{"id":666,"date":"2020-12-08T15:58:28","date_gmt":"2020-12-08T15:58:28","guid":{"rendered":"https:\/\/laboratoryinfo.com\/?p=666"},"modified":"2022-03-28T11:14:57","modified_gmt":"2022-03-28T11:14:57","slug":"gram-staining-principle-procedure-interpretation-and-animation","status":"publish","type":"post","link":"https:\/\/laboratoryinfo.com\/gram-staining-principle-procedure-interpretation-and-animation\/","title":{"rendered":"Gram Staining : Principle, Procedure, Interpretation and Animation"},"content":{"rendered":"\n<p>Gram stain is the most widely used standard procedure in microbiology that is used to classify bacteria according to their cell wall composition. <\/p>\n\n\n\n<p>It distinguishes the bacteria into two major groups; <a href=\"https:\/\/laboratoryinfo.com\/gram-positive-bacteria\/\" target=\"_blank\" rel=\"noreferrer noopener\"><strong>Gram-positive<\/strong><\/a> and <strong><a href=\"https:\/\/laboratoryinfo.com\/gram-negative-bacteria\/\" target=\"_blank\" rel=\"noreferrer noopener\">Gram-negative bacteria<\/a><\/strong>. see : <strong><a href=\"https:\/\/laboratoryinfo.com\/differences-between-gram-positive-and-gram-negative-bacteria\/\" target=\"_blank\" rel=\"noreferrer noopener\">Difference between gram-positive and gram-negative bacteria<\/a><\/strong>.<\/p>\n\n\n\n<p>It is a valuable diagnostic tool in both research and clinical settings. It helps in the characterization and identification of bacteria.\u00a0<\/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<h2 class=\"wp-block-heading\">Historical background&nbsp;<\/h2>\n\n\n\n<p>The gram staining procedure was first developed by a Danish Bacteriologist and Physician, Hans Christian Gram in 1884 while he was working in Berlin when he used the Gram staining technique to visualize Klebsiella pneumoniae in the lung tissue section of people who died because of pneumonia infection. Before this, the available staining method was designed by Robert Koch for visualizing tubercle bacilli.&nbsp;<\/p>\n\n\n\n<p>Gram didn&#8217;t use safranin as a counterstain in the original procedure. The nuclei of eukaryotic&nbsp; cells in tissue samples were left unstained, while the cocci were stained blue\/violet in the lungs of those who died of pneumonia. Later, Carl Weigert, a German Pathologist, used safranin as a counterstain.&nbsp;<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Principle of Gram staining technique<\/h2>\n\n\n\n<p>The basic principle of the gram staining technique involves the ability of the cell wall to retain the primary stain. The cell wall structure will determine whether the organism is gram-positive or gram-negative. Gram-positive bacteria have higher peptidoglycan content, whereas gram-negative bacteria have more lipid content in the cell wall.&nbsp;<\/p>\n\n\n\n<div class=\"wp-block-image\"><figure class=\"aligncenter size-full is-resized\"><a href=\"http:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-positive-vs-gram-negative-cell-wall.png\"><img decoding=\"async\" src=\"http:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-positive-vs-gram-negative-cell-wall.png\" alt=\"The difference between cell wall structure of Gram-negative and Gram-positive organisms image.\" class=\"wp-image-667\" width=\"800\" srcset=\"https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-positive-vs-gram-negative-cell-wall.png 1277w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-positive-vs-gram-negative-cell-wall-300x115.png 300w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-positive-vs-gram-negative-cell-wall-1024x393.png 1024w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-positive-vs-gram-negative-cell-wall-768x295.png 768w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-positive-vs-gram-negative-cell-wall-20x8.png 20w\" sizes=\"(max-width: 1277px) 100vw, 1277px\" \/><\/a><figcaption><strong><em>Fig 1.1: The difference between cell wall structure of Gram-negative and Gram-positive organisms.<\/em><\/strong><\/figcaption><\/figure><\/div>\n\n\n\n<blockquote class=\"wp-block-quote\"><p>When stained with a primary dye and fixed by a mordant, some bacteria can retain the primary stain by resisting decolorization, while others get decolorized by a decolorizer. Those bacteria which retain the primary color are called <strong><a href=\"https:\/\/laboratoryinfo.com\/gram-positive-bacteria\/\" target=\"_blank\" rel=\"noreferrer noopener\">Gram-positive bacteria<\/a><\/strong>, while those bacteria which get decolorized and then get counterstained are called <strong><a href=\"https:\/\/laboratoryinfo.com\/gram-negative-bacteria\/\" target=\"_blank\" rel=\"noreferrer noopener\">Gram-negative bacteria<\/a><\/strong>.<\/p><\/blockquote>\n\n\n\n<ul><li>Crystal violet (CV) molecule splits into CV+ and Cl\u2013 ions in aqueous solutions. These ions will penetrate the cell wall and cell membrane of both cells (gram-positive and gram-negative).&nbsp;<\/li><li>The CV+ ions stain the cells purple color because of their interaction with the components of <a href=\"https:\/\/laboratoryinfo.com\/various-shapes-and-arrangements-of-bacterial-cells\/\" data-type=\"URL\" data-id=\"https:\/\/laboratoryinfo.com\/various-shapes-and-arrangements-of-bacterial-cells\/\" target=\"_blank\" rel=\"noreferrer noopener\">bacterial cells<\/a> that are negatively charged.<\/li><li>Iodine (I), used as mordant, interacts with CV+ and forms large complexes of crystal violet and iodine (CV\u2013I). The process will occur in the outer layers and within the cytoplasm.&nbsp;<\/li><li>When a decolorizer such as alcohol or acetone is added, it will further interact with the lipid content of the cell membrane of both gram-negative and gram-positive bacteria.&nbsp;<\/li><li>Gram-negative bacteria will lose their outer membrane leaving the peptidoglycan layer exposed.&nbsp;<\/li><li>A thin layer of Gram-negative bacteria will leak the large crystal violet and iodine (CV\u2013I) complexes from the cell.<\/li><li>On the other hand, a Gram-positive organism will be dehydrated from alcohol treatment. This will close the pores inside the cell wall and prevent the dye from exiting the cell.&nbsp;<\/li><li>The large complexes of CV\u2013I will be trapped within the Gram-positive cells. It is due to the presence of the thick and multilayered peptidoglycan.<\/li><li>After decolorization, the Gram-negative bacteria will lose the purple dye and gram-positive bacteria will retain it.<\/li><li>Counterstain, usually safranin or fuchsin, will give these Gram-negative cells a pink or red color.<\/li><\/ul>\n\n\n\n<h2 class=\"wp-block-heading\">Difference between Gram-staining and acid-fast techniques<\/h2>\n\n\n\n<p>Following are the differences between the gram-staining and acid-fast techniques.&nbsp;<\/p>\n\n\n\n<figure class=\"wp-block-table\"><table><tbody><tr><td><strong>Gram-staining technique<\/strong><\/td><td><strong>Acid-fast technique<\/strong><\/td><\/tr><tr><td>It is a staining technique for identifying bacteria. Violet dye, decolorizing agent, and secondary dye are used.<\/td><td>It is a technique in which we use differential stains to identify acid-fast organisms like Mycobacterium.&nbsp;<\/td><\/tr><tr><td>It characterizes bacteria into two large groups.&nbsp;<\/td><td>It is used to differentiate Gram-positive bacteria.<\/td><\/tr><tr><td>Identifies in the basis of different types of the cell wall.&nbsp;<\/td><td>Identifies based on mycolic acid in the cell wall.<\/td><\/tr><tr><td>Differentiates into Gram-positive and Gram-negative.<\/td><td>Differentiates into acid-fast and non-acid-fast bacteria.<\/td><\/tr><tr><td>The primary stain is crystal violet.<\/td><td>The primary stain is carbofuchsin.<\/td><\/tr><tr><td>The counterstain is safranin.&nbsp;<\/td><td>The counterstain is methylene blue.&nbsp;<\/td><\/tr><tr><td>Gram-positive appear in blue and Gram-negative in red color.&nbsp;<\/td><td>Acid-fast bacteria in red color and non-acid fast in blue color.<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\n<div class=\"wp-block-image\"><figure class=\"aligncenter size-full\"><img decoding=\"async\" width=\"579\" height=\"277\" src=\"http:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/Gram-staining-and-Acid-fast-staining.jpg\" alt=\"Gram staining and Acid-fast staining\" class=\"wp-image-7651\" srcset=\"https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/Gram-staining-and-Acid-fast-staining.jpg 579w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/Gram-staining-and-Acid-fast-staining-300x144.jpg 300w\" sizes=\"(max-width: 579px) 100vw, 579px\" \/><figcaption><strong><em>Fig 1.2: Gram staining and Acid-fast staining<\/em><\/strong><\/figcaption><\/figure><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Specimen<\/h2>\n\n\n\n<p>Various specimens can be used in this technique. Some of the commonly used specimens are following<\/p>\n\n\n\n<ul><li>Urine<\/li><li>Ascitic fluid<\/li><li>Sputum<\/li><li>Cerebrospinal fluid (CSF)<\/li><li>Blood<\/li><li>Swabs from throat, rectum, wound, nostrils, cervix, etc<\/li><li>Synovial fluid<\/li><li>Pleural fluid<\/li><\/ul>\n\n\n\n<h2 class=\"wp-block-heading\">Reagents&nbsp;<\/h2>\n\n\n\n<p>Reagents used in this technique are following<\/p>\n\n\n\n<ul><li>Crystal-violet stain<\/li><li>Gram&#8217;s Iodine&nbsp;<\/li><li>Decolorizer (ethanol)<\/li><li>Safranin&nbsp;<\/li><li>Water<\/li><\/ul>\n\n\n\n<h2 class=\"wp-block-heading\">Clinical uses of Gram-staining<\/h2>\n\n\n\n<ul><li>Provides preliminary clue in anaerobic culture.<\/li><li>Identifies Candida and Cryptococcus Spp.<\/li><li>Classifies the bacteria into two groups, gram-positive and gram-negative bacteria.&nbsp;<\/li><li>Determines the <a href=\"https:\/\/laboratoryinfo.com\/variations-in-red-blood-cell-morphology\/\" data-type=\"URL\" data-id=\"https:\/\/laboratoryinfo.com\/variations-in-red-blood-cell-morphology\/\" target=\"_blank\" rel=\"noreferrer noopener\">cell morphology<\/a>, size, and arrangement.<\/li><li>Identifies fastidious organisms like Haemophilus.<\/li><li>Helps in initiating empirical treatment with broad-spectrum antibiotics. It can be initiated before recieving the culture report.<\/li><\/ul>\n\n\n\n<h2 class=\"wp-block-heading\">Specimens used for Gram-staining technique<\/h2>\n\n\n\n<p>The specimens used for this technique are&nbsp;<\/p>\n\n\n\n<ul><li>Stool<\/li><li>Urine<\/li><li>Blood<\/li><li>Tissue<\/li><li>Sputum<\/li><\/ul>\n\n\n\n<h3 class=\"wp-block-heading\">Four basic steps<\/h3>\n\n\n\n<ol><li>Staining a heat-fixed smear with primary crystal violet stain&nbsp;<\/li><li>Addition of a mordant (Gram\u2019s iodine)<\/li><li>Rapid decolorization with alcohol or acetone<\/li><li>Counterstaining with dye, usually safranin<\/li><\/ol>\n\n\n\n<div class=\"wp-block-image\"><figure class=\"aligncenter size-full is-resized\"><a href=\"http:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-stain-procedure.png\"><img decoding=\"async\" src=\"http:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-stain-procedure.png\" alt=\"Four basic steps of gram-staining procedure technique image\n\" class=\"wp-image-668\" width=\"800\" srcset=\"https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-stain-procedure.png 972w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-stain-procedure-300x132.png 300w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-stain-procedure-768x337.png 768w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-stain-procedure-20x9.png 20w\" sizes=\"(max-width: 972px) 100vw, 972px\" \/><\/a><figcaption><strong><em>Fig 1.3: Four basic steps of gram-staining technique<\/em><\/strong><\/figcaption><\/figure><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Steps in Gram-staining technique<\/h2>\n\n\n\n<ol><li>Take a grease-free dry slide. And wipe the bottom of the biofilm slide clean.<\/li><\/ol>\n\n\n\n<div class=\"wp-block-image\"><figure class=\"aligncenter size-full\"><img decoding=\"async\" width=\"115\" height=\"256\" src=\"http:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/clean-slide-for-gram-stain-test.jpg\" alt=\"step1 of gram stain test cleaning the slide\" class=\"wp-image-7630\"\/><\/figure><\/div>\n\n\n\n<ol start=\"2\"><li>Clean top edges of the slide about 2mm<\/li><\/ol>\n\n\n\n<div class=\"wp-block-image\"><figure class=\"aligncenter size-full\"><img decoding=\"async\" width=\"183\" height=\"256\" src=\"http:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/step2-of-gram-stain-procedure-clean-slide-for-gram-stain-test-image.jpg\" alt=\"step2 of gram stain procedure clean slide for gram stain test image\" class=\"wp-image-7632\"\/><\/figure><\/div>\n\n\n\n<ol start=\"3\"><li>Build up a ridge of petroleum jelly on the top and bottom of a cover slide<\/li><\/ol>\n\n\n\n<ol start=\"4\"><li>Cover slip with petroleum jelly<\/li><\/ol>\n\n\n\n<div class=\"wp-block-image\"><figure class=\"aligncenter size-full\"><img decoding=\"async\" width=\"259\" height=\"265\" src=\"http:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/step-4-cover-slide-with-petroleum-jelly.jpg\" alt=\"step 4 cover slide with petroleum jelly\" class=\"wp-image-7633\"\/><\/figure><\/div>\n\n\n\n<ol start=\"5\"><li>Put the biofilm on slide with cover slip<\/li><\/ol>\n\n\n\n<div class=\"wp-block-image\"><figure class=\"aligncenter size-full\"><img decoding=\"async\" width=\"299\" height=\"262\" src=\"http:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/step-5-Put-the-biofilm-on-slide-with-cover-slip.jpg\" alt=\"step 5 Put the biofilm on slide with cover slip\" class=\"wp-image-7636\"\/><\/figure><\/div>\n\n\n\n<ol start=\"6\"><li>Stain with primary crystal violet stain and incubate for almost 1 minute.&nbsp;<\/li><\/ol>\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\/12\/add-crystal-violet-and-wait-30-sec.jpg\" alt=\"add crystal violet and wait 30 sec\" class=\"wp-image-7639\" width=\"300\" srcset=\"https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/add-crystal-violet-and-wait-30-sec.jpg 453w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/add-crystal-violet-and-wait-30-sec-300x218.jpg 300w\" sizes=\"(max-width: 453px) 100vw, 453px\" \/><\/figure><\/div>\n\n\n\n<ol start=\"7\"><li>Rinse the slide with a stream of water for almost 5 seconds to remove unbound crystal violet.<\/li><\/ol>\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\/12\/wash-with-water.jpg\" alt=\"wash with water\" class=\"wp-image-7640\" width=\"300\" srcset=\"https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/wash-with-water.jpg 439w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/wash-with-water-300x205.jpg 300w\" sizes=\"(max-width: 439px) 100vw, 439px\" \/><\/figure><\/div>\n\n\n\n<ol start=\"8\"><li>Add mordant (Gram&#8217;s iodine) for 1 minute. Rinse the slide with distilled water<ul><li>It forms a complex between crystal violet and iodine.<\/li><\/ul><\/li><\/ol>\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\/12\/add-grams-iodine-wait-1.5min.jpg\" alt=\"add grams iodine - wait 1.5min\" class=\"wp-image-7641\" width=\"300\" srcset=\"https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/add-grams-iodine-wait-1.5min.jpg 467w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/add-grams-iodine-wait-1.5min-300x204.jpg 300w\" sizes=\"(max-width: 467px) 100vw, 467px\" \/><\/figure><\/div>\n\n\n\n<ol start=\"9\"><li>Wash with 95% decolorizer (acetone or ethyl alcohol) for almost 3 seconds<\/li><\/ol>\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\/12\/decolorize-with-alcohol.jpg\" alt=\"decolorize with alcohol\" class=\"wp-image-7645\" width=\"300\" srcset=\"https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/decolorize-with-alcohol.jpg 455w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/decolorize-with-alcohol-300x223.jpg 300w\" sizes=\"(max-width: 455px) 100vw, 455px\" \/><\/figure><\/div>\n\n\n\n<ol start=\"10\"><li>Rinse with a gentle stream of water.<ul><li>It will dehydrate the peptidoglycan layer, shrinking and tightening it.&nbsp;<\/li><li>The alcohol will decolorize the sample if it is Gram-negative, removing the crystal violet.<\/li><li>However, if the alcohol remains on the sample for too long, it may also decolorize Gram-positive cells.<\/li><\/ul><\/li><\/ol>\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\/12\/wash-with-water.jpg\" alt=\"wash with water\" class=\"wp-image-7640\" width=\"300\" srcset=\"https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/wash-with-water.jpg 439w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/wash-with-water-300x205.jpg 300w\" sizes=\"(max-width: 439px) 100vw, 439px\" \/><\/figure><\/div>\n\n\n\n<ol start=\"11\"><li>Add the secondary stain to the slide and incubate it for 1 minute.&nbsp;<\/li><\/ol>\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\/12\/step-11-stain-with-safranin-dye-wat-30sec.jpg\" alt=\"step 11 stain with safranin dye-wat 30sec\" class=\"wp-image-7642\" width=\"300\" srcset=\"https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/step-11-stain-with-safranin-dye-wat-30sec.jpg 460w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/step-11-stain-with-safranin-dye-wat-30sec-300x222.jpg 300w\" sizes=\"(max-width: 460px) 100vw, 460px\" \/><\/figure><\/div>\n\n\n\n<ol start=\"12\"><li>Wash with a stream of water for 5 seconds.&nbsp;<\/li><\/ol>\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\/12\/wash-with-water.jpg\" alt=\"wash with water\" class=\"wp-image-7640\" width=\"300\" srcset=\"https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/wash-with-water.jpg 439w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/wash-with-water-300x205.jpg 300w\" sizes=\"(max-width: 439px) 100vw, 439px\" \/><\/figure><\/div>\n\n\n\n<ol start=\"13\"><li>Observe under a microscope.<ul><li>Gram-positive bacteria will retain the primary stain and will not take the secondary stain, and looks violet\/purple color under a microscope.<\/li><li>If it is Gram-negative bacteria, it will not retain the primary stain. Still, it will take the secondary stain, and appears red color when viewed under a microscope<\/li><\/ul><\/li><\/ol>\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\/12\/examine-under-oil-immersion-through-the-cover-slip.jpg\" alt=\"examine under oil immersion through the cover slip\" class=\"wp-image-7649\" width=\"300\" srcset=\"https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/examine-under-oil-immersion-through-the-cover-slip.jpg 377w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2020\/12\/examine-under-oil-immersion-through-the-cover-slip-300x251.jpg 300w\" sizes=\"(max-width: 377px) 100vw, 377px\" \/><\/figure><\/div>\n\n\n\n<h2 class=\"wp-block-heading\">Errors during the gram-staining procedure<\/h2>\n\n\n\n<ul><li>Prolonged decolourisation<\/li><li>Excessive counterstaining<\/li><li>Excessive heat while fixation<\/li><li>Excessive washing between steps<\/li><li>Insufficient iodine exposure<\/li><li>Low concentration of crystal violet<\/li><\/ul>\n\n\n\n<h2 class=\"wp-block-heading\">Artifacts in gram-staining<\/h2>\n\n\n\n<ul><li>Dirty glass slides<\/li><li>Gram-staining reagents contamination<\/li><li>Contaminated water used to rinse slides<\/li><\/ul>\n\n\n\n<h2 class=\"wp-block-heading\">Requirements and preparation of reagents<\/h2>\n\n\n\n<h3 class=\"wp-block-heading\">Primary Stain : Crystal violet<\/h3>\n\n\n\n<p><strong>Solution A :<\/strong><\/p>\n\n\n\n<ul><li>Crystal violet = 2 gm<\/li><li>Ethyl alcohol= 20 ml<\/li><\/ul>\n\n\n\n<p><strong>Solution B :<\/strong><\/p>\n\n\n\n<ul><li>Ammonium oxalate = 0.8 gm<\/li><li>Distilled water = 80 ml<\/li><\/ul>\n\n\n\n<p>Mix solution A and B. Keep for 24 hours and filter. Store in an amber-colored bottle.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\">Mordant : Gram\u2019s Iodine<\/h3>\n\n\n\n<ul><li>Iodine = 1 gm<\/li><li>Potassium iodide = 2 gm<\/li><li>Distilled water = to 100 ml<\/li><\/ul>\n\n\n\n<p>Mix and Store in an amber-colored bottle.<\/p>\n\n\n\n<h3 class=\"wp-block-heading\">Decolorizer : 95% Ethanol or 1:1 acetone with ethanol<\/h3>\n\n\n\n<ul><li>Acetone = 50 ml<\/li><li>Ethanol (95%) = 50ml<\/li><\/ul>\n\n\n\n<h3 class=\"wp-block-heading\">Counterstain: safranin<\/h3>\n\n\n\n<ul><li>Safranin O = 0.34 gm<\/li><li>Absolute alcohol = 10ml<\/li><li>Distilled water = 90ml<\/li><\/ul>\n\n\n\n<p>Mix, filter, and store in an ambered colored bottle.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Interpretation of Gram staining<\/h2>\n\n\n\n<div class=\"wp-block-image\"><figure class=\"aligncenter size-full is-resized\"><a href=\"http:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-positive-vs-gram-negative.png\"><img decoding=\"async\" src=\"http:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-positive-vs-gram-negative.png\" alt=\"Gram-positive and Gram-negative bacteria staining\n\" class=\"wp-image-670\" width=\"600\" srcset=\"https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-positive-vs-gram-negative.png 1042w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-positive-vs-gram-negative-300x127.png 300w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-positive-vs-gram-negative-1024x432.png 1024w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-positive-vs-gram-negative-768x324.png 768w, https:\/\/laboratoryinfo.com\/wp-content\/uploads\/2016\/01\/gram-positive-vs-gram-negative-20x8.png 20w\" sizes=\"(max-width: 1042px) 100vw, 1042px\" \/><\/a><figcaption><strong><em>Fig 1.4: Gram-positive and Gram-negative bacteria staining<\/em><\/strong><\/figcaption><\/figure><\/div>\n\n\n\n<p>The staining results of gram stain are as follows:<\/p>\n\n\n\n<ul><li>Gram-positive will be dark-purple color<\/li><li>Gram-negative will be pale to dark red color<\/li><li>Yeasts will be dark purple color<\/li><li>Epithelial cells will be pale red color<\/li><\/ul>\n\n\n\n<h2 class=\"wp-block-heading\">Examples of Gram-positive Organisms<\/h2>\n\n\n\n<p><em>Bacillus, Nocardia, Clostridium, Propionibacterium, Actinomyces, Enterococcus, Corynebacterium, Listeria, Lactobacillus, Gardnerella, Mycoplasma, Staphylococcus, Streptomyces, Streptococcus,<\/em> etc.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Examples of Gram-negative Organisms<\/h2>\n\n\n\n<p><em>Escherichia, Helicobacter, Hemophilus, Neisseria, Klebsiella, Enterobacter, Chlamydia, Vibrio, Pseudomonas, Salmonella, Shigella,<\/em> etc.<\/p>\n\n\n\n<h2 class=\"wp-block-heading\">Animation of gram staining<\/h2>\n\n\n\n<figure class=\"wp-block-embed is-type-rich is-provider-embed-handler wp-block-embed-embed-handler wp-embed-aspect-4-3 wp-has-aspect-ratio\"><div class=\"wp-block-embed__wrapper\">\n<iframe title=\"Gram Staining\" width=\"720\" height=\"540\" src=\"https:\/\/www.youtube.com\/embed\/JvN6t8-assk?feature=oembed\" frameborder=\"0\" allow=\"accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture\" allowfullscreen><\/iframe>\n<\/div><\/figure>\n\n\n\n<h2 class=\"wp-block-heading\">Frequently Asked Questions<\/h2>\n\n\n<div id=\"rank-math-faq\" class=\"rank-math-block\">\n<div class=\"rank-math-list \">\n<div id=\"faq-question-1647930931552\" class=\"rank-math-list-item\">\n<h3 class=\"rank-math-question \">Q1. What are the primary colors used for the Gram-staining technique?<\/h3>\n<div class=\"rank-math-answer \">\n\n<p>The primary colors used for Gram-staining are crystal violet or methylene blue.<\/p>\n\n<\/div>\n<\/div>\n<div id=\"faq-question-1647930945772\" class=\"rank-math-list-item\">\n<h3 class=\"rank-math-question \">Q2. Which counterstain is used in the Gram-staining technique?<\/h3>\n<div class=\"rank-math-answer \">\n\n<p>The counterstains used for Gram-staining are fuchsin or safranin stains.<\/p>\n\n<\/div>\n<\/div>\n<div id=\"faq-question-1647930959660\" class=\"rank-math-list-item\">\n<h3 class=\"rank-math-question \">Q3. Is there any risk to doing a gram-staining test?<\/h3>\n<div class=\"rank-math-answer \">\n\n<p>No, there is no risk. There may be little risk to having a blood test and no risk to having a sputum, swab, or urine test.<\/p>\n\n<\/div>\n<\/div>\n<div id=\"faq-question-1647930973588\" class=\"rank-math-list-item\">\n<h3 class=\"rank-math-question \">Q4. How is Gram-staining performed?<\/h3>\n<div class=\"rank-math-answer \">\n\n<p>Gram-staining is a test to classify the bacteria. The Gram-staining technique uses two types of stains; a primary color and a counterstain. The cell wall of gram-positive bacteria retains primary stain, and the cell wall of gram-negative bacteria takes counterstain.<br \/>We have to introduce a dye to bacteria. If bacteria have a thick peptidoglycan cell wall, it absorbs the stain and turns purple-color. And we can say that it&#8217;s positive for peptidoglycan. If it&#8217;s not turned into purple color, the test is negative for peptidoglycan.<\/p>\n\n<\/div>\n<\/div>\n<div id=\"faq-question-1647931007313\" class=\"rank-math-list-item\">\n<h3 class=\"rank-math-question \">Q5. Can we use the gram-staining technique for Eukaryotes?<\/h3>\n<div class=\"rank-math-answer \">\n\n<p>No, we can\u2019t use the gram-staining technique for Eukaryotes or Archeae. The reason is that these both lack peptidoglycan.<\/p>\n\n<\/div>\n<\/div>\n<div id=\"faq-question-1647931026435\" class=\"rank-math-list-item\">\n<h3 class=\"rank-math-question \">Q6. Can we gram-stain a virus?<\/h3>\n<div class=\"rank-math-answer \">\n\n<p>It can not be seen with the gram-staining technique because it doesn\u2019t have a cell wall that can take up a stain.<\/p>\n\n<\/div>\n<\/div>\n<div id=\"faq-question-1647931125759\" class=\"rank-math-list-item\">\n<h3 class=\"rank-math-question \">Q7. Why can\u2019t a mycobacterium be stained with Gram-staining?<\/h3>\n<div class=\"rank-math-answer \">\n\n<p>The Gram-staining technique cannot stain it because of its high lipid content. That\u2019s why the acid-fast staining method is used to stain mycobacteria.<\/p>\n\n<\/div>\n<\/div>\n<div id=\"faq-question-1647931138760\" class=\"rank-math-list-item\">\n<h3 class=\"rank-math-question \">Q8. Which bacteria can\u2019t be stained with Gram-staining?<\/h3>\n<div class=\"rank-math-answer \">\n\n<p>The bacteria that can\u2019t be stained with the gram-staining technique are atypical bacteria. These are neither Gram-positive nor Gram-negative. Some examples of atypical bacteria are <em>Chlamydiaceae, Legionella, <\/em>and <em>Mycoplasma.<\/em><\/p>\n\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n\n\n<p><strong>References<\/strong><\/p>\n\n\n\n<ol><li><em>Coico, Richard. &#8220;Gram staining.&#8221; Current protocols in microbiology 1 (2006): A-3C.<\/em><\/li><li><em>Hucker, George J., and Harold Joel Conn. &#8220;Methods of Gram staining.&#8221; (1923).<\/em><\/li><li><em>Tripathi, Nishant, and Amit Sapra. &#8220;Gram Staining.&#8221; (2020).<\/em><\/li><li><em>MN Editors. (2022, 22 januari). Comparison Between Gram Stain and Acid Fast. Microbiology Note. Geraadpleegd op 15 maart 2022, van https:\/\/microbiologynote.com\/comparison-between-gram-stain-and-acid-fast\/<\/em><\/li><li><em>Lakna, B. (2019, 13 februari). What is the Difference Between Gram Stain and Acid Fast? Pediaa.Com. Geraadpleegd op 15 maart 2022, van https:\/\/pediaa.com\/what-is-the-difference-between-gram-stain-and-acid-fast\/<\/em><\/li><\/ol>\n","protected":false},"excerpt":{"rendered":"<p>Gram stain is the most widely used standard procedure in microbiology that is used to classify bacteria according to their cell wall composition. It distinguishes the bacteria into two major groups; Gram-positive and Gram-negative bacteria. see : Difference between gram-positive and gram-negative bacteria. It is a valuable diagnostic tool in both research and clinical settings&#8230;.<\/p>\n","protected":false},"author":4,"featured_media":668,"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":[4,3],"tags":[69,24,65,71,66,67,68,70],"acf":[],"_links":{"self":[{"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/posts\/666"}],"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=666"}],"version-history":[{"count":0,"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/posts\/666\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/media\/668"}],"wp:attachment":[{"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/media?parent=666"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/categories?post=666"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/laboratoryinfo.com\/wp-json\/wp\/v2\/tags?post=666"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}