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 detection of β-galactosidase (LacZ) activity is a mainstay in molecular biology and microbiology, especially for identifying recombinant E. coli and studying gene regulation. The three most popular systems for detecting LacZ activity are X-gal plates (for blue-white screening), ONPG assays (for quantitative measurement), and MacConkey agar (for lactose fermentation). However, misconceptions about the underlying mechanisms of these color changes persist. This article clarifies how each system works and reveals the most common mistake made when explaining these processes.

X-gal and Blue-White Screening

How it works:
X-gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside) is a colorless compound used in agar plates to detect β-galactosidase activity. When E. coli expresses LacZ, the enzyme hydrolyzes X-gal, producing a colorless intermediate that dimerizes and oxidizes to form an insoluble blue pigment (5,5′-dibromo-4,4′-dichloro-indigo)13456. Colonies with active LacZ appear blue, while those without remain white.

Correct statement:
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.

ONPG and the Yellow Color Reaction

How it works:
ONPG (ortho-nitrophenyl-β-D-galactopyranoside) is a colorless substrate used in β-galactosidase assays. When E. coli cells are permeabilized and incubated with ONPG, LacZ hydrolyzes ONPG, releasing ortho-nitrophenol, which is bright yellow. This reaction is the basis for the classic Miller assay, a quantitative method for measuring LacZ activity.

Correct statement:
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 ONPG.

MacConkey Agar and Pink Colonies

How it works:
MacConkey agar is a differential medium containing lactose and the pH indicator neutral red. When E. coli ferments lactose, acid is produced, lowering the pH. Neutral red turns pink under acidic conditions, so lactose-fermenting colonies appear pink. Crucially, the color change results from a pH shift—not from direct enzymatic hydrolysis of the dye by LacZ.

Correct statement:
E. coli growth on MacConkey agar results in pink colored colonies due to shift in pH of the medium MacConkey.

The INCORRECT Statement: Clearing Up the Confusion

Incorrect statement:
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.

Why this is wrong:
LacZ does not hydrolyze neutral red. The pink color on MacConkey agar is due to acidification from lactose fermentation, which causes the pH indicator neutral red to change color. The dye itself is not a substrate for β-galactosidase910.

Table: Mechanisms of Color Development in E. coli

Conclusion

The most common misconception is that LacZ hydrolyzes neutral red dye on MacConkey agar to produce pink colonies. In reality, the pink color results from a pH shift due to acid production during lactose fermentation. Understanding these mechanisms is essential for accurate interpretation of colorimetric assays in molecular biology and microbiology.

Correct answer:
Option (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.

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