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Bromothymol blue (also known as bromothymol sulfone phthalein and BTB) is a pH indicator. It is mostly used in applications that require measuring substances that would have a relatively neutral pH (near 7). A common use is for measuring the presence of carbonic acid in a liquid.
It was developed in 1953 by Hugh and Leifson to be utilized in microbiology to determine the way a microorganism metabolizes a carbohydrate such as glucose (dextrose). [1] OF-glucose deeps contain glucose as a carbohydrate, peptones, bromothymol blue indicator for Hugh-Leifson's OF medium or phenol red for King's OF medium, and 0.5% agar.
Universal indicator components Indicator Low pH colour Transition pH range High pH colour Thymol blue (first transition) Red 1.2 – 2.8 Yellow Methyl orange: Red 3.2 – 4.4 Yellow Methyl red: Red 4.8 – 6.0 Yellow Bromothymol blue: Yellow 6.0 – 7.6 Blue Thymol blue (second transition) Yellow 8.0 – 9.6 Blue Phenolphthalein: Colourless
The increase in pH then causes color change in the bromothymol blue indicator, turning it blue. Under neutral conditions the medium remains a green color. The color change to blue is useful because growth on Simmons' citrate agar is often limited and would be hard to observe if it were not for the color change.
Bromothymol blue was added in order to reduce false positives. The citrate agar is green before inoculation, and turns blue, because of BTB as a positive test indicator, meaning citrate is utilized. The test is also prepared on a slant to maximize bacterial growth for an even better indication of the use of citrate.
Bromothymol blue is the indicator used in the agar, it changes to yellow in case of acid production during fermentation of lactose or changes to deep blue in case of alkalinization. Lactose-positive bacteria build yellow colonies. Bacteria which decarboxylate L-cystine cause an alkaline reaction and build deep blue colonies. [1]
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Bacteria are inoculated on a medium containing sodium citrate and a pH indicator such as bromothymol blue. The medium also contains inorganic ammonium salts, which are utilized as sole source of nitrogen. Use of citrate involves the enzyme citrate lyase, which breaks down citrate to oxaloacetate and acetate.