ONPG: Difference between revisions

Latest revision as of 15:30, 29 October 2019

O-Nitrophenyl-β-D-galactopyranoside (ONPG) is structurally a modified lactose that provides a colour indicator of lactose fermentation
Tests for the presence of β-galactosidase
Positive if the media turns yellow; usually positive within an hour, but cannot be called negative until 24 hours
When positive, identifies the organism as a lactose fermenter, and may be quicker for "late" fermenters than other methods

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+* O-Nitrophenyl-β-D-galactopyranoside (ONPG) is structurally a modified lactose that provides a colour indicator of '''lactose fermentation'''
-= ONPG test for β-galactosidase =
+* Tests for the presence of β-galactosidase
+* Positive if the media turns '''yellow'''; usually positive within an hour, but cannot be called negative until 24 hours
+* When positive, identifies the organism as a lactose fermenter, and may be quicker for "late" fermenters than other methods
+[[Category:Diagnostics]]
-O-Nitrophenyl-β-D-galactopyranoside (ONPG) is structurally similar to lactose (i.e. ONPG is an analog of lactose), except that orthonitrophenyl has been substituted for glucose.
-On hydrolysis, through the action of the enzyme β-galactosidase, ONPG cleaves into two residues, galactose and o-nitrophenol. ONPG is colorless compound: O-nitrophenol is yellow, providing visual evidence of hydrolysis.
-[https://i0.wp.com/microbeonline.com/wp-content/uploads/2015/02/ONPG-Structure.png [[File:https://i0.wp.com/microbeonline.com/wp-content/uploads/2015/02/ONPG-Structure.png?resize=640%2C254|ONPG Test]]]ONPG Test
-Lactose fermenting bacteria posses both lactose permease and β-galactosidase, two enzymes required for the production of acid in the lactose fermentation test. The permease is required for the lactose molecule to penetrate the bacterial cell where the β-galactosidase can cleave the galactoside bond, producing glucose and galactose.
-Non-lactose fermenting bacteria are devoid of both enzymes and are incapable of producing acid from lactose.
-Some bacterial species appear to be non-lactose fermenters because they '''lack permease, but do possess β-galactosidase and give a positive ONPG test'''. So called late lactose fermenters may be delayed in their production of acid from lactose because of sluggish permease activity. In these instances, a positive ONPG test may provide a rapid identification of delayed lactose fermentation.
-== ONPG vs. Lactose fermentation ==
-# '''Lactose fermenter (ONPG Positive)''': ''E.coli, Klebsiella spp, Enterobacter spp'' '''produce β-galactosidase and permease'''
-# '''Late lactose fermenter (ONPG Positive)''': ''Citrobacter spp, Arizona spp'' '''produce only β-galactosidase''' so they slowly ferment lactose.
-# '''Non lactose fermenter (ONPG Negative):''' ''Salmonella'' spp; ''Shigella'' spp; ''Proteus'' spp; ''Providencia''spp and ''Morganella'' spp '''do not produce β-galactosidase''' so can not ferment lactose.
-== Media and Reagents ==
-# Sodium phosphate buffer, 1 M, pH 7.0
-# O-Nitrophenyl-β-D-galactopyranoside (ONPG), 0.75 M
-# Physiologic saline
-# Toulene
-== Quality control ==
-# Positive control: ''Escherichia coli''
-# Negative control: ''Proteus vulgaris''
-== Procedure ==
-Bacteria grown in medium containing lactose (to induce the production of the galactosidase enzyme) , such as Kligler iron agar (KIA) or [https://microbeonline.com/triple-sugar-iron-agar-tsi-principle-procedure-and-interpretation/ Triple sugar Iron (TSI) agar], produces optimal results in ONPG Test.
-'''Note:''' β-galactosidase enzyme (inducible enzyme) is made ONLY in the presence of the lactose substrate
-# A loopful of bacterial growth is emulsified in 0.05mL of physiologic saline to produce a heavy suspension
-# One drop of toluene is added to the suspension and vigorously mixed for a few seconds to release the enzyme for bacterial cells.
-# An equal quantity of buffered ONPG solution is added to the suspension.
-# The mixture is placed in a 37oC water bath
-'''When Using ONPG Tablets'''
-# A loopful of bacterial suspension is added directly to the ONPG substrate resulting from adding 1mL of distilled water to a tablet in a test tube.
-# This suspension is also placed in a 37oC water bath
-== Results and Interpretations ==
-The rate of hydrolysis of ONPG to o-nitrophenol may be rapid for some organisms; producing a visible yellow color reaction within 5 to 10 minutes.
-Most tests are positive within 1 hour; however, reactions should not be interpreted as negative before 24 hours of incubation.
-The yellow color is usually distinct and indicates that the organism has produced o-nitrophenol from the ONPG substrate through the action of β-galactosidase.
-[https://i0.wp.com/microbeonline.com/wp-content/uploads/2015/02/ONPG-test-results.png [[File:https://i1.wp.com/microbeonline.com/wp-content/uploads/2015/02/ONPG-test-results-300x270.png?resize=300%2C270|ONPG Test results]]]
-ONPG Test results