{"id":1128,"date":"2018-08-14T02:00:40","date_gmt":"2018-08-14T02:00:40","guid":{"rendered":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/?p=1128"},"modified":"2018-08-14T19:24:16","modified_gmt":"2018-08-14T19:24:16","slug":"microbiology-gramstain","status":"publish","type":"post","link":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/microiology\/microbiology-gramstain\/","title":{"rendered":"Microbiology: Gram stain."},"content":{"rendered":"

Gram Staining <\/strong>(Cappuccino & Welsh, 2017).<\/p>\n

Description<\/strong>. Gram staining is a differential staining technique<\/strong> that differentiates bacteria based on their cell wall construction. Gram-positive bacteria have a thick peptidoglycan cell wall (stains purple) and its teichoic acid cross-linkages resist the decolorizing agent (Microbe Online, 2015). Gram-negative bacteria have a thin peptidoglycan cell wall which lies between two membranous layers (outer membrane and cell membrane, or in acid-fast bacteria the lipid layer and cell membrane) (Black & Black, 2015). This test helps to classify bacteria into two major categories: gram-negative and gram-positive.<\/p>\n

Clinical significance<\/strong>. The Gram stain is the predominant differential stain\u2014especially used in the clinical setting. It quickly assesses organisms that fall into one of two categories\u2014Gram positive (purple) and Gram negative (pink\/reddish).<\/p>\n

Summary of main steps<\/strong>. It is assumed that the reader knows aseptic technique and how to heat fix.<\/p>\n

    \n
  1. Inoculate clean slide. Use aseptic technique throughout. If using a broth culture, place 2-3 loopfuls of culture onto the glass slide. If using a solid medium, first place 2 loopfuls of deionized (DI) water onto the slide. Then place 1 loopful of culture into the DI water puddle on the slide (repeat if necessary). Spread the loopful of culture around on the slide in a circular pattern.<\/li>\n
  2. Allow the slide to air dry. Then heat fix.<\/li>\n
  3. Flood slide with crystal violet<\/strong> (primary stain, Hucker’s, stains all cells purple). Wait 1 minute.<\/li>\n
  4. Flush gently with DI water.<\/li>\n
  5. Flood slide with Gram’s iodine mordant<\/strong> (intensifies primary stain by binding to the primary stain and forming an insoluble complex, crystal-violet-iodine or CV-I). Gram’s iodine is also a killing agent. Wait 1 minute.<\/li>\n
  6. Flush gently with DI water.<\/li>\n
  7. Quickly and carefully (be stingy) cover the slide with 95% ethyl alcohol decolorizer<\/strong> (selectively removes color from cell components\/structures). You should see some pale blue run-off. The decolorizer is a lipid solvent and also dehydrates protein.In Gram-negative<\/strong> cells, the decolorizer makes the cell wall more porous by dissolving the lipid components\u2014aiding in the removal of the CV-I complex.In Gram-positive<\/strong> cells, the decolorizer makes the cell wall pores smaller\u2014making the removal of CV-I more difficult (i.e. makes the CV-I “stick” better).<\/li>\n
  8. Flush immediately with DI water.<\/li>\n
  9. Flood slide with safranin<\/strong> (counterstain for contrast those structures that have been decolorized). Wait 45 seconds.<\/li>\n
  10. Flood gently with DI water.<\/li>\n
  11. Blot gently with bibulous paper.<\/li>\n<\/ol>\n

     <\/p>\n

    \"\"<\/a><\/p>\n

    Reference<\/p>\n

    Black,\u00a0J.\u00a0G., & Black,\u00a0L.\u00a0J. (2015).\u00a0Microbiology: Principles and explorations<\/em>\u00a0(9th\u00a0ed.). Hoboken, NJ: John Wiley & Sons.<\/p>\n

    Cappuccino,\u00a0J.\u00a0G., & Welsh,\u00a0C. (2018).\u00a0Microbiology: A laboratory manual<\/em>.<\/p>\n

    Microbe Online. (2015, February 2). Gram staining: Principle, procedure and results. Retrieved from http:\/\/microbeonline.com\/gram-staining-principle-procedure-results\/<\/p>\n","protected":false},"excerpt":{"rendered":"

    Gram Staining (Cappuccino & Welsh, 2017). Description. Gram staining is a differential staining technique that differentiates bacteria based on their cell wall construction. Gram-positive bacteria have a thick peptidoglycan cell wall (stains purple) and its teichoic acid cross-linkages resist the decolorizing agent (Microbe Online, 2015). Gram-negative bacteria have a thin peptidoglycan cell wall which lies […]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[36],"tags":[],"class_list":["post-1128","post","type-post","status-publish","format-standard","hentry","category-microiology"],"_links":{"self":[{"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/posts\/1128","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/comments?post=1128"}],"version-history":[{"count":7,"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/posts\/1128\/revisions"}],"predecessor-version":[{"id":1195,"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/posts\/1128\/revisions\/1195"}],"wp:attachment":[{"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/media?parent=1128"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/categories?post=1128"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/tags?post=1128"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}