Chemical Forums

Please login or register.

Login with username, password and session length

Sponsored links

Pages: [1]   Go Down

Author Topic: Stock Solution of Colistin Sulphate  (Read 1452 times)

0 Members and 1 Guest are viewing this topic.

H Mirza

  • New Member
  • **
  • Mole Snacks: +0/-0
  • Offline Offline
  • Posts: 3
Stock Solution of Colistin Sulphate
« on: December 21, 2017, 06:23:25 AM »

Hi

I have 100 mg of  colistin sulphate salt (Sigma). Potency  is > 15000 U/mg and solubility is in water  (soluble 50 mg/ml according to the label on antibiotic). I want to check the MIC (minimum inhibitory concentration) of this antibiotic against micro organisms. EUCAST has given the guidelines that microbial growth at >2mg/ml would be considered resistant. Now, I am kind of confused how to make the stock solution and in what volume should I make.

I'll appreciate if anyone can guide about it.
Logged

H Mirza

  • New Member
  • **
  • Mole Snacks: +0/-0
  • Offline Offline
  • Posts: 3
Re: Stock Solution of Colistin Sulphate
« Reply #1 on: January 11, 2018, 04:49:10 PM »

Ok, I have been reading papers and different protocols on colistin resistance. I'll be using broth microdilution for MIC determination. I'll be making a 10 mg/ml stock solution for this purpose. The potency of antibiotic is 15000 U/mg, when I used online converters, I got the information that 1U=731.7 ug.

I have also read that W=C*V/P formula does not apply on peptide antibiotics. So, should I make 14 mg/ml stock solution which would be equal to 10243.8 ug/ml? and use it as stock solution? I'll make further dilutions from its for using in assay.

Kindly guide.
Logged

Arkcon

  • Global Moderator
  • Sr. Member
  • ***
  • Mole Snacks: +515/-143
  • Offline Offline
  • Posts: 7153
Re: Stock Solution of Colistin Sulphate
« Reply #2 on: January 15, 2018, 05:15:50 AM »

Hi there.  I've read your questions but, I can't answer them, because I don't know what you're unclear on.  Let's work together to get to the bottom of it all.

I have 100 mg of  colistin sulphate salt (Sigma). Potency  is > 15000 U/mg and solubility is in water  (soluble 50 mg/ml according to the label on antibiotic).

OK.  Some facts:  how much you have (?) The potency in units your application needs, and solubility (maybe we need later.)


So far, so good.

Quote
I want to check the MIC (minimum inhibitory concentration) of this antibiotic against micro organisms. EUCAST has given the guidelines that microbial growth at >2mg/ml would be considered resistant. Now, I am kind of confused how to make the stock solution and in what volume should I make.

And this info doesn't seem to help you.  OK.  But maybe you're confused as to what you want?  You have potency, this reference is in mg.  But we can work with other info.

Ok, I have been reading papers and different protocols on colistin resistance.

OK, we can use different info.

Quote
I'll be using broth microdilution for MIC determination.


I don't recognize this term, it could be jargon for your specific application.  But we can still work with what we have.

Quote
I'll be making a 10 mg/ml stock solution for this purpose. The potency of antibiotic is 15000 U/mg, when I used online converters, I got the information that 1U=731.7 ug.

OK, that's a simple division, don't know why you use a converter.  But fine, whatever.  No question here, yet.

Quote
I have also read that W=C*V/P formula does not apply on peptide antibiotics.

OK.  You've used jargon again.  These abbreviations for units aren't used everywhere.  Also, why would they not be used for peptide antibiotics.  Some sort of relationship here with concentration and potency, that applies to things other than protein antibiotics?  I'm not following.

Quote
So, should I make 14 mg/ml stock solution which would be equal to 10243.8 ug/ml? and use it as stock solution? I'll make further dilutions from its for using in assay.

That could work, as a starting point, depending on what you want to do next.

Quote
Kindly guide.


Quote
I'll appreciate if anyone can guide about it.

We're glad to help.  And one of our rules is, you have to show your work.  Sometimes, when we tell someone that, they dump random facts, and say "There.  Sort out my work and write out my protocol."  In some ways, that's what you've done here.  But we need a better view of what you know, what you don't know, and what you want to do, in order to help.
Logged
That all depends on how reasonable we're all willing to be.  I just want my friends back, except for Cartman, you can keep him.

H Mirza

  • New Member
  • **
  • Mole Snacks: +0/-0
  • Offline Offline
  • Posts: 3
Re: Stock Solution of Colistin Sulphate
« Reply #3 on: January 17, 2018, 05:00:10 AM »

Thank you for your detailed reply. I am so glad to see your reply, I have been desperately waiting for an answer.

I should have elaborated my question. Thanks for pointing it out.
Ok, just to give a background, I am working on detection of colistin resistance in bacteria. For this purpose, I'll be determining Minimum Inhibitory Concentration (MIC) of colistin and the method which I am using is "Broth microdilution method".

Now, according to protocol, I need to make 20X solution of my antibiotic and dilute this 20X by 1:10 in growth media (growth media which will be used to grow bacteria under test). Then, I take 100 ul of this solution and mix it with 50 ul of growth media in first well of 96 well plate, then I serially dilute it by taking 50ul from first well and transferring to next well containing media and so on. After making all the dilutions of antibiotic with media, I'll add 50ul of bacteria in each well.  Final volume in each well would be 150 ul. My testing range is from 128-0.25 mg/L. So the first well should have a concentration of 128 mg/L and last well would have 0.25 mg/L (I am talking about concentration here, but volume would be 150 ul for each dilution here).

Now, the problem for me is how to make stock solution of colistin so that I reach my desired concentration in the first well?

Information given by manufacturer is that (hope, this information is helpful in calculations):
Antibiotic: Colistin Sulphate Salt
Potency: >15000 U/mg
Solubility is in water  (soluble 50 mg/ml)
Total amount: 100mg

Normally, when antibiotic are weighed, a formula is used to calculate the amount of antibiotic to be used. The formula is W=C*V/P
Where W=Weight, C=Concentration, V=Volume and P=Potency (which is given in ug/mg usually). But, in case of some antibiotics i.e colistin, potency is given in units/mg. That’s why I converted units in ug and came to know that 1U=731.7ug. But as I can’t calculate the weight according to this formula (I read in papers that this formula should not be used for peptide antibiotic), so probably this conversion becomes useless.

Now, I am confused that:
1.   How do I weigh colistin to reach my final concentration of 128mg/L in first well?
2.   If I just weigh colistin directly without going into potency. Will it be the right approach? I mean, I’ll weigh 2.56 mg/mL (First concentration:128mg/L*20=2560 mg/L) to make 20 X of colistin.

I tried not to complicate it, I hope it's clear now.

Logged
Pages: [1]   Go Up
 

Mitch Andre Garcia's Chemical Forums 2003-Present.

Page created in 0.063 seconds with 24 queries.