Methyl red test is used to identify pathogenic bacteria that produce acid from glucose phosphate. Some bacterial isolates ferment glucose through the mixed acid fermentation pathway to produce several acids including acetic acid, formic acid and lactic acid.

Production of steady amounts of acids by these microorganisms will result in a pH level that is usually below 4.4 (acidic) because the phosphate buffer of the medium have been overwhelmed by the acid produced.

Addition of methyl red to the medium result in this acidic medium and a red colouration is produced. Escherichia coli and Proteus species are both methyl red positive.


  • Prepare and dispense glucose phosphate broth in 9 ml portions of clean test tubes and sterilize the medium by autoclaving.
  • Bring the prepared medium to room temperature and allow to cool after sterilization. 
  • Inoculate the test isolate in duplicates.
  • Dissolve 0.1 g of methyl red powder in 300 ml of 95 % ethanol.
  • Make up the solution to 500 ml using distilled water.
  • Add few drops of the prepared methyl red solution (about 3-5 drops) to the broth culture of the test bacteria while leaving one of the tubes uninoculated with methyl red. This tube will serve as the negative control tube. A red colour appears in the tube(s) when the pH indicator (methyl red) is added to the broth culture, and this is possible if the pH of the broth culture medium is actually below 4.4.
  • Observe the tube(s) for a red colour which shows a positive methyl red test result. A yellow colouration shows a negative result (Figure 1).
Figure 1. Methyl red test. Tube on left is positive as indicated by a bright red colour; tube in the middle is negative while tube on the right is uninoculated tube serving as the control tube.


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