I am so glad you asked! I unfortunately lost the protocol because it was probably on my laptop, but I remember the broad strokes. So! In case anyone does end up stuck in the middle ages and can find a kindly old alchemist willing to lend you his gear, here's the revamped Penicilin (Re)Discovery Protocol!
0. WASH YOUR GODDAMN HANDS.
We're not working in a lab here, cross-conatamination WILL happen. Your job is to minimize it as much as possible. If you end up in a place where soap hasn't been invented yet, wash your hands in distilled alcohol. Your skin won't thank you, but you can afford all the nice hand creams after you cure the plague and get rich.
Find some Penicillium mushrooms!
Yes, penicilin is produced by mushrooms, though Ascomycotes are usually called moulds, it's a fungus, and it makes me laugh to call it a mushroom. Plus, in the middle ages, mushrooms were known to have medicinal properties, so you'll get a lot farther by calling them mushrooms rather than molds.
First thing you need: mouldy fruit. Oranges, or cantaloupes are preferred.
Here's the thing: mold is everywhere, so getting it will be the easiest part. The tricky part start with identifying the correct mold. You don't want to feed your patients black mold, do you?
So. Leave some fruit out. The more the better, because you want to up your chances. Then let it rot in warm and humid places. After a while, pick any fruit that looks white on the outside and green in the middle:
Not the best picture, but that's what it should look like.
2. Transplanting your (potential) Penicillium mushrooms
Until you get it on a plate it's damn near impossible to tell which mold you got. Get ready for some trial and error because you will have to sift through a lot of unwanted mold. You might want to wear a mask.
First you need something to transplant it onto. Making modern agar plates is probably impossible but thankfully not needed. You just need:
Glass plates (the kind that can be closed, you want to minimize cross contamination)
1-2 cup of Hot water (preferably distilled, ask your alchemist if he can do that)
1 cup whole milk (should be 13g of lactose per cup, if your Penicillium won't grow adjust the water-milk ration in favor of milk)
If available: Instead of milk use corn steep liquor. Unfortunately only available after America was discovered, so YMMW, but Penicillium LOVES this stuff. It will make your life SO much easier if it's available.
1 teaspoon Yeast extract (get it from a baker)
3-6 teaspoons Gelatin (get it from a butcher)
Disclaimer: The ratio of each of the ingredients will have to be adjusted depending on the purity of the ingredients and on the conventional measuring sizes of the place you end up.
Gently mix it all in and pour out into the plates, let it solidify. If you end up dumped far enough that such refinement isn't possible, make bone broth and strain it through cheesecloth several times to make it as clear as possible, then mix it 5/6 broth and 1/6 milk. Again, if available, use corn steep liquor, but if not milk is fine. Add gelatin (should still be able to get it from the butcher) as needed to solidify it. I'm afraid experimentation will be needed depending on the resources you will be working with.
When you're done, you should have something like this:
Now that you have your plates, run an inoculation loop through a flame to sterilize it.
Something like this. Wave it through the air to cool it so you don't kill your mold, grab it from your fruit and geeeeeently spread it on top of your improvised agar without breaking the surface of the gelatin!
You can see the motions on this one pretty well. Close your plates, stack them about a meter/3ft from the fireplace. Judge for yourself, but ideally somewhere you would consider comfortably warm (20-24Β°C).
3. Identifying your Penicillium Mushrooms
If all went well, you are going to have something that looks like this:
Well, realistically, it will look something like this:
We're not actually doing it in a lab, after all. But IDEALLY, it will look like the above. It doesn't have to be perfect, you just need to be able to identify Penicillium molds for now.
IDEALLY, on the plate that matches the description of the penicillium mold you'll see an exclusion zone of bacteria around the mold, like the fourth plate in the second row, so you know you have a potential winner, but if you managed to avoid bacterial growth you need to take a few extra steps.
Penicillium molds have characteristic rings of growth, grey-green-white rings. They're easy to differentiate from bacteria because the molds are fuzzy and the bacteria as smooth and slimy. In the above picture, there are four plates that potentially have what we want, and two are less certain than others. Wash out the unwanted ones, make new agar plates, sterilize your inoculation loop and transplant your best candidates. You might need to do this several times.
Two types are confirmed to produce penicilin: P. chrysogenum and P. rubens.
The former is far more widely used today, but since we're sourcing them from literally thin air, we're more likely to get P. rubens, but unless you're a mycologist you probably won't be able to tell the difference. Thankfully you won't need to, because they both produce penicillin. Which brings me to the next step.
4. Confirming it's the penicillin producing mushroom
We're gonna need more agar plates for this one, and believe it or not, you're gonna need to mix blood into your agar. Wash your hands THROUGHLY.
(Theoretically you can get away with just milk, but identifying the correct bacterial colony on white agar is going to be a nightmare, so just add some sheep blood to your agar, conventionally it's about 5% by volume but you might need more to make it)
You need some gram-positive bacteria, preferably of the Bacillota type. Please don't go out and find a patient with fucking botulism or tetanus, you need to live long enough to make the cure. Instead, if you have a vagina, scrape some of the white, mucousy stuff from there and plant it on your plate. If you don't have your own vagina, a borrowed one is fine. Penicilin also works on Treponema pallidum, so if you get a syphilis-affected prostitute that should also work. Just wear gloves.
Ideally you get something like this.
This is actually Lactobacillus brevis, but Lactobacillus colonies all look relatively the same. The important thing is that it's all gram-positive, and will therefore be affected by penicillin.
Take new plates again, plant your Penicillium mold in the middle, and the bacteria all around it, getting as close to the center as possible. You can put down a paper marker for the mold. Wait for about 20 days.
Ideally, on at least one plate, you will get something like this:
This is literally a textbook example of testing antibiotics, but the Zone of Inhibition is what you're looking for. It means the mold is releasing a compound to kill the competing bacteria for resources, in this case, Beta-lactam antibiotic, or penicillin. Make sure to pick the one with the WIDEST ZoI, because that's the one that produces most penicillin.
So now we have the root stock, but our problems have just begun. This is the part where you're absolutely going to need an alchemist's help.
The problem is that a human body is not a petri dish. It's quite a bit larger. And you want the good bacteria destroying stuff without all the nasty contaminants, so you need a SHITLOAD of mold producing a LOT of penicillin, and then you need a way to filter it. You are going to need actual lab equipment for that, or near as they had it.
Since I lost the original protocol I'm going to need to do research all over again how to do that with alchemy equipment (or at least a microbrewery), so that will be in the next installment.
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