Sciencemadness Discussion Board

How do I make a stock solution of ampicillin?

Carbon8 - 16-2-2019 at 12:00

I am preparing to grow a strain of E. coli that has a plasmid which expresses proteins that create luminescence. The plasmid also codes for ampicillin resistance. I want to make up media that includes ampicillin so that bacteria that lose (or never had) the plasmid won't grow and the culture will be enriched for plasmid-bearing bacteria.

http://www.the-odin.com/plasmid-pje202-pvib-to-create-biolum...

My question is about how to prepare a stock solution of ampicillin. A reputable organization, Addgene, suggests creating a 1000x stock solution at a concentration of 100 mg/mL, or 100 g/L.

https://www.addgene.org/mol-bio-reference/antibiotics/

So does my source for the plasmid:

http://www.the-odin.com/ampicillin-sodium/

But the solubility of ampicillin is only about 10 g/L.

https://pubchem.ncbi.nlm.nih.gov/compound/6249#section=Solub...

So how can I create a stock solution in this situation?

Note: I don't actually have any ampicillin at the moment, but I tried to make a stock solution of amoxicillin and it hardly dissolves at all. Its reported solubility is a little lower than ampicillin's (about 3 g/L vs 10 g/L), but I'm assuming that however you can get ampicillin into solution would also work for amoxicillin.

https://pubchem.ncbi.nlm.nih.gov/compound/amoxicillin#sectio...

Metacelsus - 16-2-2019 at 15:18

Where I work, we use 50 mg/mL ampicillin stock solutions and store aliquots frozen at –20 °C. It will slowly hydrolyze at room temperature (over a few weeks). Note that the solubility depends on pH: ampicillin sodium salt is much more soluble than ampicillin. (However, don't go above pH 8 or so, since the rate of hydrolysis will greatly increase.)

You could try using ethanol as a solvent for your amoxicillin if you want to make a higher concentration.

And what method are you currently using to try to dissolve your amoxicillin?

VictorMedvil - 17-2-2019 at 08:14

Buy this plasmid from Addgene or one like it, it will infinitely curn out ampicillin if merged with a cell with Ribosomes such as a harmless bacteria the L method for somatic cellular hybridization can be used to merge that plasmid with another cell with ribosomes to make your infinite Ampicillin production Microbe from the BASIC_5_Amp Plasmid and another cell.

Promoter Ampicillin Plasmid





For information on the L Method of Cellular Hybridization How to do Cellular Hybridization

[Edited on 17-2-2019 by VictorMedvil]

Tsjerk - 17-2-2019 at 12:13

This is one of the most ridiculous suggestions made on this forum, partly because much easier methods exist (the L method is used if really nothing else works), but mostly because this method will only give you ampicillin resistance, you gain absolutely nothing except for an ampicillin resistant strain...

OP is looking for a way to make an ampicillin stock because his strain carries a plasmid he wants to maintain, and the strain carries the plasmid because it also carries something else, something that is actually worth growing the strain for (luminescence genes in this case).

The reason Addgene (nice company, many nice plasmids) sells this plasmid, is because you can clone another gene just behind that lac promoter, I guess the multiple cloning site (MCS) is not shown in this image for clarity or so, that other gene would in this case be the luminescence gene. But Carbon8 already has that plasmid, has an expression strain containing that plasmid. All he has to do is to keep the coli (in this case) to maintain the plasmid by growing them on ampicillin.

Tsjerk - 17-2-2019 at 12:21

Ampicillin stocks are usually made from the sodium salt, which is highly soluble, and sterile filtered.

I guess if you don't have 200 nm filters, you can prepare the stock with thoroughly boiled water (this will also kill spores, 30 minutes or so). I actually never bothered to filter my stocks, I just prepared them with sterile water and tubes and hoped the ampicillin would kill anything that contaminated the ampicillin powder. I never ran in trouble here.

https://www.cytographica.com/lab/solutions/Amp.htm

Beginners mistake I also made: don't add any antibiotic to LB agar while still being around 100 degrees from dissolving the agar. Antibiotics degrade very fast at that temperature. Wait till it is 60 degrees, agar solidifies at around 50, but if you overshoot the cooling-down you can just redissolve the agar. We used a 60 degree stove to make sure it would be the right temperature, you can't put in a thermometer because of the contamination it would cause.

Edit: As Metacelsus suggested, make aliquots so you don't have to thaw your stock more than five times, otherwise the ampicillin will degrade. Also, if you use the sodium salt you don't have to bother about pH, the ampicillin salt will buffer itself.

[Edited on 17-2-2019 by Tsjerk]

Carbon8 - 17-2-2019 at 13:29

Thanks to all for your comments.

I think my problem is that I have the sparingly-soluble, trihydrate version of amoxicillin that's used orally for treating infections and not the more soluble, sodium salt version that's used for intravenous therapy and molecular biology.

https://www.toku-e.com/product/amoxicillin_sodium_salt/

The only recipe I could find for converting the trihydrate to the sodium salt is in an old US patent dealing with penicillin and it involves more organic chemistry than I am up for at the moment.

https://patents.google.com/patent/US2459315A

The amoxicillin/ampicillin is theoretically not essential for the growth of my bacteria, so I will see what happens without the antibiotic and I'll order some ampicillin next week.

I wonder what happens in a person's stomach that converts the trihydrate into a more soluble form so that it can enter the bloodstream. Is it just the acidity? I might try adjusting the pH of the mixture, as suggested by Metacelsus. I also plan on getting some of the 0.22-micron filters mentioned in Tsjerk's link because I want to use them to try and isolate a bacteriophage. I'll also use them to sterilize the ampicillin.

Tsjerk - 17-2-2019 at 13:34

Add equimolar NaOH to your amoxicillin, it should turn into the salt and dissolve.

In the stomach it will in as the insoluble compound, but once in the gut it dissolves because it turns back to the salt because of the higher pH there.

Carbon8 - 17-2-2019 at 13:40

Thank's Tsjerk. When you say "equimolar", do you mean the same number of moles as the amoxicillin?

Tsjerk - 18-2-2019 at 01:02

Yes, you want the mono sodium salt, equimolar means something like equal-molar ratio. But add the NaOH to the amoxicillin as I don't know how stable it is in very high pH.

Carbon8 - 18-2-2019 at 15:23

I added equimolar and double-molar doses of NaOH to the amoxicillin, but they didn't do much. However, triple-molar did eliminate the substantial amount of amoxicillin sitting on the bottom of the bottle. The solution is still pretty cloudy though.

Tsjerk - 18-2-2019 at 21:58

What is your source of amoxicillin?

Metacelsus - 18-2-2019 at 23:34

If it's pills, they probably have a lot of binders and other crap that won't dissolve.

Tsjerk - 19-2-2019 at 01:20

I'm pretty sure that if you pull off that phenolic hydrogen it will degrade before your eyes.

Carbon8 - 19-2-2019 at 13:29

My source for amoxicillin is a capsule from an old prescription for a tooth infection. I don't think there are any binders in the capsule because the contents of the capsule (a loose powder) weigh about 250 mg, which is what the dosage is claimed to be. Also, all of the undissolved material at the bottom of the bottle in which I made up the amoxicillin solution eventually dissolved when I added enough NaOH. I'm going to order the sodium salt of ampicillin from the vendor instead of fooling around with trying to get my amoxicillin into solution.

I've been successful in growing the bacteria without having added any amoxicillin, but I haven't seen any luminescence yet. The growth on the agar and in the broth is very slow because I'm incubating at approximately room temperature (18 degrees), and not E. coli's optimum of 37 degrees. The vendor's protocol says that the cells won't luminesce at all if they are grown at temperatures above about 27 degrees and that the optimal temperature is around 18 degrees. This is because the proteins that do the luminescence reaction are from Vibrio fisheri, which is found in oceanic fish. Also, the Vibrio need to be at a certain minimal concentration because they use quorum sensing to initiate luminescence.

Here are links to the vendor's instructions and to the paper that the vendor cites as the origin of the plasmid he sells.

http://www.the-odin.com/bio-luminescent-bacteria/
https://jb.asm.org/content/jb/164/1/45.full.pdf


Tsjerk - 19-2-2019 at 13:39

Quote:

I've been successful in growing the bacteria without having added any amoxicillin, but I haven't seen any luminescence yet.


of course they grow without antibiotics. You would have to reconsider if they wouldn't.

How do you know it is your E. coli that is growing? Without any selection pressure it could be any organism stuck to your hands, lets say coli from not washing your hands after wiping your ass, or bacillus from the bottom of your shoes or spores from the air.

Quote:
Also, all of the undissolved material at the bottom of the bottle in which I made up the amoxicillin solution
eventually dissolved when I added enough NaOH.




Doesn't say anything, degradation products dissolve in NaOH
Quote:
Ampicillin from the vendor instead of fooling around with trying to get my amoxicillin into solution.

Very good!
Quote:

The growth on the agar and in the broth is very slow because I'm incubating at approximately room temperature (18 degrees),
and not E. coli's optimum of 37 degrees. The vendor's protocol says that the cells won't luminesce at all if they are grown at temperatures above about 27 degrees and that the optimal temperature is around 18 degrees.



With or without antibiotics? At lower temperatures (longer time to grow) the cells have more time to kick out the plasmid.

[Edited on 19-2-2019 by Tsjerk]

[Edited on 19-2-2019 by Tsjerk]

Tsjerk - 19-2-2019 at 14:05

The fact the cells are not luminescent at less than optimal (coli optimal) means the proteins (probably... I can say this because I know the proteins are stable at higher temperatures) are toxic to coli. Therefore they kick out the plasmid. You should use amoxicillin (or another penicillin).

Did you try to make a 1000x stock with 3 equivalents of NaOH? That would give a pH of about 13. It was already mentioned penicillins don't like caustic conditions.


[Edited on 19-2-2019 by Tsjerk]

Carbon8 - 19-2-2019 at 14:30

For my first broth and plate that I'm still incubating, I did not use the amoxicillin with the three equivalents of NaOH. I should have the sodium ampicillin by next Monday and I'm going to wait for that instead of trying the amoxicillin with added NaOH.

I'm not in a hurry on the ampicillin because I'm doing other experiments. And it takes me three days to sterilize new broth and agar because I'm using tyndallization until the pressure cooker I've ordered arrives later this week.

I'm also thinking about getting another type of light-emitting bacteria to fool around with. These bacteria use kanamycin instead of ampicillin to protect their plasmid. Here are some links.

http://www.the-odin.com/pdusk-and-pdawn-light-controlled-flu...
http://www.the-odin.com/kanamycin-monosulfate/
https://docs.google.com/document/d/1mNo1gLIaepZw2vSmBcLSF9nY...
https://drive.google.com/file/d/0B_R75gIJvkFUSkVlN3RFdmgzZ2s...


[Edited on 19-2-2019 by Carbon8]

Tsjerk - 20-2-2019 at 07:59

100 degrees for an hour will kill anything, also spores.

Metacelsus - 20-2-2019 at 23:53

Quote: Originally posted by Tsjerk  
100 degrees for an hour will kill anything, also spores.


Tell that to hot spring thermophiles (for example).

Of course, those thermophiles won't grow at 37 °C so that's not a problem.

Tsjerk - 21-2-2019 at 05:55

I was thinking about mentioning that, but I didn't because indeed the don't grow at 37.

There are some species that have their optimum above 100 degrees and can actually divide at 122 degrees; Methanopyrus.