K.I.S.S.
The following method is a stupid simple and extremely effective way to deal with bacteria, no matter how bad it is.
It is very 'noobish' in its simplicity and the results show quickly. One of those things you have to try or you won't believe.
We know how dirty swabs can be sometimes. Those can easily give you the most disgusting looking plates you could ever lay your eyes on.
Maybe some spores managed to germinate and there's some helpless myc in the middle of the plate surrounded by bacterial gunk with all kinds of funny colors.
Good news is, you can save a plate like that from the trash can if it means something to you, without having to resort to costly antibiotics and all the necessary equipment required to use them.
These cultures originally came from swabbed plates that were heavily bacterial and were cleaned using this method. As you can imagine, myc looked like utter garbage:
I've tried cleaning very dirty agar cultures by transferring alone and it was useless.
I've also tried the sandwich method several times and either I didn't do it properly or it just doesn't work well when there's a lot of bacteria.
Bacteria would crawl under the sandwich, make its way up and flip me the bird from on top...
This method revolves around two principles:
- Making use of the good 'ol PF-tek substrate:
BRF, verm and water. Bacteria has a hell of a time on this substrate, It isn't just hearsay or old wives' tales. It will get slowed down to a near halt, allowing myc to get ahead.
- Taking the smallest sample possible from a contaminated culture:
And if possible, away from the areas where there's heavier contamination.
To achieve this, we are going to poke the culture with a thin needle to take a tiny biopsy.
This tek shares some things in common with
The Biopsy Method: A different way of doing LC, so I'm just going to copy some parts with a few modifications.
The ProcedureSETTING UP THE SAB
From left to right:
- The BRF puck.
It's just a little glass jar with some PF-tek substrate on the bottom. More about this later.
- The agar culture.
As you can see, the plate is upside-down to facilitate the operation. I look through the front of my SAB, so for me it's much easier to do it this way. If you work with your head over the box, simply leave the plate upright and open the lid to take the tissue sample.
- A 10ml syringe with some sterile water.
More details about this later.
All has been properly sanitized and the lids of the containers have been 'pre-opened'.
Find yourself a metal rack to raise the plates above the surface of the table or towel. I use my PC rack and cover it with a paper towel soaked in alcohol, which is not really needed but me likes it for stability.
THE 'POKE'
Now we're going to poke that nasty bacterial culture with the needle.
- First thing of course, is to flame the needle red hot. If you're using a torch 2 seconds will suffice, and always be careful not to melt the plastic hub at the base of the needle.
- In the SAB, squirt some water from the syringe to cool the needle. Sometimes a drop of water may hang from the needle after doing this. Flick the syringe with your finger to get the drop out.
- Take the agar plate, pick the spot farthest away from the more contaminated areas and poke it with the needle until you touch the bottom of the plate with the tip. Done.
To inoculate with the biopsy you have to squirt some water from the syringe into the BRF puck (see next step).
You can repeat this procedure to inoculate several BRF pucks with the same bacterial culture and using the same needle.
Now I'd like to talk a little bit about the 'poke'.
By poking I mean just that, poking a spot of the agar culture with the needle, fast and clean. It is crucial you do it this way, no fucking around needed.
There's absolutely no need to poke twice, or to do it slowly or to scrape the surface of the culture with the needle and of course,
NEVER squirt water into the plate.
The biopsy you take with the poke is literally invisible to the naked eye, so don't bother looking for it, not even with the help of a magnifying glass. There's just a clean needle, no agar, no myc, just a clean pristine needle.
It is by no means an exaggeration to say that we're inoculating with microscopic pieces of tissue, as long as you use a thin needle to do this, which you must.
This procedure works ~85% of the times on PF-tek substrate. Sometimes you will never see growth on the plate, so
you should always inoculate several pucks with the same contaminated culture.
INOCULATIONThe picture says it all.
Crack the lid and squirt some water from the syringe into the BRF puck.
A quick squirt will be more than enough to dislodge and expel the myc cells that got stuck inside the needle.
Screw the lid back on and call it done.
Now the waiting game. You should see the first signs of growth after 5 or 7 days.
This is slow, I know, but once the myc colonizes ~50% of the plate you can be damn sure there's a lot of clean and healthy myc there.
The bacteria that gets squirted into the pucks will get stuck hopelessly in one spot. It will usually end up on the bottom of the plate doing nothing.
We got some growth, now what?If the agar culture was severely contaminated I recommend waiting until the mycelium colonizes at least half of the plate. Now you can take a sample with your scalpel from the leading edge and trasnfer to agar. It can be tricky to take a sample from BRF pucks. Don't try to cut a wedge as you normally would with agar. Instead, use the tip of the blade to scoop up a small piece.
Or you could use the 'poke' method again to take a biopsy from the leading edge of the culture and use it to inoculate:
- An agar plate.
Most likely you'll see clean growth on the agar, although myc might grow ugly on the plate with all that water. I recommend taking a look at the section 'Testing a Culture for Cleanliness' from my other thread to know what to expect.
- Another BRF puck.
Just in case the culture is not clean yet.
It only took a single attempt for me to obtain clean myc every time I used this method, but in the event that it wasn't clean yet this second inoculation should take care of it.
Be careful when you poke the PF-tek substrate with the needle. Don't go all the way down to the bottom, poke it only superficially or you risk clogging the needle and when you squirt water from the syringe the jet will split in two, kinda weird.
I highly recommend keeping these BRF pucks until they pin and then transfer some pins to agar. The pins are going to be very clean and these BRF pucks pin like there's no tomorrow.
And these are the largest bastards I ever got from a BRF puck.
Prepping the PucksTHE CONTAINERSI use little glass jars that are awesome for this.
I recommend always using little glass jars with metal lids. Bulky plastic lids are to be avoided because they make inoculation more difficult.
Likewise, plastic containers like glad mini rounds aren't good for this for several reasons:
- The substrate will detach from the bottom of the plastic container very easily after sterilization. This won't happen with glass.
- They create more condensation than glass jars.
- The bulky plastic lids of mini rounds are a bitch to open if we compare it with metal lids.
- Mold spores can make their way under the plastic lids and into the plate (as crazy as it sounds) if you happen to have a heavy spore load in your house, and you need to keep these plates for long.
I drill a tiny hole through the lid and cover it with 3 layers of MP tape. Alternatively, you could use a nail to make the hole.
If you are going to be using little mason jars with 2 pieces metal lids I'd recommend pc'ing them with the metal lid placed upside down to make your life easier in the SAB.
PF-TEK SUBSTRATEAs easy as 2:1:1.
- Two parts vermiculite
- One part brown rice flour
- One part water.
I'm not going to go into the details of how to prepare PF-tek substrate because there's lots of info about it already. Simple stupid stuff.
With 80ml of vermiculite, 40ml of BRF and ~40-45ml of water you'll have enough substrate for several pucks.
Cover the bottom of the container with some substrate and pack it down just a little, enough to make the surface level. Then take a wet paper towel and clean the inside of the jars.
I like to wrap the jars in foil entirely. No need to use paper towels to cover the MP tape.
Sterilize the jars for ~70-80 minutes at 15 psi. Probably overkill but I like overkill.
Take the jars out of the PC as soon as the dialed gauge hits zero.
Prepping the SyringeI use a 10ml plastic syringe and a sharp 21G needle. Needles deteriorate very quickly after flaming them, so I'd recommend always using a new one per session.
Using a thin needle will ensure we're taking an extremely small sample, literally microscopic. This is crucial in order to reduce the amount of bacteria that gets transferred onto the PF puck.
Sharp thin needle for the win!
For anything myco and in general, luer lock syringes are always superior than luer slip syringes (google it up).
The difference is that the luer lock syringe allows a needle to be twisted onto the tip and then locked in place. This provides a secure connection and prevents the accidental removal of the needle.
Truth is, I've only used luer slip syringes for this because I can't find luer lock syringes locally. They work just fine but I feel I need to tell you what is best.
Fill the syringe with distilled water and try to get as little air in as possible (for the pic I filled the syringe carelessly and you can see a huge air pocket).
With 10ml of water you can inoculate lots of BRF pucks.
Always use freshly sterilized syringes. Don't cut corners here and never store syringes for later use.
Cap the needle.
Now you can do 2 different things before pc'ing the syringe for
15 minutes at 15 psi.
You either:
- Wrap the syringe in foil and introduce it in the pc always making sure the syringe is standing upright or the water will boil out during the pc cycle. To achieve this, you can prop the syringe against two jars or any thing that does the job.
Or:
- Find a bottle tall enough, filter it and put the syringe inside.
Bonus: Using Swabs to inoculate BRF pucksAs I mentioned before, swabs are often very dirty.
Agar is an excellent media for bacteria and they'll grow quickly and happily on it as we know all too well.
On the other hand, bacteria won't propagate so readily on PF-tek substrate, so why not use it to our advantage?
The process is explained below without pics. Fortunately and very recently, ReverendMyc very kindly offered an excellent, step-by-step pictorial of the whole procedure, click
here.
- First of all, prepare a bunch of BRF pucks. A single swab can inoculate a good amount of pucks using this method.
- Use a little filtered container to sterilize enough water to cover the bottom half an inch.
- In the SAB, take the swab and dip it in the sterile water to soften up the cotton fibers and to loosen up the spores. Don't over do it, 10 seconds is enough.
This won't work with a dry swab.
- Take your scalpel and use the tip of the blade to remove a tiny, spore-laden piece of cotton from the swab.
- Bury the piece of cotton in the BRF puck.
You can leave the swab on a clean surface between inoculations, like a clean piece of folded foil. Never put the swab back in the container with the water.
You could also sterilize an empty container and leave the swab there between inoculations with the lid cracked.
Once you see growth you can transfer to new BRF pucks using the poke to ensure a clean culture and then finish the job on agar.
That's it!