Brighteyes' Lab Notebook: Pony Mold*Update 5/31 Cleaning Results & Microscope

[brighteyes]

********Update 5/31/2016*******

I made a very simple attempt to clean up Majesty 2 which involved boiling her for 15 minutes and using a Mr. Clean eraser and lots of elbow grease to clear away the mold on her surface.  I did not attempt to remove mold inside the pony with the eraser.  I wanted to assess first a simple method of cleaning to be followed by more extensive methods like using peroxide and UV to remove any staining.  I found after my simple method there was in fact staining left behind by the mold. 

I examined the surface staining under a dissecting scope at 40x and also took chunks of affected vinyl from the neck plug and examined those under 40x as well.  I found that the surface staining mimiced the mold growth pattern and even followed the mold hyphae in some cases.  The treatment also seemed to make existing rust stains on the symbols worse.  The most serious stain began to look almost like a cyst or mini-volcano.  It was raised up and puckered, looking like it may have fluid inside.  Gross! 

When I looked at the slices I found that fungal hyphae had innervated the pores in the vinyl.  I think maybe the staining is the result of hyphae that are still inside the pores of the plastic.  These cannot be easily removed by surface cleaning.  I think they will be killed by sustained exposure to hydrogen peroxide and UV light though.  My next step will be to try this and also to grow some of the mold on slides in mold chambers so I can examine it under oil for a really good look at it.

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Just for comparison.  Here are some photos of this pony before treatment after treatment and after cleaning:

Before Treatment (note pre-existing rust marks on symbols):
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After treatment, before cleaning:
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After treatment, after simple cleaning:
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********UPDATE 5/26/16*********

Spoiler

Wow!  What a difference five months makes!  We have near confluent growth on the Majesty inoculated inside with mixed mold culture and swabbed with culture outside with 10mL sterile water added to the jar:

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Compare this with a Majesty inoculated in the same way but with no extra water added.  What a difference humidity makes in the growth of the mold!
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We are seeing growth of mold through the body of Snuzzle who was innoculated only with liquid inside and was not swabbed on the outside.  This was to test to see if mold on the inside of the pony could affect the outside.  As expected, there is mold growing out of the tail hole.  We can see some mold growth on the outside of the pony as well.  Is this mold making it's way trough the pony plastic maybe?  We will have to watch this spot and see what develops.

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The Sundance Control has only one mold spot that was likely from me holding her to put her in the jar.  I couldn't really get her completely sterile so I expected a bit of mold growth.
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Pretty crazy!  Do you guys think the mold will leave marks?  When should I take the ponies out and try and clean them to see if the mold stains the plastic or not?

To view previous results and history of the experiment click the spoiler:

Spoiler

Update!

Aaaand we've got more mold!  Here's some photos.  More detailed post on page 3.

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We talk a lot about mold in the collecting community and we also talk a lot about the various marks and discolorations that can affect ponies.  There's pindot, mold marks, cancer, regrind, highlighter etc...   Some types of marks seem to be contagious and others not so much.  We have a good handle on what causes some (like Regrind) but others are still pretty mysterious.   It can be pretty scary putting ponies into storage without knowing how they'll look when we take them out again.  Will they get moldy?  Will the mold leave permanent marks on them, turning perfect mint ponies into custom baits???

Lets play with mold and ponies! I've been wanting to perform a few experiments to determine if any of the marks we see on ponies are caused or perhaps exacerbated by mold.  Later, depending on the results of the trials with mold, I'll look at possible effects of bacteria on ponies and finally the short term and long term effects of UV light exposure.  I manage a microbiology teaching lab at a small university and have access to fun sciencey stuff like microscopes, growth media and incubators.  Let's put it to use to answer some questions about ponies 

This "study" is based on a simple line of questioning.  First, can mold grow on ponies?  Does the vinly/plastic even support mold growth?  If it doesn't then it's pretty clear the marks we see develop on ponies can't be caused by mold.  If it does, then that opens up another question- is the mold that grows on ponies responsible for any of the marks we see?  Finally, if mold is responsible for marks, what, if any, treatments will work to prevent mold growth and therefore prevent these marks?

Okay- so based on that line of questioning, here's the plan.  The Pony vs. Mold experiment will take place in three to four phases:

• Phase One: Can mold be cultured from a pony?  Do ponies support mold growth?
• Phase Two: What does Mold DO to a pony?  If an unmarked pony is innoculated with mold obtained from plastic/vinyl surface, will it result in any classic pony affliction such as pindot, cancer, etc..
• Phase Three: Microscopic Examination.  Samples of classic pony afflictions will be examined under the microscope.  If any mold is obtained from pony surfaces and cultured in petri dish, this mold will also be examined.  Physical characteristics of mold obtained from ponies will be compared to anything found in the afflicted vinyl
• Phase Four: Mold Prevention and Treatment.  If it is determined that mold causes any discoloration or physical affects to the pony, what possible treatments could be effective at killing the mold?  Try Moldzyme mold cleaner, boiling water bath, OxyClean, UV light, etc...
• Final Phase: After the conclusion of the experiment, ponies will be cleaned using most effective mold killing treatment (whatever that ends up being) and customized.
  

I started phase one today.  First, I selected four really dirty ponies: Snuzzle, Sundance, Sweet Pocket and Majesty.  Photos are in spoiler below.  They come from a really gross lot I recently one and also from a local flea market. 

Spoiler

Snuzzle:
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Sundance:
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Sweet Pocket:
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Spoiler

Majesty:
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Using a sterile cotton swap and saline, I swabbed the surface of each pony in particularly dirty areas.  Then I inoculated the surface of Sabouraud Dextrose Agar (SAB) petri dishes.  This media is designed to grow a variety of molds and inhibit bacteria. This will help us eliminate bacteria from our equations for now.  We can always go back and do these same experiments using bacteria instead of mold and see if we get any marks or discolorations.  I also took swabs from a Medly which had, to my eye, spots that look like mold and a Flutterbye that has spots that look like cancer or rust caused by metallic glitter (photos in spoiler). 

Spoiler

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Medly with prospective mold.  Notice tiny dot in center with halo of discoloration around it.  To me, this seems different than solidly discolored marks indicative of cancer.

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Does Flutterbye have discoloration from metalic glitter on her symbols?  Pony cancer?  Mold? All of the above???  Let's find out!

I then went into our lab's cold room which has it's own fine array of mold growing on plastic surfaces.  Photos in spoiler:

Spoiler

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EWWW EWWW EWWW!  Time to clean out the cold room   

I swabbed each of these molds and put them on SAB agar as well.  Now we have a nice array of petri dishes.  No growth yet, but we may see some in a few days:

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I will take a photo of the dishes each day for several weeks to determine if anything grows and I will post any interesting results here.  It is possible that mold that grows on plastic or vinyl will not be supported by standard SAB agar and so we won't get any growth even if there is mold present in the pony plastic.  My guess is that we will get growth however, because the mold is feeding not necessarily on the plastic but on the oil residue left on the ponies after have been touched.  We'll see though!

If Phase One is successful, I will move on to Phase Two and inoculate new and older ponies with mold cultures.  The new ponies will be Dollar Tree fakies and the old ponies will be three Majesty ponies- the one shown above and another two baity ones I have.   Prior to inoculation the vintage ponies will be boiled and put in an oxyclean bath and photographed to note existing body markings.  The new ponies will be swabbed to check for "native" mold.  Two new ponies and two old ponies will be swabbed with a mixture of mold cultured from ponies (if any is cultured) or cold room mold and stored in a sterile jar in a dark location to allow for mold growth.  One of each type inoculated with mold will also have a layer of sterile water placed in the bottom of the jar to create a moisture rich environment. 

As a control, a new pony and an old pony will be placed in a jar with sterile water at the bottom to create a moisture rich environment in the absence of mold.  This should help us determine what change in the pony's condition is due to moisture and what change, if any, is due to mold.  I may try and spray the ponies with Moldzyme inside and out (a commercial mold killer) to try and get rid of as much mold that may be contaminated them as possible.  All experiments will also be performed in a BioSafetyCabinet (BSC) to prevent contamination from airborne mold spores.

If we get any mold discoloration on the test ponies I will take thin sections of the affected plastic and look at it under the microscope.  I will also swab the affected area and culture the mold in special mold culture chambers and observe the mold on slides.  This will allow me to see the structure of the mold and possibly ID it.

I will post pictures of the petri dishes every few days.  If there's any suggestions or questions or input or whatever feel free!  Ooo or should we take bets?  What do you think will happen?  Will the petri dishes from the ponies grow mold?  Will mold make marks on the ponies?  I know Black Curtains has done  a lot of science with ponies before- it would be awesome to compare results!  Her study on pindot and dirt was really cool (http://mlparena.com/archive/Forums//viewtopic/t=259561.html).  Maybe we could sterilize some dirt and see if that makes a difference? 

UPDATE: 12/27/15

Here is the basic rundown of the results thus far and some of the more interesting photos of mold growing on the ponies.  So far, the only visible mold growth is on the ponies that were inoculated with a surface swab of the mold mix.  The pony that was only inoculated inside (Snuzzle) and the sterile water control (Sundance) do not show any mold growth so far.  The Majesty with the addition of water in the sterile jar is showing substantial more mold growth than the Majesty with no additional water.  She has a large patch of it near her symbol.  Neither of the fakies are showing growth yet but they also did not have a surface swab.

So, now we know given the right conditions, mold can grow on ponies.  The question will be, does this growth leave non-removable marks?  I think it's obvious the addition of water will encourage mold growth.  I wish I would have added a treatment with a descant in the jar, because adding liquid mold culture to the inside of the pony alone will increase humidity in an enclosed space.  So there is no really good control for the humidity test 

Anyway, on to photos!

Spoiler

Sterile Water Control:
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Internal inoculation only, no swab:
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Internal inoculation, swab, no extra water:
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Internal inoculation, swab, 10mL extra water (here there be mold!):
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[NigheanRuadh]

This is great!!  I'm so glad you're doing this and will be interested to see the results!!

[buttonso]

How interesting! I'll follow this with great interest! 

[Sparkle Pony]

Oh cool!  I'll definitely keep an eye on this.

[Leave a Whisper]

Wow! I'm definitely stalking this thread.

[sd_dreamcrystal]

Going to set the Notify option for the first time for me!
This is going to be interesting!

[shelvesofwhimsy]

This is awesome! I'm so glad we have a scientist/biologist in the community who has access to a lab

[Sweet_Stuff]

Whoohoo!
This is great! Thanks for sharing and I'll be looking forward to the results as well!

[archidraca]

 

[Saber-Toothed Crow]

Fascinating! I will be following this with great interest

[Snapdragon]

Oooh, SCIENCE! How exciting!! Can't wait to see what happens!!

[PJSparkles]

Did somebody say BIOLOGY? -tapdances into thread with degree-

can't wait to see the results!

[rosierjay]

yes!! love this. *follows patiently*

[BlushingBlue]

It's wonderful that you have the opportunity to do these sorts of studies, although I really hope you aren't putting your job at risk by doing so. My old lab would have come down hard on me for misappropriating university materials to do pony experiments. They were pretty uptight though.

I'll be very interested to see what new data Phase One brings. It's been established since the 1940s that fungi readily utilize various PVC plasticizers and stabilizers as a carbon source, especially DEHP/DOP and DOA, but of course, we have no idea exactly what was used in the creation of vintage MLP, so it'll be exciting to see what species you can isolate from these experiments. It seems like yeast-like fungi have an easier time colonizing PVC plasticizers than strictly-speaking molds. Maybe we can extrapolate what was used as plasticizer by the types of microbes you do (or don't) find? It'll be fun to see if you can replicate Webb et al.'s research into pPVC colonization with Aureobasidium pullulans being the primary organism found on new ponies, and other fungi taking over after A. pullulans stormed the beach, so to speak, on vintage ponies.

Phase Two sounds like it's going to take a long time to get quantifiable results, since I imagine that, much like how a chip in a windshield slowly blooms into a series of cracks, "center dot mold" marks arise from the cascading physical/chemical weakening of the vinyl from the microbial colonization and its metabolic processes, and even its eventual successors and their own appetites for destruction. I hope we don't have to wait 10-20 years to move on to Phase Three and beyond.

Personally, I'd be interested in seeing what (if anything) results from an experiment introducing prodiginine-excreting bacteria (e.g. Streptoverticullium sp.) to a pony medium. I've often suspected that certain "highlighter" marks are the result of exposure to prodigiosin-like bacterial metabolites, but of course, I have no way of proving it. If you decide to tackle bacteria next, I think it could have some interesting results, even if you don't go in for a full ASTM E1428 type test.
Can you tell I'm super excited that you get to Do Science To It?

[TwistedRiver]

A lot of the scientific terminology goes over my head, but I ill definitely be watching this thread with interest.