I have noticed that my controls containing Alamar Blue + media only are reduced by 10% (expected due to loss of buffering capacity [comparing to Alamar blue + media with cells + no test compound]). My MICs plateau at 90% and never go any lower.
When using absorbance measures, how do I factor this error into account? I get the same plateau effect when using the equation that contains Alamar blue + media + cells (test wells) in the numerator and Alamar blue + media only (negative control wells) in the denominator.
Instead of using Alamr Blue + media only for your negative control, you might try using Alamar Blue + media + cells and incorporating a compound (like tamoxifen or saponin) at a concentration that is totally inhibitory as your negative control.
Our group has used Alamar blue for MIC determinations as follows:
"Plates were read spectrophotometrically in an AD340a Beckman plate reader (Beckman-Coulter) programmed to subtract the absorbance at 600 nm from that at 570 nm; the MICs are reported as the lowest drug concentrations yielding a differential absorbance of zero or less."
So we didn't use the negative control well in the MIC calculation, only as a QC parameter for validity of the assay. I would note, however, that we have found using Alamar blue with anti-folates (and a few other unclassified inhibitors) generally results in different MIC values than evaluation by turbidity.
1: Barrow EW, Clinkenbeard PA, Duncan-Decocq RA, Perteet RF, Hill KD, Bourne PC, Valderas MW, Bourne CR, Clarkson NL, Clinkenbeard KD, Barrow WW. High-throughput screening of a diversity collection using biodefense category A and B prioritypathogens. J Biomol Screen. 2012 Aug;17(7):946-56. doi: 10.1177/1087057112448216. Epub 2012 May 31. PubMed PMID: 22653912; PubMed Central PMCID: PMC3700734.
2: Barrow EW, Dreier J, Reinelt S, Bourne PC, Barrow WW. In vitro efficacy of new
antifolates against trimethoprim-resistant Bacillus anthracis. Antimicrob Agents
Chemother. 2007 Dec;51(12):4447-52. Epub 2007 Sep 17. PubMed PMID: 17875993;
PubMed Central PMCID: PMC2167980.
3: Barrow EW, Valderas MW, Bourne PC, Barrow WW. Newly developed colorimetric
drug screening assay for Bacillus anthracis. Int J Antimicrob Agents. 2006
Feb;27(2):178-80. Epub 2006 Jan 19. PubMed PMID: 16423510.
John, I get exactly the same issue of reaching a plateau of 90% inhibition when running the experiment as describe. Thanks Christina, I hope your method can help me.
Christina, when you mention subtracting absorbance at 600nm from absorbance at 570nm, does this refer to absorbance readings obtained for the same test well? I assume you blank with media only?
Hello Shane, I read absorbance at 570 nm and 600 nm... for the test well and the blank with media only (and alamarBlue)
%Reduction Alamar Blue = C.RED test samples / C.OX blank
5.= (εOX)λ2 Aλ1 -(εOX)λ1 Aλ2 / (εRED)λ1 A´λ2 -(εRED)λ2 A´λ1
λ1 = 570 nm
λ2 = 600 nm
A = test sample Absorbance
A´ = blank absorbance(medium + AlamarBlue)
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