Anomalocaris said:

But what about that one



It looks perfect to me

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Awfullotofshrooms said:

Here’s a couple fronts and backs

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Quote:

Somebody mentioned temperature requirements for Tamps, what is it, and even for agar ?

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fahtster said:

Hate to be the bearer of bad news but just from the pics, most if not all of those pan plates are infected with bacteria.  It’s a very very common bacteria that’s probably present in most mushroom grows but is only detrimental to less hardy mycelium species like pan cyans.  It’s that orange/brown/yellow/reddish tint you see on your plates. 

I’ve spent the last 7ish months trying to get rid of it in my cultures and it will not be extinguished through normal means of transferring away or cabin sequesters or pucks or really anything “normal”

I don’t know for sure but I believe it’s Serratia marcescens which is the bacteria that produces that pinkish hue around sinks and drains in bathrooms.  From everything I’ve read, it fits the description.  It’s very light so it’s airborne.  Strains of it can be translucent so you won’t even see it unless it builds up or the only time you will know you have it is because of how the culture starts acting.  It’s motile and can do something on agar plates called “swarming”.  It’s extremely fast.  It’ll keep up with pan myc and eventually outrun it.  It’s very hard to see on low nute agar <1%

If you take ANY agar with a transfer off an infected plate.. like the tiniest bit off the very leading edge, you’ll 100% take the bacteria with it.  The best chance you have at getting any kind of culture that might give decent results is to take aerial hyphae only off the leading edge.. like the tiniest bit you can manage using either a hooked scalpel or a very sharp needle (being careful not to dip it into the culture as much as possible.)

Here’s some examples of it in action..

So you’d look at these two cultures and think, “they look pretty decent, fully grown out.. aerial surface myc the whole way”..  (you can see the transfer in the middle is the tiniest bit of myc.. I scraped that off the very top of a leading edge of aerial myc on a donor plate



But then you turn the plate(s) around..



In the first pic ^^^, the yellowish tint is very faint around transfer and in the second pic, it’s more pronounced and fills out more.. those cultures are both fully infected.  The only reason you can see it in the middle is because that’s where it started and slowly builds up to the point of being visual.

Sometimes you can’t even see it plainly like that.  Your culture will just start acting fucked up.. the leading edge will cease to grow aerial myc and the mycelium will burrow down into the agar.  Here’s the beginning of that without the obvious brown/yellow lines


Here you can see where the leading edge has stopped and growth has continued inside the agar without aerial growth.. you can see the line of brown tint (actually when you really get close up on it with a bright light IRL, it’s more of a color band with layers of orange, yellow and red)


I think this is where the bacteria has outran the leading edge and the myc is actually following the bacteria inside the agar.  Idk any of this for fact, this is just what I’ve come to after looking at 100’s of plates with this shit on it. 

More examples of the leading edge starting to crash out


Before I stopped taking pieces of agar with my transfers (like tiny pieces of leading edge agar with the smallest bit of myc on it) and switching to aerial myc only, this is what my plates would end up looking like..


The shit is brutal.  Because it’s light and airborne, it easily infects an entire plate culture and is known (Serratia marcescens) to migrate on the tops of cultures so, as you can see, even taking aerial myc isn’t fully effective.

I’ve been using gentamicin antibiotics and the first round I did, I added pre sterilization… gentamicin is heat stable but degrades at prolonged times at those high temperatures.  The next round i did, I did syringe filter “sterilizing” where I added during the cool down before pour.  I’m hoping this makes the antibiotics more effective.  I did see some improvement the first round but not enough.

I’m also trying syringe filtered h2o2 soaks of my plates and taking small transfers of myc only from the soaks.. this is showing a lot of promise but I think I may need to do multiple rounds

The good news is that aggressive varieties/cultures can manage even being infected with it.  It’s so common.. in fact, I’ve seen it on almost every pan print I’ve gotten from someone else.. it’s in my own prints, especially anything I’ve taken from the jcm, which is most of them. 

I believe it’s the reason we mostly only see aggressive varieties in circulation.  Ttbvi, MiB, Texas etc.  it’s a weak infection to these aggressive vars.  I also think it’s why poor first flushes are commonly reported and then later flushes get better.  Substrates seem to “overcome” the weak infection.  It’s why less aggressive cultures don’t recover from a shake.  Ik there’s other factors too but this bacteria is so common and ungodly persistent.

A lot of ppl say they get those brown streaks and just ignore them and they’re grows aren’t affected… I think they are affected, I just think they have very aggressive and strong genetics and if they were to rid those cultures of the infection, their cultures would perform even better. 

For example, this plate..


Gave three flushes.  First one was shit.  Second one was a little better and then the third was this


All these grows were infected with the same bacteria… all gave poor first flushes with each subsequent one getting better and better


Pretty much any grow I’ve done in the last 8 months was the same.  And those are the ones that actually made it to fruiting… I’ve thrown away so many trays that never made it to fruiting because of this facking bacteria.  The one thing that all the trays that made it had in common was that they were aggressive varieties/cultures.

Again, this is all just what I’ve come to accept in my own growing experience with pans so take it with a grain of salt but I think it’s this one bacteria that makes growing pans difficult

@OP. You see these brown spots on your transfer in this pic?


That’s a pretty good sign that plate is infected

More examples… it really likes grain water agar.  Sometimes it shows up very pronounced


Sometimes it’s only noticeable if you really look hard.  This plate is also gwa and looks aight (the pink is the reflection of my finger)


Same plate flipped (I added a loupe so you can see the color band)


Glass cover slip cabins don’t do shit


Once it’s on a plate, the whole culture is infected.  I made the mistake of putting multiple cultures on a single plate to try and save plates and ended up infecting my entire culture stock before I knew what I was dealing with

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Nice post fahtser, I am glad I saw this. I have been seeing this red bacteria clear as day on the MIB plate I am working with currently but I just went to check the BVI plate that I sent to grain recently and I am seeing it there as well, forming a border around the agar transfer wedge. It wasn't visible when I incoluated the grain, but I am using low nutrient agar, ~0.75%.

MIB, clearly fucked:


I am going to try taking a tiny sample of just the myc like you said. My BVI plate has myc growing up the side of the plate so I may be able to get a clean sample there.



What about cloning? I finally have a fruit with a thick enough stipe to attempt to clone it using tissue inside the stipe. If I can get a clean sample, that shouldn't have the bacteria right? I guess it doesnt matter because I am going to try it anyway.

Quote:

If you take ANY agar with a transfer off an infected plate.. like the tiniest bit off the very leading edge, you’ll 100% take the bacteria with it.  The best chance you have at getting any kind of culture that might give decent results is to take aerial hyphae only off the leading edge.. like the tiniest bit you can manage using either a hooked scalpel or a very sharp needle (being careful not to dip it into the culture as much as possible.)

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I might have this on a few of my pan plates. Do these narrow yellow rings look like a symptom of it? I noticed that on some of my ps. hispanica plates too, but never on cube, ochra, or tampanensis plates. I'm really new to pans, so I wasn't sure if it comes with the territory.


I'm going to pour more low-nutrient plates tonight or tomorrow, do you think it's worth trying a hot pour on top of small agar wedges transferred from that pan india plate into an otherwise empty plate?