Graphical method/ conc-response curve/others?

Dear Prabhat,

The minimum inhibitory concentration (MIC) value is defined as the lowest sample concentration that inhibited visible growth, as indicated by the MTT staining. Only living microorganisms could convert MTT to formazan and a blue color appeared in the well [1]. The minimum bactericidal concentration (MBC) is determined as the highest dilution at which no growth occurred in medium. 

To  determine the median inhibitory concentration (IC50) value.

 The percentage (%) of the bacterial growth inhibition is determined as [(Ac–At)/Ac] × 100, where Ac is an average of six replicates of light absorption values at wavelength --- nm of the negative controls, and At was an average of six replicates of light absorption values at wavelength --- of the samples. The IC50

value is calculated using the linear relation between the inhibitory probability and concentration logarithm according to the method of Sakuma [2]. The IC50 value is expressed as the mean ± standard deviation of three independent experiments.

[1]. Abe, K.; Matsuki, N. Measurement of cellular 3 -(4,5 - dimethylthiazol - 2 - yl) - 2,5 –diphenyl tetrazolium bromide (MTT) reduction activity and lactate dehydrogenase release using MTT. Neurosci. Res. 2000, 38, 325-329.

[2]. Sakuma, M. Probit analysis of preference data. Appl. Entomol. Zool. 1998, 33, 339-347.

I suggest that you read the attached file for better clarification.

Hoping this will be helpful,

Rafik

In my experience, IC50 is not used to measure inhibition of bacterial cell growth. It may be used for other microorganisms. The reason is that the antibacterial compound is normally tested as a series of 2-fold dilutions, and the growth inhibitory effect of the compound is usually observed over a very narrow concentration range, one or two dilutions. A plot of bacterial growth versus concentration of compound often is too steep to calculate IC50 meaningfully. That may be why the MIC is used instead with bacteria. For bacteria, the MIC is more of an observation than a calculation. In a broth microdilution assay, one simply observes the lowest compound concentration in the set of 2-fold serial dilutions at which the bacteria do not grow to stationary phase in one day from a dilute starter culture. MICs are normally reported in µg/ml.

By the way, MBC, minimal bactericidal concentration, is the lowest concentration of the compound that kills a culture of bacteria. This is distinct from MIC, which is the minimal concentration that inhibits growth of a dilute culture of bacteria.

Thanks for wonderful information

hi guys...i have 1 question regarding MIC (minimum inhibitory concentration ) for antibacterial assay...lactobacillus vs pathogens..to analyse this assay..what is the suitable unit of lactobacillus concentration to get the IC50?log cfu /ml or microlitre/ml?

log10CFU/ml is usually used to express the concentration of bacteria in a suspension capable of forming colonies when plated on agar. When measuring the killing kinetics of an antibacterial compound in broth, one plots log10CFU/ml versus time of exposure to the compound.

Is it possible to convert  IC50 to MIC values or vice versa?

Do you mean the IC50 for bacterial growth? If so, then you should have a graph of bacterial growth (CFU/ml or OD) versus compound concentration. On this curve, you would have to pick a point at which bacterial growth was not evident. That would be the MIC. Now compare the IC50 to this MIC for several experiments to see if there is a consistent ratio between them. If there is, then that ratio can be used as a conversion factor.

Can anyone help please

I am working on calculating MIC by OD method.

If concentration of compound increases then OD will decrease or increase and what does this condition says in each case?

The OD600 of the cells should decrease as the compound concentration increases. For MIC measurements, it is usual to make 2-fold serial compound dilutions. There is usually a sharply defined transition from no growth inhibition to complete growth inhibition in 1 or 2 dilutions. If the compound has an absorbance at 600 nm, its absorbance will increase with concentration. In that case, you should subtract the compound absorbance from the compound+bacteria absorbance. This requires preparing a second set of compound dilutions to which you add medium but no bacteria.

Great Adam and thanks

I have introduced solid compounds in the broth medium containing bacterial and fungal strains and each sample had been checked for its OD after filtering the compound.

Does it sounds a proper protocol? Or if you have any other suggestions please?

When you filter out the compound, do the bacteria or fungi pass through the filter?

Hello, Adam B Shapiro, I have a similar problem like Nelofer Jamil. I could not develop a protocol of microtiter broth dilution method for MIC of plant extracts with measuring OD and regression analysis. Could you give an article reference or a short procedure? Thank you

IC50 values are not determined for antimicrobial agents rather MIC (minimum concentration that inhibits the visible growth), MIC50 and MIC90 are determined to assess antimicrobial activity and spectrum of an antimicrobial agent against a microbial species. Because a single species can have numerous strains/isolates with varying susceptibilities to an agent MIC testing is performed against large number of clinical and laboratory isolates. MIC50 is the concentration that inhibits 50% of the strains tested and MIC90 is the concentration that inhibits 90% of the strains tested (typically 100 or more isolates are tested for determining MIC50 and MIC90, but it can be calculated based on data from lower numbers of strains tested too). Similarly, MBC (minimum bactericidal concentration resulting in 99.9% kill, i.e. over 3-log kill of strain after overnight treatment), MBC50 and MBC90 are used to assess bactericidal activity of an antimicrobial agent. MIC and MBC are done at the same time, but occasionally bactericidal activity is assessed by monitoring log reduction in a test organism using 0.5X, 1X, 2X, 4X MIC value over a short treatment/contact times such as 5, 10, 15, 30, 60, 120, and 300 seconds to show rapid bactericidal activity of an antimicrobial agent.

The broth microdilution MIC measurement (for a single strain; I'm not talking about MIC90) does not require any mathematical treatment, such as regression, and it is not even necessary to measure the optical density (OD) of the culture. The culture is started with a highly diluted bacterial culture that is not visibly turbid, and the test substance is added at the same time at a range of concentrations. (If the test substance is visibly turbid, you are going to have a problem.). After the culture is incubated overnight, you can identify the MIC by eye, simply as the well containing the lowest concentration of test substance at which the culture did not become visibly turbid.

Sometimes, you will see trailing. This means that there is a little bit of growth of the culture over a range of concentrations, making it difficult to decide on the MIC. You should take note of this and report it. There is a rule about deciding on the MIC in the presence of trailing. The bacteria tend to settle to the bottom of the U-shaped wells, forming a "button." If the button is less than 1 mm in diameter, it is considered to be lack of growth, but if the buttton is larger than 1 mm, then growth is said to have occurred.

Because MIC measurements are always made on 2-fold serial dilutions, the resolution of an MIC is in steps of 2-fold. If your highest tested concentration is 32 mg/L, for example, then the possible MIC values are >32, 32, 16, 8, 4, 2,1, 0.5, 0.25, 0.125, 0.063, 0.031, and

Adam B Shapiro, the comment you made on the 16th of February 2016 on the use of MIC for antibacterial assays rather than IC50 is very helpful. I am writing a paper and I agree with the statement but can't find a reference. Do you maybe know of a reference I can have a look at?

You could refer to the Clinical Laboratory Standards Institute (CLSI) publications on antibacterial susceptibility testing methods, such as broth microdilution and agar dilution.

Adam B Shapiro thank you for your help, much appreciated.

MIC is the best way to assess the potency of an anti bacterial drug. Adam B Shapiro thanks for explaining the critical points in evaluating the MIC values. With my experience one can go for microdilution method using 96-well plate for determining MIC instead of macro dilution (tube dilution) method.

*Image from Google

Hi Adam B Shapiro, your answers really helped in understanding the concept.

I have a ques, I am working on some herbal plant extracts therapeutic formulations.

Whenever I go for micro broth dilution method for MIC, My blank i.e. compound+media gives higher absorbance as compared to Test i.e. Compound+cells.

This is creating a lot of confusion, can you please help?

It is important to distinguish between absorbance and optical density (OD). An absorbance measurement is made on a transparent sample and measures the intensity of the color (as in Beer's Law). The OD measurement used for MIC measurements represents the turbidity (cloudiness) of the sample, but would also include absorbance if the sample is colored at the wavelength measured.

Is your herbal plant extract, diluted in medium, colored (in addition to the color of the medium)? Is it turbid, which is an indication of insolubility?

Why would the OD be reduced when the bacteria are included? One reason might be metabolism of the colored material by the bacteria, changing its absorbance. If the plant extract is turbid due to insolubility, the bacteria may have consumed it, thereby removing the insoluble material. Alternatively, the insoluble material may have stuck to the bacterial surface and precipitated with the bacteria, lowering the turbidity.

Obviously, it is problematic to perform broth microdilution assays using an OD measurement when the test material is colored at the measured wavelength, or if it is insoluble.

You can compensate for the color of the material by reading the turbidity of the bacteria by eye, rather than using a plate reader. The MIC is just the lowest test concentration at which the cells don't grow, resulting in a well without visible turbidity.

There is no point trying to compensate for turbidity caused by insolubility of the test substance. If the substance is not in solution, then it isn't likely to have any effect on the cells. It's essentially inert.