Hi,
In running bacterial MIC with antibiotics, do we actually need to measure OD values in spectrophotometer and after getting values at particular emission and excitation wavelength how we get the final output?
Thanks.
You don't "need" OD but it helps.
It allow you to move forward from grow/no grow and get a feeling as to partial growth. If you don't care for such you can use +/-
Yoram Gerchman
Thanks for the answer.
But if I am using it how I can calculate it. What wavelength and calculation I should proceed with?
Hi shivangi
for calculating IC50, IC90 value we need to measured OD. IC50 value is the lowest conc. of your drug that can inhibit or killing 50% of your bacterial cultural.
Shivangi ..
Assuming you are talking bacteria or yeasts we usually use 600nm although its not a must (see discussion in
https://www.researchgate.net/post/Why-is-bacterial-density-measured-at-OD-600)
If you are talking other cells it could vary a lot and in some cases depends on you metabolic assay (i.e. how do you measure cell livelihood/death).
If you explain IN DETAILS what you are to do and what is your system we can try and help more.
Hi Actually use the spectrophotometer to measure OD is not necessary, but it is better to do it. In this method, you must measure OD in 600 nm and then compare your results in treatment groups with control group. If OD increases that means bacteria could growth and if OD doesn’t change that means bacterial could not growth, so that you can calculate the MIC value.
Pouria Mohammadparast Tabas
Thanks for the answer.
But in this case we are not measuring fluorescence but the scattering (growth).
Can we measure it by fluorescence (how much resazurin is concerted to resofurin) and get some quantitative number.
Yoram Gerchman
Thanks for the answer.
I want to quantify my MIC data by getting some values in fluorescence measurement. How can I correlate the values I am getting at particular excitation and emission with the effect of compound on bacterial cells.
Shivangi, it is very hard to answer vague questions. Please write here or im private email, exectly what are trying to do and I will try to help.
It depends on the information that you need,
If you only wants to determine the MIC then it is not mandatory to do OD measurement of MIC plate if you are using a dye which will give a color change with bacterial growth that can be detected visually, with these protocols you will decide on the lowest concentration that didn’t show color change (bacterial growth) as the MIC.
But if you want to obtain more information as conducting a concentration response curve for the tested substance and determining the maximum inhibitory effect (Emax), IC50 and IC90, then you will need to measure the optical density change in the plates (you will measure the optical density before incubation and after incubation and calculate the change as % of inhibition of bacterial growth)
Another case is if you are doing microdilution assay for highly water insoluble compounds, in this case depending on OD measurement since after incubation the turbidity due to the presence of the drug in aqueous medium may be confused with bacterial growth, and sometimes in these cases you will need to make an uninoculated plate (contains everything as the MIC plate except for the organism) and measure the difference in the optical density between the inoculated and uninoculated plates after incubation.
Though I myself didn’t conduct resazurin plate-based antibacterial assay but many articles in indexed journals conducted the experiment depending on visual assessment of color change without measuring OD.
On the other hand I tried using microdilution assay depending on optical density without including a dye and it was convenient method that gives good results.
So eventually deciding to measure the OD depends on what is the information that you need, the characteristics of your tested antimicrobial agents and the availability of the equipments.
The attached video could help with the method:
https://www.jove.com/v/53028/a-reference-broth-microdilution-method-for-dalbavancin-vitro
It is better to take 10 or 20 microlitre from each wells and plate on appropriate solid culture medium.incubate for appropriate time and look for growth. Record the wells with no growth. The minimal concentration well with NO GROWTH should be considered as the well with MIC.
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