{"id":2775,"date":"2020-03-14T14:57:07","date_gmt":"2020-03-14T14:57:07","guid":{"rendered":"https:\/\/laboratoryinfo.com\/?p=2775"},"modified":"2022-03-07T19:36:56","modified_gmt":"2022-03-07T19:36:56","slug":"onpg-test","status":"publish","type":"post","link":"https:\/\/laboratoryinfo.com\/onpg-test\/","title":{"rendered":"ONPG Test (for \u03b2-galactosidase) &#8211; How to do (Principle), Results Interpretation and Uses"},"content":{"rendered":"\n<p>There are different types of bacteria and each has different abilities and reactions to certain organisms. The lactose-fermenting ability of bacteria\u2019s ability primarily depends on the enzyme&#8217;s permease and beta-galactosidase.<\/p>\n\n\n<style>.kb-table-of-content-nav.kb-table-of-content-id6113_3b9ebd-8c .kb-table-of-content-wrap{padding-top:var(--global-kb-spacing-sm, 1.5rem);padding-right:var(--global-kb-spacing-sm, 1.5rem);padding-bottom:var(--global-kb-spacing-sm, 1.5rem);padding-left:var(--global-kb-spacing-sm, 1.5rem);background-color:#ecf2f7;}.kb-table-of-content-nav.kb-table-of-content-id6113_3b9ebd-8c .kb-table-of-contents-title-wrap{padding-top:0px;padding-right:0px;padding-bottom:0px;padding-left:0px;}.kb-table-of-content-nav.kb-table-of-content-id6113_3b9ebd-8c .kb-table-of-contents-title{font-weight:regular;font-style:normal;}.kb-table-of-content-nav.kb-table-of-content-id6113_3b9ebd-8c .kb-table-of-content-wrap .kb-table-of-content-list{font-weight:regular;font-style:normal;margin-top:var(--global-kb-spacing-sm, 1.5rem);margin-right:0px;margin-bottom:0px;margin-left:0px;}.kb-table-of-content-nav.kb-table-of-content-id6113_3b9ebd-8c .kb-toggle-icon-style-basiccircle .kb-table-of-contents-icon-trigger:after, .kb-table-of-content-nav.kb-table-of-content-id6113_3b9ebd-8c .kb-toggle-icon-style-basiccircle .kb-table-of-contents-icon-trigger:before, .kb-table-of-content-nav.kb-table-of-content-id6113_3b9ebd-8c .kb-toggle-icon-style-arrowcircle .kb-table-of-contents-icon-trigger:after, .kb-table-of-content-nav.kb-table-of-content-id6113_3b9ebd-8c .kb-toggle-icon-style-arrowcircle .kb-table-of-contents-icon-trigger:before, .kb-table-of-content-nav.kb-table-of-content-id6113_3b9ebd-8c .kb-toggle-icon-style-xclosecircle .kb-table-of-contents-icon-trigger:after, .kb-table-of-content-nav.kb-table-of-content-id6113_3b9ebd-8c .kb-toggle-icon-style-xclosecircle .kb-table-of-contents-icon-trigger:before{background-color:#ecf2f7;}<\/style>\n\n\n<p>What permease does is it enables lactose to get through the cell wall of the microorganism and allowing<em><strong> beta-galactosidase<\/strong><\/em> to break it down into galactose and sugar. The bacteria will metabolize galactose and glucose. Some bacteria do not have permease and classified as non-lactose fermenters.<\/p>\n\n\n\n<p>To find out the lactose fermenting ability of bacteria, a test has to be performed \u2013 the ONPG Test. It is an extremely sensitive test used to check the ability of the bacteria to ferment lactose.<\/p>\n\n\n\n<p>It uses an artificial substrate called O-nitrophenyl-beta-D-galactopyranoside to ascertain enzymatic activity which helps in the identifying and differentiating various types of organisms. <sup><em><strong>(1, 2, 3, 4, and 5)<\/strong><\/em><\/sup><\/p>\n\n\n\n<div class=\"wp-block-image\"><figure class=\"aligncenter size-full is-resized\"><img decoding=\"async\" src=\"http:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/02\/Two-tubes.jpg\" alt=\"Two tubes\" class=\"wp-image-2844\" width=\"500\" srcset=\"https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/02\/Two-tubes.jpg 600w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/02\/Two-tubes-300x157.jpg 300w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/02\/Two-tubes-20x10.jpg 20w\" sizes=\"(max-width: 600px) 100vw, 600px\" \/><\/figure><\/div>\n\n\n\n<p class=\"has-text-align-center\"><b><strong>Image 1:<\/strong><\/b> Two tubes &#8211; the one on the left with no obvious changes in color while the one on the right turned yellow, which is an indicator of the presence of enzymes.<b><br><strong>Picture Source:<\/strong><\/b> i2.wp.com\/mltgeeks.com<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">What is the purpose of the ONPG test?<\/h2>\n\n\n\n<p>The purpose of the ONPG test is to check for the organism\u2019s ability to produce the <em><strong>\u03b2-galactosidase enzyme<\/strong><\/em>.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">What are the uses of the ONPG test?<\/h2>\n\n\n\n<ul><li>&nbsp;It is performed to help differentiate the members of Enterobacteriaceae and other microorganisms according to their beta-D-galactosidase activity.<\/li><li>It helps in distinguishing Enterobacteriaceae that are late lactose fermenters non-lactose fermenters. <sup><em><strong>(4, 5, and 6)<\/strong><\/em><\/sup><\/li><\/ul>\n\n\n\n<div class=\"wp-block-image\"><figure class=\"aligncenter size-full is-resized\"><img decoding=\"async\" src=\"http:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/02\/ONPG-test-of-Streptococcus-bacteria-as-seen-on-two-test-tubes.jpg\" alt=\"ONPG test of Streptococcus bacteria as seen on two test tubes\" class=\"wp-image-2845\" width=\"400\" srcset=\"https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/02\/ONPG-test-of-Streptococcus-bacteria-as-seen-on-two-test-tubes.jpg 400w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/02\/ONPG-test-of-Streptococcus-bacteria-as-seen-on-two-test-tubes-300x300.jpg 300w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/02\/ONPG-test-of-Streptococcus-bacteria-as-seen-on-two-test-tubes-150x150.jpg 150w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/02\/ONPG-test-of-Streptococcus-bacteria-as-seen-on-two-test-tubes-20x20.jpg 20w\" sizes=\"(max-width: 400px) 100vw, 400px\" \/><\/figure><\/div>\n\n\n\n<p class=\"has-text-align-center\"><b><strong>Image 2:<\/strong><\/b> An ONPG test of Streptococcus bacteria as seen on two test tubes.<b><br><strong>Picture Source:<\/strong><\/b> encrypted-tbn0.gstatic.com<b><\/b><\/p>\n\n\n\n<h2 class=\"wp-block-heading\">What is the principle of the ONPG test?<\/h2>\n\n\n\n<p>The test uses an artificial substrate called O-nitrophenyl-beta-D-galactopyranoside, which is similar in structure to that of lactose. The sugar is replaced with the o-nitrophenyl group, which has the ability to penetrate the cell in the absence of permease.<\/p>\n\n\n\n<p>When using the broth way, the organism to be tested is taken out from a medium that contains a high level of lactose. The organism is inoculated using the ONPG broth. You will know if it contains beta-galactosidase if the beta-galactoside bond splits allowing the release of o-nitrophenol; a compound characterized by its yellow color.<\/p>\n\n\n\n<p>Once you notice these changes, it means that the organism being tested produces the \u03b2-galactosidase enzyme.<\/p>\n\n\n\n<p>If the disk method is used, the subject of the test is taken out from a medium that contains a high level of lactose. A suspension is prepared wherein an ONPG disk is added to about 0.5 ml of suspension.<\/p>\n\n\n\n<p>Watch out for changes in color, specifically yellow color as it indicates that the organism being tested has beta-galactosidase.<\/p>\n\n\n\n<p>The changes in color usually take place within 24 hours. If the organism has a high level of beta-galactosidase, the changes in color is rapid. It would only take a few minutes\u2019 post ONPG medium inoculation. <sup><em><strong>(4, 5, 6, 7, and 8)<\/strong><\/em><\/sup><\/p>\n\n\n\n<h2 class=\"wp-block-heading\">How is the ONPG test done?<\/h2>\n\n\n\n<p>There are two methods of performing the ONPG test: the broth method and the disk method.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\"><strong>Broth Method<\/strong><\/h3>\n\n\n\n<p>Media \u2013 ONPF broth which consists of ONPG, Na2HPO4, KH2PO4 per 1 liter and a pH of 8.0.<\/p>\n\n\n\n<p><strong>Method:<\/strong><\/p>\n\n\n\n<ol><li>The test medium should be brought into room temperature.<\/li><li>The test medium should be inoculated with heavy inoculum from a pure culture 18 hour to 24 hours.<\/li><li>Incubate with a loose cap at the desired temperature of 35 to 37-degree centigrade.<\/li><li>After an hour, look for any changes in color.<\/li><li>If after an hour and no changes in color are detected, then continue the incubation for 24 hours.<\/li><\/ol>\n\n\n\n<h3 class=\"wp-block-heading\"><strong>Disk Method<\/strong><\/h3>\n\n\n\n<p>Media \u2013 To prepare the disk, you have to impregnate a controlled concentration of ONPG onto a filter paper disk, which is around0.25-inch in diameter.<\/p>\n\n\n\n<p><strong>Method:<\/strong><\/p>\n\n\n\n<ol><li>Put the ONPG disk into the test tube and slowly add a saline solution (0.2 ml).<\/li><li>Inoculate the tube using a loop that is full of test isolate.<\/li><li>Incubate for 4 hours at a temperature of 35 to 37 degrees Celsius.<\/li><li>Examine the disk for any changes in color. <sup><em><strong>(8, 9, and 10)<\/strong><\/em><\/sup><\/li><\/ol>\n\n\n\n<h2 class=\"wp-block-heading\">Interpreting Results<\/h2>\n\n\n\n<ul><li><b>Positive Results \u2013&nbsp;<\/b>if the color changes to yellow, it is an indicator of the presence of \u03b2-galactosidase. It is easy to tell the changes in color as the disc and the fluid turn yellow.<\/li><li><b>Negative Results \u2013&nbsp;<\/b>If the color remains the same, it means that the organism being tested does not contain the \u03b2-galactosidase enzyme.<\/li><\/ul>\n\n\n\n<h2 class=\"wp-block-heading\">Are there any limitations?<\/h2>\n\n\n\n<ol><li>It is not ideal to use in cultures that have naturally producing yellow pigment.<\/li><li>ONPG test alone is not reliable for complete identification of microorganisms. Other tests have to be done such as biochemical, molecular, immunological, and mass spectrometry.<\/li><li>The organism subject for testing should be inoculated from a lactose-containing medium. <sup><em><strong>(2, 5, and 9)<\/strong><\/em><\/sup><\/li><\/ol>\n\n\n\n<p><strong>References<\/strong><\/p>\n\n\n\n<ol><li><em>https:\/\/microbeonline.com\/onpg-test-\u03b2-galactosidase-principle-procedure-results\/<\/em><\/li><li><em>https:\/\/microbiologyinfo.com\/onpg-test\/<\/em><\/li><li><em>https:\/\/catalog.hardydiagnostics.com\/cp_prod\/content\/hugo\/onpgbroth.htm<\/em><\/li><li><em>https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC480699\/<\/em><\/li><li><em>https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC477897\/<\/em><\/li><li><em>https:\/\/bio.libretexts.org\/Bookshelves\/Ancillary_Materials\/Laboratory_Experiments\/Microbiology_Labs\/Microbiology_Labs_I\/42%3A_Beta-Galactosidase_Test_(ONPG)<\/em><\/li><li><em>https:\/\/jcp.bmj.com\/content\/jclinpath\/26\/11\/821.full.pdf<\/em><\/li><li><em>https:\/\/mltgeeks.com\/onpg-test-principlescomposition-and-cultural-response\/<\/em><\/li><li><em>http:\/\/www.medical-labs.net\/onpg-test-o-nitrophenyl-\u03b2-d-galactoside-1424\/<\/em><\/li><li><em>https:\/\/assets.fishersci.com\/TFS-Assets\/LSG\/manuals\/IFU62030.pdf<\/em><\/li><\/ol>\n","protected":false},"excerpt":{"rendered":"<p>There are different types of bacteria and each has different abilities and reactions to certain organisms. The lactose-fermenting ability of bacteria\u2019s ability primarily depends on the enzyme&#8217;s permease and beta-galactosidase. What permease does is it enables lactose to get through the cell wall of the microorganism and allowing beta-galactosidase to break it down into galactose&#8230;<\/p>\n","protected":false},"author":4,"featured_media":2845,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"rank_math_lock_modified_date":false,"_kad_post_transparent":"","_kad_post_title":"","_kad_post_layout":"","_kad_post_sidebar_id":"","_kad_post_content_style":"","_kad_post_vertical_padding":"","_kad_post_feature":"","_kad_post_feature_position":"","_kad_post_header":false,"_kad_post_footer":false,"footnotes":""},"categories":[4],"tags":[],"acf":[],"_links":{"self":[{"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/posts\/2775"}],"collection":[{"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/users\/4"}],"replies":[{"embeddable":true,"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/comments?post=2775"}],"version-history":[{"count":0,"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/posts\/2775\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/media\/2845"}],"wp:attachment":[{"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/media?parent=2775"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/categories?post=2775"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/tags?post=2775"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}