:thumbup:

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Invisiblehamloaf
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Re: Mephisto's Master Slant Tek [Re: Mephistophelian]
    #12969579 - 07/28/10 10:23 PM (13 years, 7 months ago)

Like OMG!  This is just what I was looking for.  :super:
HL


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Invisiblehamloaf
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Re: Mephisto's Master Slant Tek [Re: hamloaf]
    #12970750 - 07/29/10 07:51 AM (13 years, 7 months ago)

Morning bump.  Yup.  :yesnod:
HL


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Re: Mephisto's Master Slant Tek [Re: hamloaf]
    #12970809 - 07/29/10 08:26 AM (13 years, 7 months ago)

sweet tek Mephistophelian, i just started doing slants to save my cultures, been to lazy before, strange thing is some come out perfect and the odd few just dont set right, i.e like wet jelly and no good to use/

Not sure if this cause im using small ammounts i.e PDA mix for only 125 ml water usually using premade mix,

I mix up real good using off boiled water before PC'ing.

Not sure what Im doing wrong?

Is it worth giving them a little shake once out the pc to make sure its mixed properly?


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Edited by FractalXplora (07/29/10 08:26 AM)

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OfflineMephistophelian
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Re: Mephisto's Master Slant Tek [Re: FractalXplora]
    #12970866 - 07/29/10 09:03 AM (13 years, 7 months ago)

Well the one thing you want to make absolute sure of is that your calculations are right (by that I mean you have the proper nute/agar ratio to water).

I think that there is the chance that your powders weren't mixed entirely before pouring into each slant. Each time I've done these like this, I've always made sure not to let my powders get too settled before putting into each. That being said, when doing larger batches, there is always a chance that you get the odd one that exploded though I've never had one not set. Occasionally I'll get some that are cloudy and others clear.

I think next time around...stir/mix your agar slurry very well before drawing the syringe or pouring into the funnel, but personally I would think that if you do pouring there is a higher chance of getting a poor mix of powders where as with a syringe you can draw it from the bottom/center while its all still swirling and agitated. That's just IMHO.


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Invisibleandymc
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Re: Mephisto's Master Slant Tek [Re: Mephistophelian]
    #12970953 - 07/29/10 09:42 AM (13 years, 7 months ago)

I've had trouble with boil-over when PCing slants.  Naturally I started out loosening the lids, but I've had a whole rack of slants end up empty that way.  Tightening the lids seems to stop it, but it just seems like a dumb thing to do.  You reckon it's because they're cooling down too quickly?  I let them cool naturally in the PC..


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Re: Mephisto's Master Slant Tek [Re: Mephistophelian]
    #12970961 - 07/29/10 09:44 AM (13 years, 7 months ago)

Nice teknology, also with pouring youre much more likely to make a mess. Do you use these for your long haul cultures?

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Re: Mephisto's Master Slant Tek [Re: badman]
    #12970981 - 07/29/10 09:53 AM (13 years, 7 months ago)

Cheers for the advice Mephisto.

I've just ordered a 100ml measuring clylinder as i been using a crappy kitchen jug. I like the idea of using the syringe to fill the slants, giving it an extra shake. Hopefully this will iron out my problems.


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OfflineMephistophelian
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Re: Mephisto's Master Slant Tek [Re: FractalXplora]
    #12971027 - 07/29/10 10:04 AM (13 years, 7 months ago)

@Andy:

The water filled inside the pint jars to provide a 'heat jacket' on the slants is what will help substantially with the cooling end of it. Just like when you heat a bottle/flask of agar, you need the water to sit 2/3 (at least) as high as your agar to reduce the cooling time as agar is extremely volatile. Even taking off the weight before your steam has finished venting (even though the PSI gauge has dropped off) can make a few explode. Using a rack (I don't own one so I'm just going out on a limb here) but I have seen them, and I don't have a clue how your supposed to have a water jacket around them to cool them down slower unless you just screw the lid on real tightly (kinda scary :shiftyeyes:).

@Badman:

I use these yes, the last handful I had used up was Vac-Tainers but I've moved to these as they are cheaper and easier to use the lids (Vac-tainers are a PITA for they're lids and very temperamental as you can see in the picture for the G/E hold). I'll take a picture of all my slants at lunch so you can see the different varieties. I plan on using Sterile water with tissue submerged with 0 GE (not to be mistaken with LC or Grain LC as those both still have nutrients in the water) as experimental for room temperature storage as well, though I would recommend against banking everything on that method since some species will die using that.

Edited by Mephistophelian (07/29/10 10:10 AM)

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Re: Mephisto's Master Slant Tek [Re: Mephistophelian]
    #12971274 - 07/29/10 10:57 AM (13 years, 7 months ago)

You gonna scratch a plate or submerge wedges in Eppendorf tubes? I checked up on some of my wedges in tubes and few of them have dried up but water has collected in the bottom most of them are fine. Im gonna start adding SDW to all now not only my own stock better in the long run.

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Re: Mephisto's Master Slant Tek [Re: badman]
    #12971572 - 07/29/10 12:15 PM (13 years, 7 months ago)

:yesnod:

:scaryshroom::thumbup:

:respect:


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Re: Mephisto's Master Slant Tek [Re: 13shrooms]
    #12971634 - 07/29/10 12:26 PM (13 years, 7 months ago)

Very nice Meph! Very detailed and the pics are really helpful.

Two questions- Does the shape of the wood really matter? I've been using a few long round 2mm dowels.

And- How many slants of each culture do you like to keep around in storage?

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Re: Mephisto's Master Slant Tek [Re: lazy bones]
    #12971847 - 07/29/10 01:07 PM (13 years, 7 months ago)

Great writeup.  Even took the time to include pictures.  Great work. :mushroom2: ~P~


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OfflineMephistophelian
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Re: Mephisto's Master Slant Tek [Re: lazy bones]
    #12972474 - 07/29/10 02:55 PM (13 years, 7 months ago)

Quote:

lazy bones said:
Very nice Meph! Very detailed and the pics are really helpful.

Two questions- Does the shape of the wood really matter? I've been using a few long round 2mm dowels.

And- How many slants of each culture do you like to keep around in storage?




Wood shape isn't terribly significant, as long as it doesn't severely hinder your agar slant shape and the like. (you know, like it being 1/2 wood 1/2 agar or something odd)

I actually keep 4 copies of each...I plan on making copies that 'library' and keeping it in another fridge off-site of my house/yard if anything were to happen all is not lost. I haven't gotten there yet since its such a massive under taking but...soon. Very soon.


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