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alyks
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[*] posted on 18-5-2006 at 23:49
Penicillin cultivation from mold?


How is this done? I think it was discovered by the mold with a cultivation medium.
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[*] posted on 4-6-2006 at 19:36


Penicillin mold is a common mold which can be isolated from the molds that grow on bread. It was discovered when some bacteriological plates got moldy, and the bacteria wouldn't grow near the mold.

That being said, I think that the actual extraction of Penicillin is a PITA, and needs relatively large amounts of the mold. Thus, for extraction, the mold is grown in an aerated nutrient broth, which gives you much better yields in terms of penicillin/cm^3.
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[*] posted on 5-6-2006 at 06:20


In principle you could grow bacteria on agar plates, and infect these with various samples from soil, old woods, waste waters, mouldy bread and so on, until you see some fungal mycelia growing, while killing bacteria nearby. Should be a neat and fairly simple experiment, as you wouldnt even need to be worried about sterility.

Penicillin was discovered in 1929 by Alexander Fleming.




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[*] posted on 8-11-2006 at 09:04


Penicillin is extracted from Penicillium notatum
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[*] posted on 8-11-2006 at 18:25


it is simply separated from the mold after it has stopped growing -- the actual strain of mold that produces useful yields of the drug was found after years of searching so synthesis is not trivial at all - google it..

[Edited on 9-11-2006 by jimmyboy]
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[*] posted on 2-10-2010 at 15:07


I added some mno2 (probably it didnt have any effect) and a lot of citric acid into the water. AFter staying like 2 weeks outside on the whole top of solution there is green mold, probably the one which produces penicillin. I will now get it to grow on calcium citrate, just how could I then isolate pure penicillin from that?
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[*] posted on 6-10-2010 at 09:56


Penicillin, or at least the activity of the mold, was discovered in yee olden times by 'wise women' who used to feed people moldy bits of bread as cures. This was at the time when doing that kind of thing meant you'd be burnt as witch (see; Ignaz Semmelweis Vs. Lewis Pasteur, check the dates).

Commercially, they use skimmers for this kind of thing. They have a gigantic tank of it, molding away, and a spinning drum half submerged in the broth picks up the layer of scum on the surface of the tank. Then a blade scrapes it back off and out into a collection bin or conveyor, where it goes off to be extracted.

The same idea is used to skim oil off water, fish tanks, ponds and various other films off other things. Some of them use belts instead of the drum.

Here's the drum kind;






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[*] posted on 6-10-2010 at 10:40


sadly, the witchhunt mentallity is alive and well in modern times too.
ask George Bush and his cronies and, your former prime minister.
it's just human nature, don't people just suck?
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[*] posted on 19-1-2011 at 18:42


It is still alive.

Today, it's genetics and subatomic physics.

A hundred years from now, everyone will be laughing it up at how silly people were when they didn't eat their cornflakes with added stem cells. And those would be the same people who'd be throwing shit storm grenades into the research labs now.

Science is fundamentally and factually beyond the written law, because it is not based on society. Society needs to adapt to what science says. People don't like being told what to do with their own lives and opinions at such a deep, controversial level. And scientists are the human shield between it and the general public.

***If you're talking about Gordon Brown, he wasn't ever actually elected into the role of prime minister by the public. GO DEMOCRACY, YAY! :D

-----------------------------------------------------------------------
To the topic at hand!

I think the easiest way would be to follow one of the methods for science projects online, which is to simply sterilise some glass and leave some bread in there, in the dark - waiting for it to go mouldy.

The citrus peel method is likely suggested because the spectrum of mould growth changes based on pH, as some are not fond of low pH's at all, whereas others prefer slightly alkaline conditions.

I would then produce some plates, cut out bits of filter paper, dip them in the broth and breath all over the plates to get them nicely contaminated, then incubate and watch for clear spots. These plates would be easy due to them needing to be contaminated.

If you see an exclusion spot around the filter paper, you have likely have the antibiotic.

If you want to scale up production, you would then inoculate a sample of the culture onto some sterile plates. A good method for this is to only spot certain zones of the plates, or the centre. You then incubate the plate until it's well colonised with mould.

You can now look through the plates for those showing zoning of the moulds. For instance, some areas of the plate will be pristine white and fully, others will be dirty green and so on, showing boarders between them where they appear to change in a small space. These is a board between a different culture or network.

Penicillium tends to be greeny colours, and these can sometimes only appear once the mycelium has had a chance to colonise a decent amount of plate space and age, as it is the network going into spore production - these networks release spores from the 'roots' of the mould, whereas button mushrooms you have for dinner have a more advanced fruiting stage that involves gills and a cap to protect them.

You then cut a sample out of that zone and put it on a sterile plate - you should do this with a couple of samples, each on their own plate.

Then watch again. Now, you don't want to see any zoning, you want a pure culture. You may need to repeat this two or three times to get the sample away from the other forms on there.

This process is much like re-crystallisation in chemistry, you are attempting to 'crystallise' a pure sample away from contaminants. And it does work, surprisingly well for the amount of effort and cost involved.

It is the method by which mycologists culture fungi that they've collected outdoors. When they take spore prints or tissue samples, they will inevitably be covered in bacteria, contaminating spores, doggy piddle and various other gems of life*. The samples go onto plates and, hopefully, on one of those plates, a small, clean spot of mould will appear amongst the rubbish; where the mould has managed to established a little home for it's self in the short term. A small chunk of the clean spot is cut out with a sterile scalpel, moved to a new sterile plate and re-incubated. This time, the plate should have a much larger or full colonisation of that mould alone.

Once you have a pure culture, you redo the broth test and check you have the antibiotic activity.

If it's green for go, you can proceed to do a big batch. As the mould will tend to float on the surface, you may be best off choosing something with a large surface area to grow it in; rather than tall and thin.

You will likely only be able to do batches and have to empty it all out to extract the cake.

One would need to be aware that in biology labs, the cultured dishes are usually of inert strains of bacteria. If you breath and cough all over the plates to inoculate yours, then incubate them at body temperature, you have a potential pathogen risk and could get very sick if not careful.

Also, when growing commercial broths, there are often many species and strains of an organism that will produce the desired product, the goal is to find one that produces a lot of it and that is easy to maintain.

Numerous forms of penecillium are added to cheeses and meats with the specific aim being to prevent colonisation by other moulds or bacteria.

The bacteria part means potential antibiotic activity and the colonisation part means the genus is a strong competitor, meaning it will be easier to swamp a plate with it as opposed to contaminations - where as some other moulds, bacteria and yeasts are weak competitors, and need constant care and maintenance or they'll get bullied to death by the others.

*Fly Agaric mushrooms, Amanita Muscaria, are often full of maggots eggs or the grown up version. Yum yum sez the hippy! :P

**John setup and ran a HEPA filtered mycology lab, bunny suits, laminar bench, autoclaves and all.

[Edited on 20-1-2011 by peach]




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[*] posted on 20-1-2011 at 05:50


Try citric acid solution with just a little bit of calcium acetate. Leave it outside and it will make very nice penicillin mold on the surface of the solution. Then you can make pure cultures from that. I think its easiest way to make not contaminated solutions.
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[*] posted on 29-7-2011 at 12:34


Technically, you can't cultivate penicillin, penicillin is a chemical compound. You can cultivate Penicillium, which is a genus, and an umbrella term for over 300 fungi species that have the capacity to make penicillin and related compounds. Of those 300 species I would choose Penicillium chrysogenum because otherwise you will end up making cheese.
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[*] posted on 29-7-2011 at 14:48


The article below mentions something about a strain found growing on a grapefruit in Peoria, Illinois. As an aside, I got about 500 grapefruits off my 4 trees last year, the ones I didn't get because they were too high fall later and turn an almost solid blue with mold. I don't like the mold smell.

http://books.google.com/books?id=utN05OmYOF8C&pg=PA231&a...

[Edited on 29-7-2011 by Morgan]
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[*] posted on 29-7-2011 at 15:04


Historical tidbit.

On February 1, 1941, the first test of penicillin in humans was conducted. A policeman, who had an advanced staphylococcus bacterial infection, was treated with penicillin for five days, after which the penicillin supply ran out. Almost as soon as the treatment started, the man began to show steady signs of improvement until the treatment stopped. Although he later died, it was shows that penicillin could be used even in advanced stages of an infection. It also showed that much more penicillin would be needed to fully carry out a treatment. That same year, the group decided to test children, because children would need smaller doses of the drug. From February to June, five children were treated with penicillin in stages were other medicines failed. Of the five, only one died, and an autopsy revealed that it was a result of an unrelated cause (McGrew 248).
http://library.thinkquest.org/21798/data/tqmainsite/penicill...
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[*] posted on 11-8-2011 at 22:27
Different Varieties of the mold


The mold you want with the highest yield of penicillin is the kind that grows on over ripe citrus. The one that grows on bread is rather low in the amount of penicillin it produces
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[*] posted on 15-9-2011 at 06:05


There's quite a bit of good information in one of the original patents, especially if you're looking to make up a batch of 'ye olde' benzylpenicillin.


Quote:

It has now been found according to this invention that substances having antibiotic activity can be obtained by reacting with a suitable chemical reagent, the fermentation, liquor obtained ‘by growing a penicilline producing mould preferably in the absence of an added precursor...The present invention therefore provides 6aminopenicillanic acid having a structural formula: [img][/img]

which is capable of reacting with phenylacetyl chloride
to produce benzylpenicillin, and which gives a negative
Bratten-Marshall test and a negative ninhydrin test, and its šalts






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[*] posted on 28-6-2012 at 17:23


Quote: Originally posted by peach  
It is still alive.

Today, it's genetics and subatomic physics.

A hundred years from now, everyone will be laughing it up at how silly people were when they didn't eat their cornflakes with added stem cells. And those would be the same people who'd be throwing shit storm grenades into the research labs now.

Science is fundamentally and factually beyond the written law, because it is not based on society. Society needs to adapt to what science says. People don't like being told what to do with their own lives and opinions at such a deep, controversial level. And scientists are the human shield between it and the general public.

***If you're talking about Gordon Brown, he wasn't ever actually elected into the role of prime minister by the public. GO DEMOCRACY, YAY! :D

-----------------------------------------------------------------------
To the topic at hand!

I think the easiest way would be to follow one of the methods for science projects online, which is to simply sterilise some glass and leave some bread in there, in the dark - waiting for it to go mouldy.

The citrus peel method is likely suggested because the spectrum of mould growth changes based on pH, as some are not fond of low pH's at all, whereas others prefer slightly alkaline conditions.

I would then produce some plates, cut out bits of filter paper, dip them in the broth and breath all over the plates to get them nicely contaminated, then incubate and watch for clear spots. These plates would be easy due to them needing to be contaminated.

If you see an exclusion spot around the filter paper, you have likely have the antibiotic.

If you want to scale up production, you would then inoculate a sample of the culture onto some sterile plates. A good method for this is to only spot certain zones of the plates, or the centre. You then incubate the plate until it's well colonised with mould.

You can now look through the plates for those showing zoning of the moulds. For instance, some areas of the plate will be pristine white and fully, others will be dirty green and so on, showing boarders between them where they appear to change in a small space. These is a board between a different culture or network.

Penicillium tends to be greeny colours, and these can sometimes only appear once the mycelium has had a chance to colonise a decent amount of plate space and age, as it is the network going into spore production - these networks release spores from the 'roots' of the mould, whereas button mushrooms you have for dinner have a more advanced fruiting stage that involves gills and a cap to protect them.

You then cut a sample out of that zone and put it on a sterile plate - you should do this with a couple of samples, each on their own plate.

Then watch again. Now, you don't want to see any zoning, you want a pure culture. You may need to repeat this two or three times to get the sample away from the other forms on there.

This process is much like re-crystallisation in chemistry, you are attempting to 'crystallise' a pure sample away from contaminants. And it does work, surprisingly well for the amount of effort and cost involved.

It is the method by which mycologists culture fungi that they've collected outdoors. When they take spore prints or tissue samples, they will inevitably be covered in bacteria, contaminating spores, doggy piddle and various other gems of life*. The samples go onto plates and, hopefully, on one of those plates, a small, clean spot of mould will appear amongst the rubbish; where the mould has managed to established a little home for it's self in the short term. A small chunk of the clean spot is cut out with a sterile scalpel, moved to a new sterile plate and re-incubated. This time, the plate should have a much larger or full colonisation of that mould alone.

Once you have a pure culture, you redo the broth test and check you have the antibiotic activity.

If it's green for go, you can proceed to do a big batch. As the mould will tend to float on the surface, you may be best off choosing something with a large surface area to grow it in; rather than tall and thin.

You will likely only be able to do batches and have to empty it all out to extract the cake.

One would need to be aware that in biology labs, the cultured dishes are usually of inert strains of bacteria. If you breath and cough all over the plates to inoculate yours, then incubate them at body temperature, you have a potential pathogen risk and could get very sick if not careful.

Also, when growing commercial broths, there are often many species and strains of an organism that will produce the desired product, the goal is to find one that produces a lot of it and that is easy to maintain.

Numerous forms of penecillium are added to cheeses and meats with the specific aim being to prevent colonisation by other moulds or bacteria.

The bacteria part means potential antibiotic activity and the colonisation part means the genus is a strong competitor, meaning it will be easier to swamp a plate with it as opposed to contaminations - where as some other moulds, bacteria and yeasts are weak competitors, and need constant care and maintenance or they'll get bullied to death by the others.

*Fly Agaric mushrooms, Amanita Muscaria, are often full of maggots eggs or the grown up version. Yum yum sez the hippy! :P

**John setup and ran a HEPA filtered mycology lab, bunny suits, laminar bench, autoclaves and all.

[Edited on 20-1-2011 by peach]


Very interesting, Peach.

Suppose that you found a viable mold sample. If you wanted to preserve a sample of the mold to use in the future, how would you do that?

[Edited on 29-6-2012 by radagast]
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[*] posted on 27-9-2012 at 09:32


I actually just went through a multi week process of trying to isolate pencillium from citrus fruit and test for the presence of pencillin.

www.basementbiotech.org

It failed but...I learned a few things

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[*] posted on 17-11-2012 at 14:10


Quote: Originally posted by Oppenheimer  
I actually just went through a multi week process of trying to isolate pencillium from citrus fruit and test for the presence of pencillin.

www.basementbiotech.org

It failed but...I learned a few things



you can grow penicillin in a plastic box with big surface area and only citric acid

leave citric acid for few weeks and it should be full of penicillinum mold
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[*] posted on 3-12-2012 at 16:57


Decided to photograph a fallen grapefruit in the back yard.
http://www.uoguelph.ca/~gbarron/MISCELLANEOUS/penicill.htm

DSC_0018.JPG - 110kB Grapefruit Mold.JPG - 68kB

[Edited on 4-12-2012 by Morgan]
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magnus454
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[*] posted on 3-12-2012 at 19:28


There ya go, ton of penicillin right there! Sterilize some fruit peals, and inoculate it with some of that.



History is repeating itself.
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