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 to lactose.
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 colourless.
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
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 beta-galactosidase.
(c) Costin Stoica
|ONPG test by impreganted-discs method,
positive (left) and negative (right)