Agar for testing

[SkorpiusLabs]

If you are home brewing and want to ensure your batches are clear and contamination free, then I strongly suggest becoming familiar with agar.

I understand many may have no clue what I’m referring to but I am happy to guide anyone that may be interested.

It’s a very simple and effective solution in any home lab for determining any contam issues present either within your product or in your lab in general.

If this interests anyone just let me know and I will build out this thread with much more detail on how to begin.

Cheers!

 

[Qualityoflife86]

I'm up for learning new things

 

[Zebedee]

I'm up for learning new things

Yeah, I’d be interested in further information too.

 

[SkorpiusLabs]

My pleasure guys

So agar in general is a wonderful tool once mastered, just in life in general for knowing what’s lingering in your spaces , on your tooth brush or utensils etc.

My experience stems from mycology where agar is the only real way to keep varieties alive and healthy. On an average week I would pour and use about 2-500 plates just maintaining my varieties. Needless to say during this time I have encountered endless types of molds, bacterias and other various contamination.

The reason I mention this is because I use to sell liquid culture and before I would sign a batch as acceptable for sale it needed to be triple tested and contam free.

The way this was done was simple
2 drops per plate infront of hepa and then seal , label and wait. Within 2-3days growth would be in full swing and if anything was present other than healthy mycelium it would stand out like a sore thumb.

The same goes for gear, after processing your batch randomly select 1-3 vials pending the size of the batch and simply extract 1cc from each. Lightly squeeze 1-2 drops onto your agar and then seal with para film and label with details of sample.

Since there is no mycelium (hopefully lol) you are looking for a clear plate, however if anything appears then your batch is likely contaminated

I can go into greater detail of types of contam but long story short in gear if anything grows please don’t stick it in you lol



Now for the boring stuff

Tools needed
Petri dish ( preferably 100mmx15mm but 90 is fine)

Media bottle (size pending volume of pour , I do 5ltr at a time so I stick with 1ltr bottles)

Parafilm

Hepa or SAB

Gloves

Iso

Pressure cooker

Large pot

Agar

Extra light malt extract

———-

Combine 10g agar / 8 g malt and 500 ml water into pot and warm til dissolved while stiring

Once dissolved (about 15min) pour into media bottle(s)

Cap and turn back a quarter turn so it’s just slightly loose

Cover with tinfoil

Load into pc on rack and ensure water is up 4”

Bring PC up to 15psi before starting timer, once at 15psi time for 35mins

Follow proper PC safety during the cook , I can elaborate if needed

When 35min is up turn off heat and let pressure reduce naturally

When pressure is gone move the pc to your clean area (pref infront of hepa) open and remove media bottle and let cool

When bottle has cooled to 100-120 it is good to pour

Fill each dish in the stack from bottom to top

Let cool and solidify (3-5 hrs) and then can bag for future use in the sleeve they came out of or can use right away


I apologize as I’m used to teaching people that are familiar with mycology so if I need to elaborate on anything just ask and I would be happy to do so

Cheers

 

[deeeznuts]

Thank you for this @SkorpiusLabs

 

[hijoshiki]

The problem with using agar is that you'll need an incubator with 10% CO2 at 36-37C for bacteria.

Of course you can incubate agar slants at room temp for fungus, but that's probably not going to be what you're after and that takes too long to grow before you can be sure it's negative.

There's lots of ways to investigate sterility, but it requires lab equipment if you want to do it correctly.

If I wanted to see if there were bacteria in my oil I would first dilute the sample (10ml vial) with sterile saline, centrifuge for 10min at 3000rpm using sterile conical centrifuge tubes, then removing that supernatant leaving 1ml of fluid in the bottom and using that to charge a cytospin slide adapter for a hemostainer adding a drop of albumin for good adhesion to a glass slide for a gramstain. If you don't see anything on the gramstain in 20 fields of high power 50x then you can probably be safe that you're okay to inject.

Obviously if you have a microscope you could concentrate the sample and charge a hemocytometer as you can see bacteria that are 2um in size fairly easy using phase contrast that make it easy to see bacteria and one of these microscopes for a cheap will cost you around $1000, the Olympus I used cost $17,000.

The problem with all of this is that you're gonna have to be rich or have lots of expendable income.

You're best bet is to invest in really good filters and double filter using 0.2um. And if you don't trust it then heat it at 200 F for 30 minutes. That should eliminate and kill most microorganisms both bacteria and viral. My guess is the biggest risk to most is deep injections and poorly cleaning the injection site which causes a staph infection.

 

[peedamn]

The problem with using agar is that you'll need an incubator with 10% CO2 at 36-37C for bacteria.

Of course you can incubate agar slants at room temp for fungus, but that's probably not going to be what you're after and that takes too long to grow before you can be sure it's negative.

There's lots of ways to investigate sterility, but it requires lab equipment if you want to do it correctly.

If I wanted to see if there were bacteria in my oil I would first dilute the sample (10ml vial) with sterile saline, centrifuge for 10min at 3000rpm using sterile conical centrifuge tubes, then removing that supernatant leaving 1ml of fluid in the bottom and using that to charge a cytospin slide adapter for a hemostainer adding a drop of albumin for good adhesion to a glass slide for a gramstain. If you don't see anything on the gramstain in 20 fields of high power 50x then you can probably be safe that you're okay to inject.

Obviously if you have a microscope you could concentrate the sample and charge a hemocytometer as you can see bacteria that are 2um in size fairly easy using phase contrast that make it easy to see bacteria and one of these microscopes for a cheap will cost you around $1000, the Olympus I used cost $17,000.

The problem with all of this is that you're gonna have to be rich or have lots of expendable income.

You're best bet is to invest in really good filters and double filter using 0.2um. And if you don't trust it then heat it at 200 F for 30 minutes. That should eliminate and kill most microorganisms both bacteria and viral. My guess is the biggest risk to most is deep injections and poorly cleaning the injection site which causes a staph infection.

You can make an at home incubator that works for less than 10 dollars

 

[hijoshiki]

You can make an at home incubator that works for less than 10 dollars

How without light?

How do you keep the temperature regulated, most pathogens have a window where they grow best?

How to introduce CO2 at 5%-10% and control for relative humidity around 95%?