19. Which one of the following statements relating to the mechanism of color development in response to LacZ expression in Escherichia coli is INCORRECT?
(1) E. coli growth on LB agar with X-gal results in blue colored colonies because LacZ produced in the cell hydrolyses X-gal present in the medium into a blue colored product. (2) When the membranes of the cells harboring LacZ are permeabilized and cells incubated in a buffer with ONPG, the solution turns yellow because LacZ encoded protein hydrolyzes
(3) E. coli growth on MacConkey agar results in pink colored colonies because LacZ encoded protein produced in the cell hydrolyzes the neutral red dye present in the medium into a pink colored product.
(4) E. coli growth on MacConkey agar results in pink colored colonies due to shift in pH of the medium MacConkey

Colorimetric assays are essential tools in molecular biology for detecting β-galactosidase (LacZ) activity in Escherichia coli. These methods rely on substrates like X-gal, ONPG, and pH-sensitive dyes in MacConkey agar. However, misconceptions about their mechanisms are common. This article clarifies the science behind these color changes and identifies the incorrect statement about LacZ-mediated color development.

Key Mechanisms of LacZ Activity Detection

1. X-gal (5-Bromo-4-Chloro-3-Indolyl-β-D-Galactopyranoside)

• Principle: X-gal is a chromogenic substrate cleaved by β-galactosidase into galactose and an insoluble blue pigment (5,5′-dibromo-4,4′-dichloro-indigo).

• Application:

  • E. coli colonies expressing LacZ turn blue on LB agar containing X-gal.

  • Used in blue-white screening to identify recombinant clones.

• Correct Statement:
  “E. coli growth on LB agar with X-gal results in blue-colored colonies because LacZ hydrolyzes X-gal into a blue product.”

2. ONPG (Ortho-Nitrophenyl-β-D-Galactopyranoside)

• Principle: ONPG is a colorless substrate cleaved by β-galactosidase into galactose and ortho-nitrophenol (yellow).

• Application:

  • Permeabilized cells release β-galactosidase, which reacts with ONPG to produce a yellow solution.

  • Used in quantitative assays (e.g., Miller assay).

• Correct Statement:
  “Permeabilized cells incubated with ONPG turn yellow because LacZ hydrolyzes ONPG.”

3. MacConkey Agar

• Principle: A differential medium containing lactose, bile salts, crystal violet, and neutral red dye.

  • Lactose Fermentation: E. coli fermenting lactose produce acidic byproducts, lowering the pH.

  • Neutral Red Dye: Turns pink/red under acidic conditions (pH < 6.8).

• Key Point: The pink color arises from pH changes, not direct LacZ interaction with the dye.

• Correct Statement:
  “E. coli growth on MacConkey agar results in pink colonies due to pH shifts from lactose fermentation.”

• Incorrect Statement:
  “E. coli growth on MacConkey agar results in pink colonies because LacZ hydrolyzes neutral red.”

Identifying the Incorrect Statement

Option (3) is incorrect because:

• MacConkey agar’s pink color results from acidic pH (due to lactose fermentation), not enzymatic cleavage of neutral red by LacZ.

• Neutral red is a pH indicator, not a substrate for β-galactosidase.

Summary Table: LacZ Detection Methods

Conclusion

The incorrect statement is Option (3). LacZ does not hydrolyze neutral red; the pink color on MacConkey agar arises from pH changes caused by lactose fermentation. Understanding these mechanisms is critical for accurate interpretation of bacterial assays in molecular biology and microbiology.

Keywords: LacZ, β-galactosidase, X-gal, ONPG, MacConkey agar, neutral red, lactose fermentation, pH indicator, blue-white screening, E. coli, colorimetric assay.