| 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.
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.
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.
| Antibiogram |
| Encyclopedia |
| Culture media |
| Biochemical tests |
| Stainings |
| Images |
| Movies |
| Articles |
| Identification |
| Software |
(c) Costin Stoica
| R E G N U M PROKARYOTAE |
| Back |
| ONPG test by impreganted-discs method, positive (left) and negative (right) |