Lactose fermentation requires the presence of permease and beta-galactosidase enzymes. Some lactose fermenting organisms do
not have permease but do possess beta-galactosidase, which hydrolyses lactose to form galactose and glucose. The test is used to
determine the presence beta-galactosidase.
ONPG (ortho-nitrophenyl-β-D-galactopyranoside) is used for it's similarity in structure
ONPG solution preparation: dissolve 80 mg ortho-nitrophenyl-ß-galactopyranosid in
15 ml distilled water then add 5 ml NaH2PO4 1.0M. Adjust pH to 7. Solution must be
Use 18 hours culture from 1% lactose-agar medium. Harvest culture and make a
suspension in 0.25 ml saline solution. Add 1 drop of toluene and stir well for enzyme
releasing. Incubate 5 minutes at 37 ˚C. Add ONPG solution 0.75M and incubate at 37
˚C. Examine colour changes hourly. If no changes appear in 4-6 hours then continue
incubation up to 18-24 hours.
Positive: yellow color.
If beta-galactosidase is present, ONPG (colourless) is split into galactose and o-nitrophenol (yellow).
An alternative method, using commercially available ONPG-impreganted discs may be taken into consideration, as is more simple
and easy to do.
Bacteria grown on glucose containing medium show less reactivity than those grown on lactose containing media. Glucose inhibits
(c) Costin Stoica
|ONPG test by impreganted-discs method,
positive (left) and negative (right)