ONPG
The ability of
bacteria to ferment lactose depends on two enzymes,beta-galactopermease and
beta-galactosidase. beta-galactopermease allows lactose to enter the bacterial cell wall,
where it is then broken down into glucose and galactose by
beta-galactosidase. The glucose and galactose can then be metabolized by
the bacteria. However, some organisms lack beta-galactopermease and appear as late or non-lactose-fermenters.
Principle
O-nitrophenyl-beta-D-galactopyranoside
(ONPG) is an artificial substrate structurally similar to lactose with the
exception that glucose is substituted with an o-nitrophenyl group. Unlike
lactose, the substrate O-nitrophenyl-beta-D-galactopyranoside (ONPG) is capable
of penetrating the bacterial cell without the presence of beta-galactopermease.
Media:
- ONPG broth: Na2HPO4 (9.46 g), phenylalanine (4 g), ONPG (2 g),
KH2PO4 (0.907 g), per 1000 mL, pH 8.0
- ONPG disk: ONPG Differentiation Disk is prepared by
impregnating carefully controlled concentrations of ONPG onto a 0.25 inch
diameter filter paper disk.
Method
A. For ONPG disk method
1. Place an ONPG disk into a sterile tube and add
0.2 mL saline.
2. Heavily inoculate the tube with a loopful of
the test isolate.
3. Incubate at 35-37°C for up to 4 hours.
4. Examine for color change of the disk.
B. For broth method
1. Bring test medium to room temperature.
2. Inoculate the test medium with heavy inoculum
from a pure 18-24 hour culture.
3. Incubate aerobically, with loose caps, at 35-
37ºC.
4. Examine for a yellow color development at 1
hour.
5. If no color change seen after an hour of
incubation, continue incubation for up to 24 hours.
Results
- Positive: Development of a yellow colouration (presence of
β-galactosidase)
- Negative: No colour development (absence of enzyme)
References
www.time2026end.com
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