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42: Beta-Galactosidase Test (ONPG)

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    Learning Objectives

    • Identify the ability of the bacterium to make the enzyme beta - galactosidase


    Lactose utilization requires a couple of enzymes, one of which is beta - galactosidase. In this test, you use a molecular decoy called ONPG (Ortho-nitrophenyl-b-D-galactopyranoside) that will turn to a yellow color in the presence of this enzyme. ONPG is an analog of lactose that the enzyme can break down to produce a yellow colored end-product, O-nitrophenol. Since this enzyme is made ONLY in the presence of the lactose substrate, you need to be sure to grow this organism on media high in lactose (e.g., from lactose broth or on TSIA agar).

    ONPG_structure.png 75px-P-Nitrophenol.svg.png

    Chemical structure of (left) Ortho-Nitrophenyl-β-galactoside and p-Nitrophenol . from Wikipedia (credit: Edgar181 and NEUROtiker respectively)


    This test is generally for gram – bacteria (also to identify some Staphylococcus species)


    • ONPG discs
    • 0.85-0.9% NaCl 1 ml
    • pipettes and pi-pumps
    • sterile small tubes and caps


    1. FIRST, your organism needs to be growing in/ on any medium with lactose (to induce the production of the galactosidase enzyme)
    2. Pipet 0.5 ml of the saline into a sterile tube.
    3. Inoculate with the bacterium and add the ONPG disc in a sterile manner (forceps dipped in alcohol and flamed) to the tube.
    4. Incubate at 37º C for 4 hours. There is so little fluid in the tube, it will dehydrate if you do not read it in a few hours (alternatively, you can cover it tightly with parafilm).


    The fluid and disc will turn any shade of yellow if + for galactosidase enzyme.



    1. The enzyme, galactosidase, tests for the breakdown of the sugar_______.
    2. Why do you want to grow the bacterium on a high-lactose medium to run this test?

    Contributors and Attributions

    This page titled 42: Beta-Galactosidase Test (ONPG) is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Jackie Reynolds.

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