Wort Contamination?

[emccoy]

I'm attempting to propagate yeast in our lab for the first time as detailed by: https://discussions.probrewer.com/fo...-of-yeast-sops

We're doing a 1bbl batch instead though, so it's smaller scale. We also don't have an incubator/shaker, so my flasks with wort that are waiting to be inoculated are just sitting in the lab.

I made the wort using DME (10.1p), and I pasteurized it in the flasks for 30 minutes. If I needed to open them again (except to inoculate), I re-pasteurized them. (I originally prepared 16p wort, opened them to dilute and re-distribute 10.1p wort, and then re-pasteurized everything again). The flasks were kept sealed during pasteurization with foil and parafilm on top and around the neck.

Additionally, when I open the flasks to inoculate them/take cell counts, I am wearing an N-95 mask, I have sprayed the outside of the flasks and the table with isopropanol, and I have a flame going for 10 minutes on the benchtop before I open anything. Before I start, I spray my gloves with isopropanol.

However, despite all of this I have an odd film on top of my un-inoculated wort. It looks concerning, so I was wondering if everyone else thinks its contamination too.

[dick murton]

Looks horribly like wild yeast or bacterial contamination.

When you say pasteurise - what temperature?

The only (almost) foolproof way is to boil for 15 - 20 minutes, rapidly cool with filtered air on top - in practice this is sterile cotton wool and as soon as cool, pitch. If you are forced to take of wort and keep it for a few days, sterilise, cool and keep as cold as possible in a fridge and just before use, resterilise.

Pasteurisation, assuming you mean heating up to circa 65 C, is simply not hot enough. Pasteurisation does not guarantee sterility.

[emccoy]

Ahh, I see

I was using the wrong terminology-- I made the DME wort in a pot and boiled it for 15 minutes before pouring off into the flasks and sealing with foil/parafilm. I then put the flasks in water, and tried to keep the water around 180-190 F (82-88 C) for 30 minutes, but since I'm using an out-door style open flame setup, it's hard to get lower temps, so it was actually around 200F (93 C) for 30 minutes.

I'll try looking into sterile cotton wool, thanks! Maybe my foil/parafilm seal isn't as good as I originally thought.

I also didn't think of keeping the flasks in the fridge. I'll try this too!

[Lallemand Eric]

Seems like your main issue was temperature not being high enough. Boiling will be much better than 93'C. If you invest in a small hot plate or heated stir plate you could boil the wort in the flask itself and avoid having to transfer it from a pot to the flask. 15-20 min of boiling should be good, but don't start the clock until you reach an active boil.

You might also consider investing in a small autoclave/pressure cooker. A few hundred dollars goes a long way for peace of mind, especially if you will propagate regularly. The next time your contamination might not be so visible and could result in a spoiled brew.