| ONPG Test |
DESCRIPTION
Lactose fermentation requires the presence of permease and ß-galactosidase enzymes. Some lactose fermenting
organisms do not have permease but do possess ß-galactosidase, which hydrolyses lactose to form galactose and
glucose. The test is used to determine the presence ß-galactosidase.
Lactose fermentation requires the presence of permease and ß-galactosidase enzymes. Some lactose fermenting
organisms do not have permease but do possess ß-galactosidase, which hydrolyses lactose to form galactose and
glucose. The test is used to determine the presence ß-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 30 minutes, 1 hour and 24 hours at 37 ˚C.
RESULTS
Positive: yellow color.
Negative: colourless.
If ß-galactosidase is present, ONPG (colourless) is split into galactose
and o-nitrophenol (yellow).
Also, commercially ONPG discs may be used.
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 30 minutes, 1 hour and 24 hours at 37 ˚C.
RESULTS
Positive: yellow color.
Negative: colourless.
If ß-galactosidase is present, ONPG (colourless) is split into galactose
and o-nitrophenol (yellow).
Also, commercially ONPG discs may be used.
NOTES:
Bacteria grown on glucose containing medium show less reactivity than those grown on lactose containing media.
Glucose inhibits ß-galactosidase.
Bacteria grown on glucose containing medium show less reactivity than those grown on lactose containing media.
Glucose inhibits ß-galactosidase.
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