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BAM R53: ONPG Test

January 2001

Bacteriological Analytical Manual



Monosodium phosphate solution. 1.0 M, pH 7


NaH2PO4 H206.9 g
Distilled water45 ml
NaOH solution, 30% (w/v)3 ml


Dissolve NaH2PO4.HOO in distilled water. Add 30% NaOH solution and adjust to pH 7. Bring volume to 50 ml with distilled water and store in refrigerator (about 4°C).


0.0133 M o-Nitrophenyl--D-galactoside (ONPG)


ONPG80 mg
Distilled water, 37°C15 ml
Monosodium phosphate solution, 1.0 M, pH 75 ml


Dissolve ONPG in distilled water at 37°C. Add 1.0 M NaH2PO4 solution. Solution should be colorless. Store in refrigerator (about 4°C). Before use, warm appropriate portion (sufficient for number of tests) of ONPG solution to 37°C.




Inoculate cultures to be tested onto triple sugar iron agar slants and incubate for 18 h at 37°C (or other appropriate temperature, if required). Nutrient (or other) agar slants containing 1.0% lactose may also be used. In sterile 13 x 100 mm tube containing 0.25 ml of physiological saline, emulsify large loopful of culture growth to form heavy suspension. Add 1 drop of toluene to each tube and shake well to liberate enzyme. Let tubes stand 5 min at 37°C. Add 0.25 ml buffered 0.0133 M ONPG solution to each suspension to be tested. Incubate tubes at 35-37°C. Examine tubes at intervals up to 24 h. Yellow color is positive result.


ONPG disks are available commercially. Emulsify growth from 18 h lactose-containing medium in 0.2 ml sterile physiological saline in sterile 13 x 100 mm tube. Add disk and agitate gently. Incubate and read tubes as above.


Original Source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998.