17 replies to this topic

[Severian]

Howdy agar heads

Been upping my skill level in the agar game! Woo! Still no expert but I'm it's becoming more routine

However, I'm finding the continual transfer of contamination- specifically bacterial- when transferring between plates to be irksome- so I'm investigating the use of antibacterial agents in the agar mix.

I'm wondering about your experience here- I've read that many folk use gentamyacin- how much do you add, and in what form?


Edit: according to info found in this thread https://mycotopia.ne...antibiotic-agar
It's gentamiacin sulfate which is added to the agar prior to sterilization because it can withstand pc temps. And, an internet search , turned up 25g for 200$ ... I'm imagining that means it's likely the other internet search that turned up 250 g of Gentamicina Oxitetraciclina in powder form for agricultural use for about 20 dollars - is probably not going to be effective- but hey at that price an experiment may be worth it.

Also, are there downsides from the perspective of colony health when using antibiotics?

Edit: Coors Mikey - No matter what though when I use antibiotic agar I always notice slower germination times.


First photo- an example of the type of contamination which most commonly enters/contaminates my blank plates during pouring


Another contam photo- this is transfer #4 from a clone of gt-. First time ive ever seen that red I think

Edited by Severian, 23 November 2022 - 11:02 AM.

[Arathu]

Storage was a problem for me...I lost the last batch of gentamyacin I bought (probably to humidity I suspect) That's not chump change right there either....

It did work to clean up some fungus from dirty prints, and I have also seen bacterial infections that are likely beneficial (I suspect symbiotic) too.....

Perhaps keep in mind that things turning colors MAY be sporulating....I tend to bleach things that look like that last photo (but I will try to coax a mycelium to leap upwards onto a piece of agar suspended above the contaminated plate then transfer)

I've also seen bacteria that must be inside the fungus come along and never get "cured".....it's an incredibly connected world in which we play....

A

Edited by Arathu, 23 November 2022 - 07:16 PM.

[Myc]

I've been warned that red and pink organisms can be pathogenic. Be sure to wear an N-95 and use a still-air glovebox to handle samples. Wear your mask while Lysol-ing the hell out of the glovebox once you're done. Lysol - even with its bad rep in use for fungi - kills nearly everything.

 

It looks like your sterile technique may be suffering a bit. Once the mycelium begins to run you should make a transfer. You may have to make several of these transfers before the sample you snag is free of contamination - but it can be done. Always try to grab material from the growing edge as opposed to the center mass. I didn't see from your post as to your method of starting the culture - spores, live culture, field-collected sample, etc.

 

Your first photo is most likely a wild yeast. I propagate yeast deliberately and that's exactly what it looks like on agar. If you like hooch you could try inoculating some wort and see if a fermentation happens. ;)

 

The second plate is hopelessly contaminated. Destroy the sample and take care in the process so as not to inhale any spores from the red stuff (I'm guessing it's pseudomonis bacteria).

 

Best of luck on your next round. Sourcing gentamycin sulfate is difficult. Sporeworks used to carry it in small quantities.

You can also research aureomycin as a post-sterilization additive.

[Severian]

Major agreeance with the observation that my sterile teq is suffering- still getting the feel for this still air box- and absoluty the second plate is hopelessly contam- I generally don't even attempt transfers from anything that looks like that or /mold/trich....

Parte of the issue I've been having I guess is because of the yeast you identified- in many plates, it takes over the blank within 2 days- not even giving then mycellium a chance to run

Thanks for the advice re.when to transfer- I've also been waiting too long, trying to look for good a growth before transferring- as opposed to taking a sample as soonas it starts running.

[WelcomeUniversal]

Hello! I use levomycetin and Gentamicin sulfate in every msh growing.
Gent is useless for agar/grain, becouse u can not boil it. Its awesome works for LC and multispore water. I add 1ml (40 mg) to 10ml of LC.
Levo is cool for grain and agar, i add 500mg tablet to 2 liters of water, then boil it and add what i want.

About damage and storing:
Cubensis - doesnt care about levo and gent with this dosage. U can add everywhere. U can store during 1 year without any problems.
Panaeolus - dont like them. If u use gent, make sure, that spores contact with gent 30 minutes, NOT MORE. Spores really can die couse of this. I didnt test it with myc, so dont know.
Galindoi/tamp - in this dosage spores and myc become more slow, but i didnt see any negative effects. But i didnt test storing long time for sclerotia. I add regular dosages to grain and spores, about 20 days for colonization (twice slower than cubs), than i case and got fruits and sclerotias from cakes without any problems.

U could use hydrogen peroxide too, awesome thing on agar transfering, cloning or g2g - it kills all spores and bacterias, but doesnt touch myc. Just put a myc part to hydrogen peroxide 3% for 30 seconds and go!

Good luck, i hope it helps.

Edited by WelcomeUniversal, 25 November 2022 - 02:20 PM.

[Severian]

Mega awesome thanks welcome

I have a feeling the peroxide trick might be a lifesaver! Simply dip the agar wedge with the mycellium in peroxide before transferring- and voila?

[Myc]

Not so much.

You'll want to pour peroxidated agar - which is the addition of H2O2 after pc-ing your agar. You have to wait until temps are below 100*F to add the H2O2 or it will be destroyed.

You cannot start spores on this medium. They will be destroyed by the H2O2.

[WelcomeUniversal]

Mega awesome thanks welcome
I have a feeling the peroxide trick might be a lifesaver! Simply dip the agar wedge with the mycellium in peroxide before transferring- and voila?

Yeah. But be sure, that:
- u dont take contam mic(tricho, mucor and so on) from agar. It doesnt works with myc, only spores and bacterias.
- ur airbox/sab is sterile. Peroxide(when contacts with air) immidiatly destroys to h2o and o2. U still have a chance to get bacterias from air on ur sterile agar and sterile msh myc.

Edited by WelcomeUniversal, 26 November 2022 - 07:29 PM.

[Ferather]

I've used black tea agar plenty of times to know it works, but I can't compare to gentamyacin. The reason I use it is because it contains no carbohydrates, and its easy and cheap to get.

 

                    

 

As a test, you can leave out some exposed black tea agar and see what happens.

 

 

Jemds.com -- You can Google more, key words: Black tea antibacterial.

Edited by Ferather, 27 November 2022 - 05:12 AM.

[Severian]

Thanks for all info

Got a round of green tea agar mix in the PC now

25 g green tea steeped in boiling water for 10 minutes
6 grams- dextrose
10 g agar
500 ml water

@ a pH of 6. According to test strips.

We shall see!

[smellitstinknot]

Another method to clean a bacterial culture which is very simple and doesn't require antibiotics/h202 is the sandwich tek as it's known. Just cut out a wedge of agar from a fresh plate large enough to cover your contaminated mycelium and carefully place it on top. Within a few days you'll see fuzz growing through the wedge. You then scrape the surface with an inoculating loop or scalpel and streak a fresh plate. You should soon see visible growth and have a perfectly clean culture.

[Ferather]

@Severian, I have not tried green tea, only black. Let me know how that works out. There are a few reasons I use black over green, black tea has undergone oxidation, which turns it brown and more acidic.

This is much the same as green wood over brown wood (see here). Many fungi can breakdown and convert plant phenols, polyphenols (example lignin) into sugar and other materials.

 

The black tea recipe I am using, and posted images, is just black tea, no malt extract or any other sources of carbohydrates (sugars), keeping it sugar free.

[Severian]

So update-

The green tea agar worked to eliminate what I now understand to be most likely yeast- the super fast spreading contam mentioned in op- a few of my plates carried over bacteria from infected plates- but it seemed this was pretty strong bacteria to me- coming from outdoor clones-

Had plates contam with trich as well- or some other variety of green mold)



A further question as something I saw you say ferather in another thread made me think of it-

Does firmness of the agar (which is essentially a measure of moisture to agar ratio right) affect contamination growth?

A less firm agar being more 'wet' - this more inviting to contams? I noticed this round I used less agar and my plates were much less firm than normal

[Severian]

Next round I'll go carb free

[Severian]

Well after 2 days on the carb free green tea- zero signs of contamination of any sort.

[Arathu]

Boom! Agar and experimentation RULES!

 

A

[Ferather]

I am glad to hear the results. The firmness should reduce the chances of contamination, I see more contamination from liquid cultures of the same recipe as agar (minus the agar), cant say I have directly tested that though.

To my knowledge, bacteria don't do as well on firm-solid surfaces, however at the same time, the same recipes used to grow fungi can also be used to grow yeast and other micro organisms.

 

I usually add a little extra agar when making black tea agar (no carbs), also my water is hard water (contains calcium).

 

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Gluconeogenesis - Wikipedia

 

[Ferather]

Here are some images where you can see the oxidative enzymes-reactants being secreted by the mycelium, into black tea agar, turning the tea even darker (black ring).

Reminder, when tea oxidizes it turns from green to brown-black, like wood. Reminder 2: Phenolic content in tea - Wikipedia.

 

Notable post: Cubensis and gluconeogenesis from plant phenols