{"id":877,"date":"2023-05-30T10:51:46","date_gmt":"2023-05-30T05:06:46","guid":{"rendered":"https:\/\/microbenotes.com\/?p=877"},"modified":"2023-05-30T10:52:00","modified_gmt":"2023-05-30T05:07:00","slug":"beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results","status":"publish","type":"post","link":"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/","title":{"rendered":"Beta (\u03b2) Lactamase Test: Principle, Types, Procedure, Results"},"content":{"rendered":"\n<p><strong>Beta (\u03b2) Lactamase Test is used to detect <a href=\"https:\/\/microbenotes.com\/beta-lactamase-and-ambler-classification\/\" data-type=\"post\" data-id=\"1670\">beta-lactamase<\/a> enzymes produced by different bacteria.<\/strong><\/p>\n\n\n\n<ul>\n<li>Beta-lactamase is a plasmid-encoded or chromosomal-encoded enzyme that hydrolyzes the beta-lactam ring of the beta-lactam class of antibiotics resulting inactivation of these drugs. In addition, the beta-lactam gene frequently resides on integron.&nbsp;<\/li>\n\n\n\n<li>The first beta-lactamase was described in 1940 as a \u201cpenicillinase\u201d that is capable of hydrolyzing penicillin in <em>E. coli.<\/em><\/li>\n\n\n\n<li>Beta-lactamase enzyme production may be constative or induced by exposure to antimicrobials.<\/li>\n\n\n\n<li>According to the Bush-Medeiros Classification System, beta-lactamase enzymes divide into 4 molecular classes (A-D) based on their amino acid structure.<\/li>\n\n\n\n<li>Beta-lactamase enzymes are detected by rapid beta-lactamase tests. This test gives results earlier than MIC or Disk diffusion test.<\/li>\n<\/ul>\n\n\n\n<div id=\"ez-toc-container\" class=\"ez-toc-v2_0_65 counter-hierarchy ez-toc-counter ez-toc-custom ez-toc-container-direction\">\n<div class=\"ez-toc-title-container\">\n<p class=\"ez-toc-title \" >Table of Contents<\/p>\n<span class=\"ez-toc-title-toggle\"><a href=\"#\" class=\"ez-toc-pull-right ez-toc-btn ez-toc-btn-xs ez-toc-btn-default ez-toc-toggle\" aria-label=\"Toggle Table of Content\"><span class=\"ez-toc-js-icon-con\"><span class=\"\"><span class=\"eztoc-hide\" style=\"display:none;\">Toggle<\/span><span class=\"ez-toc-icon-toggle-span\"><svg style=\"fill: #0a0a0a;color:#0a0a0a\" xmlns=\"http:\/\/www.w3.org\/2000\/svg\" class=\"list-377408\" width=\"20px\" height=\"20px\" viewBox=\"0 0 24 24\" fill=\"none\"><path d=\"M6 6H4v2h2V6zm14 0H8v2h12V6zM4 11h2v2H4v-2zm16 0H8v2h12v-2zM4 16h2v2H4v-2zm16 0H8v2h12v-2z\" fill=\"currentColor\"><\/path><\/svg><svg style=\"fill: #0a0a0a;color:#0a0a0a\" class=\"arrow-unsorted-368013\" xmlns=\"http:\/\/www.w3.org\/2000\/svg\" width=\"10px\" height=\"10px\" viewBox=\"0 0 24 24\" version=\"1.2\" baseProfile=\"tiny\"><path d=\"M18.2 9.3l-6.2-6.3-6.2 6.3c-.2.2-.3.4-.3.7s.1.5.3.7c.2.2.4.3.7.3h11c.3 0 .5-.1.7-.3.2-.2.3-.5.3-.7s-.1-.5-.3-.7zM5.8 14.7l6.2 6.3 6.2-6.3c.2-.2.3-.5.3-.7s-.1-.5-.3-.7c-.2-.2-.4-.3-.7-.3h-11c-.3 0-.5.1-.7.3-.2.2-.3.5-.3.7s.1.5.3.7z\"\/><\/svg><\/span><\/span><\/span><\/a><\/span><\/div>\n<nav><ul class='ez-toc-list ez-toc-list-level-1 ' ><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-1\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#objectives\" title=\"Objectives\">Objectives<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-2\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#chromogenic-cephalosporin-test-nitrocefin-test-of-beta-%ce%b2-lactamase-test\" title=\"Chromogenic cephalosporin test (Nitrocefin test) of Beta (\u03b2) Lactamase Test \">Chromogenic cephalosporin test (Nitrocefin test) of Beta (\u03b2) Lactamase Test <\/a><ul class='ez-toc-list-level-3' ><li class='ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-3\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#principle-of-nitrocefin-test\" title=\"Principle of Nitrocefin test\u00a0\">Principle of Nitrocefin test\u00a0<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-4\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#requirement-of-nitrocefin-test\" title=\"Requirement of Nitrocefin test\">Requirement of Nitrocefin test<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-5\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#procedure-of-nitrocefin-test\" title=\"Procedure of Nitrocefin test\">Procedure of Nitrocefin test<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-6\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#results-and-interpretation-of-the-nitrocefin-test\" title=\"Results and interpretation of the Nitrocefin test\">Results and interpretation of the Nitrocefin test<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-7\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#positive-and-negative-controls\" title=\"Positive and negative controls\">Positive and negative controls<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-8\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#applications-of-nitrocefin-test\" title=\"Applications of Nitrocefin test\">Applications of Nitrocefin test<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-9\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#limitations-of-the-nitrocefin-test\" title=\"Limitations of the Nitrocefin test\">Limitations of the Nitrocefin test<\/a><\/li><\/ul><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-10\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#acidimetric-method-of-beta-%ce%b2-lactamase-test\" title=\"Acidimetric method\u00a0of Beta (\u03b2) Lactamase Test\">Acidimetric method\u00a0of Beta (\u03b2) Lactamase Test<\/a><ul class='ez-toc-list-level-3' ><li class='ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-11\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#principle-of-acidimetric-method\" title=\"Principle of Acidimetric method\u00a0\">Principle of Acidimetric method\u00a0<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-12\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#requirement-of-acidimetric-method\" title=\"Requirement\u00a0of Acidimetric method\u00a0\u00a0\">Requirement\u00a0of Acidimetric method\u00a0\u00a0<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-13\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#procedures-for-disk-test-of-acidimetric-method\" title=\"Procedures (for disk test) of Acidimetric method\u00a0\">Procedures (for disk test) of Acidimetric method\u00a0<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-14\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#procedure-for-tube-method-of-acidimetric-method\" title=\"Procedure for tube method\u00a0of Acidimetric method\u00a0\">Procedure for tube method\u00a0of Acidimetric method\u00a0<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-15\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#results-and-interpretation-of-acidimetric-method\" title=\"Results and interpretation of Acidimetric method\u00a0\">Results and interpretation of Acidimetric method\u00a0<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-16\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#applications-of-acidimetric-method\" title=\"Applications of Acidimetric method\u00a0\u00a0\">Applications of Acidimetric method\u00a0\u00a0<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-17\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#limitations-of-acidimetric-method\" title=\"Limitations of Acidimetric method\u00a0\u00a0\">Limitations of Acidimetric method\u00a0\u00a0<\/a><\/li><\/ul><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-18\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#iodometric-method-of-beta-%ce%b2-lactamase-test\" title=\"Iodometric method\u00a0of Beta (\u03b2) Lactamase Test\">Iodometric method\u00a0of Beta (\u03b2) Lactamase Test<\/a><ul class='ez-toc-list-level-3' ><li class='ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-19\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#principle-of-iodometric-method\" title=\"Principle\u00a0of Iodometric method\u00a0\">Principle\u00a0of Iodometric method\u00a0<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-20\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#requirement-of-iodometric-method\" title=\"Requirement\u00a0of Iodometric method\">Requirement\u00a0of Iodometric method<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-21\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#procedure-of-iodometric-method\" title=\"Procedure of Iodometric method\">Procedure of Iodometric method<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-22\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#results-and-interpretation-of-iodometric-method\" title=\"Results and Interpretation of Iodometric method\">Results and Interpretation of Iodometric method<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-23\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#applications-of-iodometric-method\" title=\"Applications\u00a0of Iodometric method\">Applications\u00a0of Iodometric method<\/a><\/li><li class='ez-toc-page-1 ez-toc-heading-level-3'><a class=\"ez-toc-link ez-toc-heading-24\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#limitations-of-iodometric-method\" title=\"Limitations\u00a0of Iodometric method\">Limitations\u00a0of Iodometric method<\/a><\/li><\/ul><\/li><li class='ez-toc-page-1 ez-toc-heading-level-2'><a class=\"ez-toc-link ez-toc-heading-25\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/#reference\" title=\"Reference&nbsp;\">Reference&nbsp;<\/a><\/li><\/ul><\/nav><\/div>\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"objectives\"><\/span><strong>Objectives<\/strong><span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<p>To detect the enzyme beta-lactamase by chromogenic, iodometric, and acidometric tests, which confers beta-lactam resistance to various bacterial organisms.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"chromogenic-cephalosporin-test-nitrocefin-test-of-beta-%ce%b2-lactamase-test\"><\/span><strong>Chromogenic cephalosporin test (Nitrocefin test) of Beta (\u03b2) Lactamase Test<\/strong> <span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"principle-of-nitrocefin-test\"><\/span><strong>Principle of <strong>Nitrocefin test<\/strong>\u00a0<\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<p>Nitrocefin disks are impregnated with nitrocefin, a chromogenic cephalosporin.\u00a0 Bacteria produced a significant amount of \u03b2-lactamase enzyme resulting in hydrolyzed amide bond in a \u03b2- lactam ring. Color of the nitrocefin disk changed from yellow to red. The color change of the disk indicates a positive test, and the colors that remain the same indicate a negative test. These beta-lactamases are capable of inactivating \u201cpenicillinase-labile-penicillins\u201d such as amoxicillin, ampicillin, penicillin, carbenicillin, mezlocillin, and piperacillin.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"requirement-of-nitrocefin-test\"><\/span><strong>Requirement <strong>of <strong>Nitrocefin test<\/strong><\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<ul>\n<li>Nitrocefin disk (commercially available, follow manufacture instructions) stored at 2-8 \u2103.<\/li>\n\n\n\n<li>Sterile distilled water&nbsp;<\/li>\n\n\n\n<li>Glass slide or empty petri dish&nbsp;<\/li>\n\n\n\n<li>Sterile Pasteur pipette<\/li>\n\n\n\n<li>Sterile wooden stick and inoculating loop<\/li>\n\n\n\n<li>The test organism colony was grown overnight (18-24 hrs) on non-selective media.<\/li>\n<\/ul>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"procedure-of-nitrocefin-test\"><\/span><strong>Procedure <strong>of <strong>Nitrocefin test<\/strong><\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<ol>\n<li>Using sterile forceps, dispense the required number of disks onto a clean microscope slide or an empty petri dish.<\/li>\n\n\n\n<li>Before inoculation, allow the nitrocefin disk to be brought to room temperature.&nbsp;&nbsp;&nbsp;&nbsp;<\/li>\n\n\n\n<li>&nbsp;Moisten each disk with 1 drop of sterile distilled water.&nbsp;<\/li>\n\n\n\n<li>&nbsp;With a sterile loop or applicator stick, smear several colonies onto the disk surface and also smear the positive control strain colony and negative control strain colony.&nbsp;<\/li>\n\n\n\n<li>&nbsp;Observe the disk for color change. Positive results usually appear within 15 s to 5 min. If no color change occurs within 5 min, the test is negative. However, positive reactions for some staphylococci may take up to 1 h.&nbsp;<\/li>\n<\/ol>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"results-and-interpretation-of-the-nitrocefin-test\"><\/span><strong>Results and interpretation <strong>of the <strong>Nitrocefin test<\/strong><\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<p>Positive: yellow color changes to red when the culture inoculates.&nbsp;<\/p>\n\n\n\n<p>Negative: no change in the color of the disk.<\/p>\n\n\n\n<figure class=\"wp-block-image size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"800\" height=\"420\" src=\"https:\/\/microbenotes.com\/wp-content\/uploads\/2018\/07\/Result-Interpretation-of-Beta-\u03b2-Lactamase-Test.jpg\" alt=\"Beta (\u03b2) Lactamase Test\" class=\"wp-image-878\" srcset=\"https:\/\/microbenotes.com\/wp-content\/uploads\/2018\/07\/Result-Interpretation-of-Beta-\u03b2-Lactamase-Test.jpg 800w, https:\/\/microbenotes.com\/wp-content\/uploads\/2018\/07\/Result-Interpretation-of-Beta-\u03b2-Lactamase-Test-300x158.jpg 300w, https:\/\/microbenotes.com\/wp-content\/uploads\/2018\/07\/Result-Interpretation-of-Beta-\u03b2-Lactamase-Test-768x403.jpg 768w\" sizes=\"(max-width: 800px) 100vw, 800px\" \/><figcaption class=\"wp-element-caption\">Beta (\u03b2) Lactamase Test<\/figcaption><\/figure>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"positive-and-negative-controls\"><\/span><strong>Positive and negative controls<\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<p>Bacteria that give positive results: <em>Staphylococcus aureus<\/em> ATCC 29213 (Quality control)&nbsp;<\/p>\n\n\n\n<p>Bacteria that give negative results: <em>Haemophilus influenza<\/em> ATCC 10211 (Quality control), <em>S aureus<\/em> ATCC 25923.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"applications-of-nitrocefin-test\"><\/span><strong>Applications <strong>of <strong>Nitrocefin test<\/strong><\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<ul>\n<li>The chromogenic cephalosporins test (Nitrocefin) is a biochemical test that is a sensitive method for detecting beta-lactamase-producing strains of <em>N. gonorrhoeae<\/em>, <em>H. influenza, Staphylococcus<\/em> spp<em>, Enterococcus <\/em>spp<em>,<\/em> and <em>Moraxella catarrhalis<\/em>. It is the most reliable test for detecting beta-lactamase-producing<em> Enterococcus <\/em>spp<em>.<\/em><\/li>\n\n\n\n<li>Easy to perform and gives results earlier than other methods.<\/li>\n\n\n\n<li>The chromogenic method is quicker and more convenient than the acidimetric and iodometric methods.<\/li>\n\n\n\n<li>The sensitivity and efficiency of the chromogenic method are high due to the detection of both penicillinase and cephalosporinase enzymes produced by isolates.<\/li>\n\n\n\n<li>Nitrocefin test is only reliable test for detecting \u03b2 -lactamase production by <em>Enterococcus<\/em> spp.&nbsp;<\/li>\n\n\n\n<li>The chromogenic cephalosporin compound nitrocefin test was also used for detecting \u03b2-lactamase produced by <em>Achromobacter.<\/em><\/li>\n\n\n\n<li>Nitrocefin has a wide susceptibility and sensitivity to the commercially available beta-lactam.<\/li>\n<\/ul>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"limitations-of-the-nitrocefin-test\"><\/span><strong>Limitations <strong>of the <strong>Nitrocefin test<\/strong><\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<ul>\n<li>Moisten disk is critical to the development of the color, if the disk begins to dry out, it may be necessary to rehydrate the disk with a small amount of water.<\/li>\n\n\n\n<li>The indistinct and weak reaction was obtained for strains grown on <a href=\"https:\/\/microbenotes.com\/blood-agar-composition-principle-preparation-and-uses\/\" data-type=\"post\" data-id=\"580\">blood agar<\/a> plates.<\/li>\n\n\n\n<li>&nbsp;Beta-lactamase detection with the nitrocefin disk should not entirely replace conventional susceptibility test methods, as other factors also influence the results of such tests, and on occasion, intrinsic resistance to beta-lactam antimicrobials has not been correlated with the production of beta-lactamase.<\/li>\n\n\n\n<li>Do not over-saturate the tip, as it could dilute the reagent.<\/li>\n\n\n\n<li>Detection of beta-lactamase activity in staphylococci may take up to one hour. Induction of the enzyme may also be required. This can be done by testing growth from the zone margin around an oxacillin disk.<\/li>\n\n\n\n<li>A negative result does not rule out resistance due to other mechanisms.<\/li>\n\n\n\n<li>Nitrocefin disk method cannot be used to test the members of\u00a0<a href=\"https:\/\/microbenotes.com\/enterobacteriaceae-cultural-characteristics\/\" data-type=\"post\" data-id=\"28386\">Enterobacteriaceae<\/a>,\u00a0<em>Pseudomonas\u00a0<\/em>species, or other aerobic, gram-negative bacilli because the results may not be predictive of susceptibility to the beta-lactams most often used for therapy.<\/li>\n\n\n\n<li>The Nitrocefin disk cannot be used for organisms where penicillin resistance is not due to beta-lactamase production, such as&nbsp;<em>Streptococcus pneumoniae&nbsp;<\/em>and Streptococci.<\/li>\n<\/ul>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"acidimetric-method-of-beta-%ce%b2-lactamase-test\"><\/span><strong>Acidimetric method\u00a0of Beta (\u03b2) Lactamase Test<\/strong><span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"principle-of-acidimetric-method\"><\/span><strong>Principle of <strong>Acidimetric method\u00a0<\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<p>In acidimetric test, penicillin-phenol red substrate reacts with beta-lactamase enzyme resulting in penicilloic acid produced. The color of the disk or solution (in tube test) changes from violet or red to yellow due to the pH decreases. Color change into yellow indicates a positive test or no change in color indicates a negative test.&nbsp;<\/p>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"requirement-of-acidimetric-method\"><\/span><strong>Requirement\u00a0of <strong>Acidimetric method\u00a0<\/strong>\u00a0<\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<p><strong>For disk or strip test\u00a0<\/strong><\/p>\n\n\n\n<ul>\n<li>Acidimetric disk (commercially available, following manufacturer instruction) stored at 2-8 \u2103.<\/li>\n\n\n\n<li>Sterile distilled water&nbsp;<\/li>\n\n\n\n<li>Glass slide or empty petri dish&nbsp;<\/li>\n\n\n\n<li>Sterile Pasteur pipettes<\/li>\n\n\n\n<li>Sterile wooden stick and inoculating loop<\/li>\n\n\n\n<li>The test organism colony was grown overnight (18-24 hrs) on non-selective media.<\/li>\n<\/ul>\n\n\n\n<p><strong>For tube test\u00a0<\/strong><\/p>\n\n\n\n<ul>\n<li>0.5% Phenol red solution (add 0.5 g of phenol red to small amount of water, dissolved properly dissolved heat may be needed to dissolve dye and make up volume 100ml then store at 25\u00b0C. Shelf life is 6 months.)<\/li>\n\n\n\n<li>&nbsp;Crystalline potassium penicillin G (vial containing 20 million U). Store as indicated by the manufacturer.<\/li>\n\n\n\n<li>&nbsp;1 N NaOH (add 4 g of NaOH crystals to 100 ml of water). Caution: This will cause heat production. Store at 25\u00b0C.<\/li>\n\n\n\n<li>&nbsp;Sterile 1- and 10-ml pipettes and pipette bulb&nbsp;<\/li>\n\n\n\n<li>&nbsp;Sterile polystyrene capped tubes (12 by 75 mm)&nbsp;<\/li>\n\n\n\n<li>&nbsp;Sterile wooden applicator sticks or inoculating loops.<\/li>\n<\/ul>\n\n\n\n<p><strong>\u00a0Preparation of penicillin-phenol red substrate reagent\u00a0<\/strong><\/p>\n\n\n\n<ul>\n<li>Add 2 ml of the 5% phenol red solution to 16.6 ml of sterile distilled water.<\/li>\n\n\n\n<li>&nbsp;Add the phenol red-water solution (18.6 ml) to the vial of crystalline benzylpenicillin G.&nbsp;<\/li>\n\n\n\n<li>&nbsp;Remove the solution from the vial and place it in a sterile container.&nbsp;<\/li>\n\n\n\n<li>&nbsp;Add 1 N NaOH dropwise to this acidic solution until it develops a violet color (pH 8.5).&nbsp;<\/li>\n\n\n\n<li>Dispense in 0.1-ml aliquots into sterile tubes and freeze at &#8211; 20\u00b0C or lower in a non-frost-free freezer.&nbsp;<\/li>\n<\/ul>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"procedures-for-disk-test-of-acidimetric-method\"><\/span><strong>Procedures (for disk test) of <strong>Acidimetric method\u00a0<\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<ol>\n<li>Using sterile forceps, dispense the required number of disks onto a clean microscope slide or an empty petri dish, and the remaining unused disk immediately place into the freezer.<\/li>\n\n\n\n<li>Before inoculation, allow the nitrocefin disk to be brought to room temperature.&nbsp;&nbsp;&nbsp;&nbsp;<\/li>\n\n\n\n<li>&nbsp;Moisten each disk with 1 drop of sterile distilled water.&nbsp;<\/li>\n\n\n\n<li>&nbsp;With a sterile loop or applicator stick, smear several colonies onto the disk surface and also smear the positive control strain colony and negative control strain colony.&nbsp;<\/li>\n\n\n\n<li>&nbsp;Observe the disk for color change. Positive results usually appear within 10 min. If no color change occurs within 10 min, the test is negative. However, positive reactions for some staphylococci may take up to 1 h. When dry, the color remains for up to 24 h.<\/li>\n<\/ol>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"procedure-for-tube-method-of-acidimetric-method\"><\/span><strong>Procedure for tube method\u00a0of <strong>Acidimetric method\u00a0<\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<ol>\n<li>&nbsp;Remove the desired number of reagent tubes from the freezer and allow them to thaw at room temperature (one tube per organism).&nbsp;<\/li>\n\n\n\n<li>&nbsp;With a sterile loop or applicator stick, add four or five colonies to the test solution to make an opaque, milky suspension.<\/li>\n\n\n\n<li>&nbsp;Observe for color change. A positive reaction will occur in less than 15 min. If no color change occurs within 15 min, the test is negative. A color change after 15 min usually indicates deterioration of the substrate not related to the presence of beta-lactamase and should not be considered positive. Since positive reactions for some staphylococci may take up to 1 h, results that turn positive after 15 min may not be reliable for these bacteria.<\/li>\n<\/ol>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"results-and-interpretation-of-acidimetric-method\"><\/span><strong>Results and interpretation of <strong>Acidimetric method\u00a0<\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<ul>\n<li>Positive: Violet or red color changes to yellow.&nbsp;<\/li>\n\n\n\n<li>Negative: no change in color occurs.<\/li>\n<\/ul>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"applications-of-acidimetric-method\"><\/span><strong>Applications of <strong>Acidimetric method\u00a0<\/strong>\u00a0<\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<ul>\n<li>Acidometric method is more effective than iodometric method for detection of coagulase positive Staphylococcal \u03b2-lactamase.<\/li>\n\n\n\n<li>Rapid acidimetric method used for performing a beta-lactamase test on <em>Haemophilus <\/em>spp<em>,<\/em> <em>Neisseria gonorrhoeae,<\/em> and <em>staphylococcus<\/em> spp.&nbsp;<\/li>\n\n\n\n<li>The test is easy to perform and interpret.<\/li>\n<\/ul>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"limitations-of-acidimetric-method\"><\/span><strong>Limitations of <strong>Acidimetric method\u00a0<\/strong>\u00a0<\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<ul>\n<li>This test applies only to aerobic bacteria.<\/li>\n\n\n\n<li>Acidometric test don\u2019t differentiate between acylase and \u03b2-lactamase activity.<\/li>\n\n\n\n<li>Only detect penicillinase, not cephalosporinase enzyme.<\/li>\n<\/ul>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"iodometric-method-of-beta-%ce%b2-lactamase-test\"><\/span><strong>Iodometric method\u00a0of Beta (\u03b2) Lactamase Test<\/strong><span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"principle-of-iodometric-method\"><\/span><strong>Principle\u00a0of <strong>Iodometric method\u00a0<\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<p>This method is based on the fact that \u03b2 -lactamase can hydrolyze penicillin G and release a reducing product (penicilloic acid), which reduces iodine and prevent it from combining with starch. Discoloration of dark blue iodine starch complex indicates positive results.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"requirement-of-iodometric-method\"><\/span><strong>Requirement\u00a0<strong>of <strong>Iodometric method<\/strong><\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<ul>\n<li>Penicillin: (6,000 \u00b5g\/ml) dissolved in phosphate buffer (pH 6.0, 0.05 to 1 M) Store at 2 to 8\u00b0C. Shelf life is 24 h.&nbsp;<\/li>\n\n\n\n<li>Starch reagent: Add 1 g of soluble starch to 100 ml of distilled water and heat in a boiling water bath until starch dissolves. Store at 2 to 8\u00b0C. The shelf life is 1 week.<\/li>\n\n\n\n<li>Iodine reagents: Dissolve 2.03 g of iodine and 53.2 g of potassium iodide in a small volume of distilled water and make the final volume to 100 ml. Store at 2 to 8\u00b0C in a dark bottle; replace if precipitate is apparent. Shelf life is 2 months.<\/li>\n\n\n\n<li>&nbsp;&nbsp;Empty sterile microdilution tray or small test tube<\/li>\n\n\n\n<li>&nbsp;Sterile 1 ml pipettes and pipette bulb&nbsp;<\/li>\n\n\n\n<li>&nbsp;Sterile wooden applicator sticks or inoculating loops<\/li>\n<\/ul>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"procedure-of-iodometric-method\"><\/span><strong>Procedure <strong>of <strong>Iodometric method<\/strong><\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<ol>\n<li>&nbsp;Dispense 0.1 ml of the penicillin solution into a well of a microdilution tray (or a small test tube).&nbsp;<\/li>\n\n\n\n<li>Add the test organism to make an opaque, milky suspension.&nbsp;<\/li>\n\n\n\n<li>Add 2 drops of the starch solution and mix. Let sit at room temperature (approximately 25\u00b0C) for 30 to 60 min. Add 1 drop of the iodine reagent. Shake or stir the mixture for 1 min.&nbsp;<\/li>\n\n\n\n<li>Observe for color change. Decolorization (to white) in less than 10 min indicates a positive reaction. If no color change occurs within 10 min, the test is negative. However, positive reactions for some staphylococci may take up to 1 h.<\/li>\n<\/ol>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"results-and-interpretation-of-iodometric-method\"><\/span><strong>Results and Interpretation <strong>of <strong>Iodometric method<\/strong><\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<ul>\n<li>Positive: fading of blue to colorless.&nbsp;<\/li>\n\n\n\n<li>Negative: blue or purple color.<\/li>\n<\/ul>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"applications-of-iodometric-method\"><\/span><strong>Applications\u00a0<strong>of <strong>Iodometric method<\/strong><\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<ul>\n<li>Iodometric slide test results have been reported to be similar to the Nitrocefin disk test and thus, can be used in detecting Staphylococcal \u03b2-lactamase when chromogenic methods are not available.&nbsp;<\/li>\n\n\n\n<li>Iodometric method is more sensitive and accurate compared to acidometric method for the detection of beta-lactamase production by<em> staphylococcus<\/em>.&nbsp;<\/li>\n\n\n\n<li>Most reliable method for testing beta-lactamase production by <em>N. gonorrhoeae<\/em>.<\/li>\n<\/ul>\n\n\n\n<h3 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"limitations-of-iodometric-method\"><\/span><strong>Limitations\u00a0<strong>of <strong>Iodometric method<\/strong><\/strong><\/strong><span class=\"ez-toc-section-end\"><\/span><\/h3>\n\n\n\n<ul>\n<li>Iodometric test is specific to the \u03b2-lactamase test but is not a chromogenic method for the detection of enzymes from cephalosporinase activity.<\/li>\n\n\n\n<li>Acquisition of freshly prepared starch and iodine solution may hinder its routine use.<\/li>\n<\/ul>\n\n\n\n<h2 class=\"wp-block-heading\"><span class=\"ez-toc-section\" id=\"reference\"><\/span><strong>Reference&nbsp;<\/strong><span class=\"ez-toc-section-end\"><\/span><\/h2>\n\n\n\n<ol>\n<li>Kuo, S. S., &amp; Feng, T. Y. (1989). Iodometric method for detection of \u03b2-lactamase activity in yeast cells carrying ampicillin resistance gene in chimeric plasmids.&nbsp;<em>Analytical biochemistry<\/em>,&nbsp;<em>177<\/em>(1), 165-167.<\/li>\n\n\n\n<li>Leber, A.L. (2016). Clinical Microbiology Procedure Handbook. Vol.1-3, American Society for Microbiology, ASM Press.<\/li>\n\n\n\n<li>Livermore, D. M. (1995). beta-Lactamases in laboratory and clinical resistance.&nbsp;<em>Clinical microbiology reviews<\/em>,&nbsp;<em>8<\/em>(4), 557-584.<\/li>\n\n\n\n<li>Bidya, S., &amp; Suman, R. S. (2014). <a href=\"https:\/\/www.scirp.org\/journal\/paperinformation.aspx?paperid=44037\" target=\"_blank\" data-type=\"URL\" data-id=\"https:\/\/www.scirp.org\/journal\/paperinformation.aspx?paperid=44037\" rel=\"noreferrer noopener\">Comparative study of three \u03b2 lactamase test methods in Staphylococcus aureus isolated from two Nepalese hospitals<\/a>.\u00a0<em>Open Journal of Clinical Diagnostics<\/em>,\u00a0<em>2014<\/em>.<\/li>\n\n\n\n<li>Petersson, A. C., Eliasson, I., Kamme, C., &amp; Mi\u00f6rner, H. (1989). Evaluation of four qualitative methods for detection of beta-lactamase production in Staphylococcus and Micrococcus species.&nbsp;<em>European Journal of Clinical Microbiology and Infectious Diseases<\/em>,&nbsp;<em>8<\/em>(11), 962-967.<\/li>\n\n\n\n<li>Levesque, R., Letarte, R., &amp; Pech\u00e8re, J. C. (1983). Comparative study of the beta-lactamase activity found in Achromobacter.\u00a0<em>Canadian Journal of Microbiology<\/em>,\u00a0<em>29<\/em>(7), 819-826.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"<p>Beta (\u03b2) Lactamase Test is used to detect beta-lactamase enzymes produced by different bacteria. Objectives To detect the enzyme beta-lactamase by chromogenic, iodometric, and acidometric tests, which confers beta-lactam resistance &#8230; <a title=\"Beta (\u03b2) Lactamase Test: Principle, Types, Procedure, Results\" class=\"read-more\" href=\"https:\/\/microbenotes.com\/beta-%ce%b2-lactamase-test-objectives-principle-procedure-and-results\/\" aria-label=\"Read more about Beta (\u03b2) Lactamase Test: Principle, Types, Procedure, Results\">Read more<\/a><\/p>\n","protected":false},"author":28,"featured_media":878,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[702],"tags":[1628,1629,1630,1635,1636,1631,1632,1633,1637,1634],"custom":{"td_video":"","featured_image":"https:\/\/microbenotes.com\/wp-content\/uploads\/2018\/07\/Result-Interpretation-of-Beta-\u03b2-Lactamase-Test.jpg","author":{"name":"Babita Sharma","avatar":"https:\/\/secure.gravatar.com\/avatar\/b546a1fa5bb00031dd12fe643aa0fb5d?s=96&d=mm&r=g"},"categories":[{"term_id":702,"name":"Biochemical Test","slug":"biochemical-test","term_group":0,"term_taxonomy_id":702,"taxonomy":"category","description":"<strong>Biochemical tests are the tests that are performed on different bacteria for their identification on the basis of their biochemical activities towards different biochemical compounds.<\/strong>\r\n<ul>\r\n \t<li>Biochemical tests are one of the traditional methods for the identification of microorganisms, usually performed with phenotypic identification.<\/li>\r\n \t<li>For many years these methods were employed extensively, and they continue to be used nowadays, especially in some laboratory routines where a particular type of microorganism has to be identified rapidly.<\/li>\r\n \t<li>The ability of microorganisms to utilize certain biomolecules, resulting in useful organic compounds for themselves forms the basis of various biochemical tests.<\/li>\r\n \t<li>Biochemical tests are of different types, where the identification or distinction between different microorganisms is made on various bases.<\/li>\r\n \t<li>One of the traditional methods commonly used is a simple visual detection of the growth of the organism in the presence of essential nutrients by increased turbidity in the liquid medium.<\/li>\r\n \t<li>In other tests, however, the results are based on the change in color of the medium as a result of the change in the pH of the medium.<\/li>\r\n \t<li>Microorganisms can be classified into different groups on the basis of their reaction to such tests. Some tests even allow the distinction of microorganisms to the species level.<\/li>\r\n \t<li>Biochemical tests are thus, essential as they are inexpensive and relatively simple to perform.<\/li>\r\n \t<li>The physiology of bacteria and other microorganisms differs from one another, which allows for the differentiation of such microorganisms.<\/li>\r\n \t<li>Biochemical tests, however, have some disadvantages. Despite being inexpensive and allowing both quantitative and qualitative information about the diversity of microorganisms present in a sample, these methods are laborious and time-consuming, and results are only observed after several days.<\/li>\r\n \t<li>In some cases, false positives are obtained, especially when considering similar microbial species.<\/li>\r\n<\/ul>","parent":0,"count":114,"filter":"raw","cat_ID":702,"category_count":114,"category_description":"<strong>Biochemical tests are the tests that are performed on different bacteria for their identification on the basis of their biochemical activities towards different biochemical compounds.<\/strong>\r\n<ul>\r\n \t<li>Biochemical tests are one of the traditional methods for the identification of microorganisms, usually performed with phenotypic identification.<\/li>\r\n \t<li>For many years these methods were employed extensively, and they continue to be used nowadays, especially in some laboratory routines where a particular type of microorganism has to be identified rapidly.<\/li>\r\n \t<li>The ability of microorganisms to utilize certain biomolecules, resulting in useful organic compounds for themselves forms the basis of various biochemical tests.<\/li>\r\n \t<li>Biochemical tests are of different types, where the identification or distinction between different microorganisms is made on various bases.<\/li>\r\n \t<li>One of the traditional methods commonly used is a simple visual detection of the growth of the organism in the presence of essential nutrients by increased turbidity in the liquid medium.<\/li>\r\n \t<li>In other tests, however, the results are based on the change in color of the medium as a result of the change in the pH of the medium.<\/li>\r\n \t<li>Microorganisms can be classified into different groups on the basis of their reaction to such tests. Some tests even allow the distinction of microorganisms to the species level.<\/li>\r\n \t<li>Biochemical tests are thus, essential as they are inexpensive and relatively simple to perform.<\/li>\r\n \t<li>The physiology of bacteria and other microorganisms differs from one another, which allows for the differentiation of such microorganisms.<\/li>\r\n \t<li>Biochemical tests, however, have some disadvantages. Despite being inexpensive and allowing both quantitative and qualitative information about the diversity of microorganisms present in a sample, these methods are laborious and time-consuming, and results are only observed after several days.<\/li>\r\n \t<li>In some cases, false positives are obtained, especially when considering similar microbial species.<\/li>\r\n<\/ul>","cat_name":"Biochemical Test","category_nicename":"biochemical-test","category_parent":0}]},"_links":{"self":[{"href":"https:\/\/microbenotes.com\/wp-json\/wp\/v2\/posts\/877"}],"collection":[{"href":"https:\/\/microbenotes.com\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/microbenotes.com\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/microbenotes.com\/wp-json\/wp\/v2\/users\/28"}],"replies":[{"embeddable":true,"href":"https:\/\/microbenotes.com\/wp-json\/wp\/v2\/comments?post=877"}],"version-history":[{"count":0,"href":"https:\/\/microbenotes.com\/wp-json\/wp\/v2\/posts\/877\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/microbenotes.com\/wp-json\/wp\/v2\/media\/878"}],"wp:attachment":[{"href":"https:\/\/microbenotes.com\/wp-json\/wp\/v2\/media?parent=877"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/microbenotes.com\/wp-json\/wp\/v2\/categories?post=877"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/microbenotes.com\/wp-json\/wp\/v2\/tags?post=877"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}