Biochemical identification: Which test detects strong mixed-acid fermentation of glucose by showing a stable pH drop below approximately 4.5 in the medium?

Introduction / Context:
Biochemical tests differentiate Enterobacterales and other Gram-negative rods. Two companion assays, methyl red (MR) and Voges–Proskauer (VP), assess contrasting glucose fermentation pathways: mixed-acid versus butanediol fermentation. Correctly linking a significant, stable pH drop to the MR test is foundational in identification schemes.

Given Data / Assumptions:

• Organism ferments glucose vigorously, producing acids that reduce pH below ~4.5.
• Indicator dye methyl red turns red at low pH.
• Medium is MR-VP broth (buffered peptone–glucose).

Concept / Approach:
The MR test detects mixed-acid fermentation yielding stable acidic end products (lactic, acetic, formic acids). When pH < 4.4–4.5, methyl red remains red, indicating MR-positive (e.g., Escherichia coli, Proteus, Salmonella). VP detects neutral end products (acetoin/2,3-butanediol) typical of Enterobacter/Klebsiella, often MR-negative.

Step-by-Step Solution:
Inoculate MR-VP broth and incubate 24–48 h. Aliquot for MR, add a few drops of methyl red indicator. Observe a persistent red colour → stable pH < ~4.5 → MR positive. Conclude the test that detects the strong acid drop is MR, not VP.

Verification / Alternative check:
Run parallel VP: an organism is often MR-positive/VP-negative or vice versa. Use known controls (E. coli MR+, Enterobacter cloacae VP+).

Why Other Options Are Wrong:

• Indole: Detects tryptophanase activity, not pH stability.
• Citrate: Detects citrate as sole carbon source, alkalinization turns bromothymol blue to blue.
• VP: Detects acetoin (neutral products), not strong acid end-point.
• Urease: Ammonia raises pH; opposite direction.

Common Pitfalls:
Reading MR too early can miss a late acidification pattern; ensure adequate incubation and mixing before adding indicator.

Final Answer:
Methyl red (MR) test detects a stable pH below ~4.5 from mixed-acid fermentation.