Methyl red and Voges–Proskauer tests

Principle

• Both are done in MR-VP broth (buffered peptone, glucose, phosphate) and show different glucose fermentation pathways.
• Methyl red (MR): mixed acid fermentation produces strong, stable acids that drop the pH to about 4.4 or lower, overcoming the buffer.
• Voges–Proskauer (VP): butanediol fermentation produces acetoin, which is oxidized in alkali to diacetyl; diacetyl reacts with guanidine groups from peptone to give a red color, enhanced by α-naphthol.

Specimen

• Pure colony of a Gram-negative rod, lightly inoculated into two MR-VP broths (or one broth split).

Reagents and equipment

• MR-VP broth; methyl red indicator.
• Barritt's reagents: A = 5% α-naphthol in ethanol; B = 40% KOH.

Procedure

• MR: incubate at 35–37 °C for at least 48 h (up to 5 days), then add about 5 drops methyl red to an aliquot.
• VP: incubate at least 24–48 h; to about 2.5 mL of broth add 6 drops (0.6 mL) α-naphthol first, then 2 drops (0.2 mL) 40% KOH.
• Shake the VP tube to aerate and let stand; read within about 15–30 minutes.

Results

• MR positive: red color immediately; negative: yellow; orange is equivocal (re-incubate and repeat).
• VP positive: pink-red color; negative: yellow or copper-brown.
• E. coli: MR positive, VP negative; Klebsiella pneumoniae and Enterobacter: MR negative, VP positive.

Quality control and pitfalls

• Controls: E. coli (MR+/VP−) and K. pneumoniae or Enterobacter aerogenes (MR−/VP+).
• Reading MR before 48 h may give false positives because early acid is later neutralized.
• Adding reagents in the wrong order or reading VP after about 1 hour gives false positives (copper color).

Clinical use

• Differentiation of Enterobacterales (IMViC) and some other genera (e.g. VP for Streptococcus anginosus group, Staphylococcus spp.).