| ONPG Test |
DESCRIPTION
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.
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.
PROCEDURE
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.
RESULTS
Positive: yellow color.
Negative: colourless.
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.
PROCEDURE
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.
RESULTS
Positive: yellow color.
Negative: colourless.
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.
NOTES:
Bacteria grown on glucose containing medium show less reactivity than those grown on lactose containing media. Glucose inhibits
beta-galactosidase.
An alternative method, using commercially available ONPG-impreganted discs may be taken into consideration, as is more simple
and easy to do.
NOTES:
Bacteria grown on glucose containing medium show less reactivity than those grown on lactose containing media. Glucose inhibits
beta-galactosidase.
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| ONPG test by impreganted-discs method, positive (left) and negative (right) |