Mold in my Agar Slants?

[jnr1005]

So my first batch of slants, I did 20 minutes of pressure cooking, cooled, then slanted. I innoculated all but 2...and left the 2 in the cubboard. Went to inocculate the last 2 and one had this huge nasty growth in it. (Like a sooty fungus.) Cleaned and tossed that one.

Made a 2nd batch of 12 a week ago and left them in a container on top of the fridge. (These are sealed in vials with screw on lids. ) Today I checked on them, and 2 of the 12 have a white spot of what looks like soon to be hairy mold growing.

I thought pressure cooking for 20 minutes was long enough to kill off all bacteria? How am I ending up with stuff growing in my agar slants? And now I am a little concerned about my first slants that I did a day after making them. What if they have that spore in them, but by putting them in the fridge I am inhibiting growth, but not eliminating the possibility of minute bacteria being in them...

Ugh.

 

[Riot]

Do you have a pressure gauge on your pressure cooker, or is it a single setting one? Most of those only get about 8-10 psi, which won't get to the 250F required to destroy spores. Your best bet is to make slants a week or so ahead of time and let them incubate at 70+. That way you will know if they are contaminated before you inoculate them with a yeast sample.

 

[jnr1005]

I guess I will just have to do that then. I bought a pressure cooker from Fagor that is supposed to be a 1 setting (high 15 psi) setting only. There is no gauge so I can't gaurentee it hits it, but I would think it would come close since it is supposed to be set for that.

 

[Riot]

Maybe try leaving it on heat for longer, might give you a lower contamination rate.

 

[Begin2Brew]

I have been struggling with this as well. I made 8 plates my first batch and only 2 survived a week. The next three batches all yielded no useful plates. I have tried baking and pouring the plates without putting them in a pressure cooker. I have done the pressure cooker and wrapped in electrical tape. Almost every process I tried did not end well, until my last attempt.

I took and made the agar, poured the plates and wrapped them in aluminum foil around the edges. I then tossed them in the pressure cooker at 15psi for 30 minutes. So far it's been a week and they are all good.

 

[Riot]

I'm kind of surprised you guys are having such a high failure rate. I lose about %15 with just a non pressurized boil because I don't have a pressure cooker. I would think even 7psi would be an improvement on that. Maybe try boiling just the water the day before you mix your substrate, it's possible your water has an usually high spore concentration

 

[Bsquared]

Mold spores can be very tough to kill, If you can get some autoclave tape and and put it on you plates, it will change color if the time and pressure was sufficient to sterilize your medium. Typically an Autoclave runs at 15psi and 250ºF for 15-30min.

 

[jnr1005]

I'm going to try for 30 minutes next time and see where that gets me. (They gotta die sometime..)

 

[kombat]

My first batch of slants, I had an 80% contamination rate. Like you, I used a pressure cooker, but for 15 minutes instead of 20.

The second time, I had much greater success, but I did several things differently:

• I pressure-cooked them for 20 minutes instead of 15
• After the pressure cooker cycle completed, I wore gloves instead of using my bare hands, and I dipped my gloved hands in StarSan before opening the pressure cooker and touching anything
• I affixed the caps onto the vials while they were still in the pressure cooker, rather than lifting them out and putting the caps on out in the open air.

With those changes, my success rate jumped to 100%. I always leave my slants for at least 2 weeks at room temperature to see if any are contaminated before innoculating them with yeast. I am equally meticulous regarding sanitation during innoculation (gloves, dipped in StarSan, flaming the lip of the vials before and after streaking, working near an alcohol lamp, etc.).

Since then, I've bought a proper pressure canner that will go to the required 15 psi, but I haven't used it yet.

 

[EarlyAmateurZymurgist]

I have been struggling with this as well. I made 8 plates my first batch and only 2 survived a week. The next three batches all yielded no useful plates. I have tried baking and pouring the plates without putting them in a pressure cooker. I have done the pressure cooker and wrapped in electrical tape. Almost every process I tried did not end well, until my last attempt.

I took and made the agar, poured the plates and wrapped them in aluminum foil around the edges. I then tossed them in the pressure cooker at 15psi for 30 minutes. So far it's been a week and they are all good.

That's the wrong way to make plates. Plates should never be placed in a pressure cooker. That's a sure-fire way to make plates that contain so much condensation that they are almost guaranteed to grow mold.

When making plates, one should dry sterilize one's petri dishes in the oven at 350F for 90 minutes before allowing them to cool to 150F (dry sterilization kills live microflora and spores via oxidation). The media that will be poured into the plates should be autoclaved (pressure cooked) for 15 minutes in a covered container and allowed to cool to between 120 and 140F. The petri dishes are then removed from the oven and poured while they are still hot. It takes a little practice to be able to lift a lid and pour a plate in one smooth motion, but anyone who is willing to invest a little time can master the process.


Here's what properly poured plates look like:

Did you notice that the plates in the photo shown above only have a thin film of condensation?

All plates should be "proofed" at room temperature for several days. Any plate that makes it through the proofing step should be sealed with parafilm and refrigerated upside down in a sealed container.

 

[EarlyAmateurZymurgist]

If one is using proper screw-cap culture tubess, autoclaving (pressure cooking) slants for fifteen minutes at 250F @ 15 PSI is all that it takes to kill live microflora and spores. I have never had a slant go bad in over twenty years.

 

[MichaelBrock]

[*]I affixed the caps onto the vials while they were still in the pressure cooker, rather than lifting them out and putting the caps on out in the open air.

I pressure cook mine with the caps fitting loosely to the vials (a few I accidentally screwed down with no issues) and have never had a contaminated slant.

 

[Begin2Brew]

I pressure cook mine with the caps fitting loosely to the vials (a few I accidentally screwed down with no issues) and have never had a contaminated slant.

And here is the reason I went with wrapping the plates in tin foil. I made slants as well and they are all clean after 6 months. So I wanted to have my plates covered when I sterilize them. I have almost no condensation after a couple of days and none of the plates have any mold or anything else growing in them.