Pangea Hatch Boil Warning

[Natural]

Hey everyone sorry to spam the forums I was just asking for help about an egg the other day and now I'm back with some info. In my other post (https://ball-pythons.net/forums/show...672-Egg-Advice) I noted that my egg was dying and moldy and some of my other eggs had mold. This was super weird to me since I've incubated ~10 clutches before with no issues even close to the ones I experienced here. I realized that the only thing I did different with this clutch compared to my previous ones is that I decided to reuse Pangea Hatch for the substrate instead of using new. For those that don't know, Pangea Hatch is a clay incubation medium that advertises that it can be boiled and reused (https://i.imgur.com/pGIek0l.png). I brought it to a rolling boil for 10 minutes stirring frequently, gave it a rinse, and then used that for my substrate. About a month later my eggs had mold on them and problems continued to escalate until sadly the entire clutch was lost by day 65. I decided to investigate if perhaps reusing the substrate was the problem. I purchased sterile applicator swabs and dextrose agar plates and set to work.

I swabbed the substrate currently in my incubator (top plate), some older and dried substrate that had been used for a previous clutch (second plate), and then I boiled those two substrate samples for 10 mins in the same way I had done prior to reusing and swabbed it a few minutes after boiling (third plate). Lastly, I opened a fresh bag of Pangea Hatch and gave a sample a swab (last dish). The then placed these dishes into my incubator at 88 degrees F and allowed them to culture for 3 days.



Unsurprisingly the top two plates were teeming with bacteria after having eggs hatch in it. Unfortunately, you can see several spotty bacterial cultures and a large mold culture managed to survive the boiling. The fresh bag of Pangea Hatch shows no signs of contaminants, the only thing visible in the plate is the markings from the applicator dragging along the dextrose.

This little experiment showed me that boiling and reusing Pangea Hatch can certainly introduce pathogens into an incubation environment. I take full responsibility for what happened to my eggs, and I will obviously never do this again in the future. Hopefully this will also help guide anyone who stumbles upon this post that is considering reusing Pangea Hatch.

[Lord Sorril]

Eggs, and even entire clutches die all the time. Don't take it personal.
There is going to be bacteria and mold in the wild all over the ball python eggs anyway.

As for your experiment--I commend your effort to use science!
Speaking as a Microbiologist:
-If you did not collect and plate those samples using aseptic technique in a sterile biological fume/flow hood then your results will be inherently inaccurate.
-If you had opened and cultured control plates that monitored the environment for the same duration that you were collecting your samples and they were *miraculously clean* (which they wouldn't be) then you could make a subjective argument.
-In addition there is various types of media for growth plates. I am not aware if the same bacteria that grow on TSA or SDA media are also capable of harming snake eggs...1 colony or 1 million, it doesn't make a difference if it is wrong type of mold or bacteria

Anyhow, my two cents!

[Natural]

Eggs, and even entire clutches die all the time. Don't take it personal.
There is going to be bacteria and mold in the wild all over the ball python eggs anyway.

As for your experiment--I commend your effort to use science!
Speaking as a Microbiologist:
-If you did not collect and plate those samples using aseptic technique in a sterile biological fume/flow hood then your results will be inherently inaccurate.
-If you had opened and cultured control plates that monitored the environment for the same duration that you were collecting your samples and they were *miraculously clean* (which they wouldn't be) then you could make a subjective argument.
-In addition there is various types of media for growth plates. I am not aware if the same bacteria that grow on TSA or SDA media are also capable of harming snake eggs...1 colony or 1 million, it doesn't make a difference if it is wrong type of mold or bacteria

Anyhow, my two cents!

I appreciate your input on what I did! I definitely agree the experiment was not what I would consider high science, between my frustration at losing the clutch and what I was able to get my hands on I did the best I could to get myself an answer. I guess most of all I was just surprised that anything at all could survive the boiling. I was certain that at the very least that plate should have nothing culturing. I suppose it could be contamination, but until I find myself a hood I'll probably never know haha. I also do agree that I have no idea if the same mold that harmed my eggs was the same that was growing on the dextrose and I'm definitely in no rush to run any tests to see if what's growing on the plates could harm eggs . I guess I could maybe scrape some mold off the eggs and see if it grows on the dextrose but for all I know a separate mold that had spores on the egg could grow and then I'll never know anyway. If you have any ideas for how I could tease any additional answers out of this please let me know! I think even with the potential pitfalls of my little experiment I'll still probably shy away from reusing the substrate ever again. I reused substrate for three clutches and all three ended up having pretty severe mold. Two have already died (they were in the same egg bin from last post) and now a third that's meant to hatch this week has several eggs covered in the same black mold. It's just a very very frustrating time for me.

[Malum Argenteum]

Is Pangea Hatch just Turface? Sure looks like it.

[Armiyana]

I also wonder if rinsing it with a non-sterile liquid could also be a factor. Like... fully rinsing it free of debris before boiling instead of rinsing afterwards.

definitely a neat experiment. Oddly I've just had terrible luck with gecko eggs the last few years so I have stuck to my BPs for now. I might do cheap sponges in the future with egg crate as far as for the humidity though... my cats apparently love trying to find the vermiculite or pearlite and make a mess playing with it. X_x

[Lord Sorril]

I reused substrate for three clutches and all three ended up having pretty severe mold. Two have already died (they were in the same egg bin from last post) and now a third that's meant to hatch this week has several eggs covered in the same black mold. It's just a very very frustrating time for me.

Ahhh two clutches dead. I would be super upset too.

If I thought hard enough about it I could give you schematics to build a Walter White style microbiology setup in your basement. Of course that is a bit of overkill, and by the time you were done you would have spent an epic level of effort. It would easier to send a sample of the mold that is causing you issues to a laboratory then they could ID it using their equipment. IDEXX charges my BioPharma $400/sample to do that-they would probably charge you more. Not sure if there is any smaller private options...I have never checked.

The way I see it: Three clutches and all three with mold narrows down your options considerably:
-The substrate is contaminated. (solution: use different substrate)
-The water is contaminated. (solution: use only distilled water)
-The incubation housing is contaminated. (solution: disinfect the housing prior to use with 1:9 Sodium Hypochlorite (Liquid Bleach):Regular Water
-The eggs are contaminated by the environment immediately when laid. (solution: ?)
-Your hands are contaminated. (solution: put on latex gloves and then coat hands in 70% Isopropyl alcohol and let them air dry prior to handling eggs)
-Incubation failed and the eggs died as a result. (solution: review incubation design)

The benefit of knowing the identification of the mold is that you can figure out exactly where it came from and how to prevent/destroy it.

[Natural]

Ahhh two clutches dead. I would be super upset too.

If I thought hard enough about it I could give you schematics to build a Walter White style microbiology setup in your basement. Of course that is a bit of overkill, and by the time you were done you would have spent an epic level of effort. It would easier to send a sample of the mold that is causing you issues to a laboratory then they could ID it using their equipment. IDEXX charges my BioPharma $400/sample to do that-they would probably charge you more. Not sure if there is any smaller private options...I have never checked.

The way I see it: Three clutches and all three with mold narrows down your options considerably:
-The substrate is contaminated. (solution: use different substrate)
-The water is contaminated. (solution: use only distilled water)
-The incubation housing is contaminated. (solution: disinfect the housing prior to use with 1:9 Sodium Hypochlorite (Liquid Bleach):Regular Water
-The eggs are contaminated by the environment immediately when laid. (solution: ?)
-Your hands are contaminated. (solution: put on latex gloves and then coat hands in 70% Isopropyl alcohol and let them air dry prior to handling eggs)
-Incubation failed and the eggs died as a result. (solution: review incubation design)

The benefit of knowing the identification of the mold is that you can figure out exactly where it came from and how to prevent/destroy it.

I definitely agree with what you noted as my options. I think my major deciding factor is that I have had 5 clutches this year, 3 went poorly and 2 went great. The only difference in procedure between the successful clutches and the failed clutches was new vs. used Pangea Hatch. My first two clutches I had new stuff leftover so just used that since I had it, then I switched to the boiled substrate once my new ran out thinking it was a good way to reduce waste and save some money. That seems to have backfired majorly. I have one last clutch that just got laid a few days ago that I have on new substrate so curious to see how that one turns out, but I have hope that it will go well. If it doesn't I might very well spend the money to get the mold identified and commit myself to war against it for the rest of my life. It's just been a very very frustrating last few months dealing with the fallout from this.

[Natural]

I also wonder if rinsing it with a non-sterile liquid could also be a factor. Like... fully rinsing it free of debris before boiling instead of rinsing afterwards.

definitely a neat experiment. Oddly I've just had terrible luck with gecko eggs the last few years so I have stuck to my BPs for now. I might do cheap sponges in the future with egg crate as far as for the humidity though... my cats apparently love trying to find the vermiculite or pearlite and make a mess playing with it. X_x

The risk of rinsing is a very good point that I had also considered. I actually decided not to rinse the substrate post boiling for this experiment for that reason. I figured my sink head would almost certainly have some bacteria on it so I ended up boiling for 10 mins, and then took a small strainer that I had soaked in some 70% ethanol and dumped a sample into the strainer. Then I swabbed the center of the sample and moved forward with that. Still possibly could be contamination from the top of the pot while I was dumping it out or something similar, but I did the best I could to really just isolate the sample.

The only thing I can think of it that the clay is super porous which is kind of the selling point of it because it allows it to absorb a ton of water. So I'm wondering if those pours allow for bacteria or spores to sit safely away from the super lethal temperatures and gives them a chance to survive. I wonder if boiling for 20 mins or 30 mins would make an improvement, but at that point boiling for so long is almost just not worth it.