{"id":35,"date":"2007-02-22T11:53:28","date_gmt":"2007-02-22T18:53:28","guid":{"rendered":"http:\/\/www.bigroom.org\/wordpress\/?p=35"},"modified":"2007-02-22T11:53:28","modified_gmt":"2007-02-22T18:53:28","slug":"making-the-great-leap-out-of-the-19th-centuryacid-fast-staining","status":"publish","type":"post","link":"https:\/\/www.bigroom.org\/wordpress\/?p=35","title":{"rendered":"Making the great leap out of the 19th Century&#8230;&#8221;Acid-Fast&#8221; staining"},"content":{"rendered":"<p>The &#8220;standard&#8221; acid-fast differential staining process for the &#8220;High G+C Gram-Positive&#8221; bacteria[1] as we learned it is pretty <span class=\"moreinfo\" title=\"Invented in 1882, 2 years *before* the Gram stain.\">archaic<\/span>.<\/p>\n<p>It goes something like this:<\/p>\n<ul>\n<li>Smear and heat-fix the slide<\/li>\n<li>flood the slide with <span class=\"moreinfo\" title=\"A red dye dissolved with phenol ('Carbolic Acid')\">Carbolfuschin<\/span><\/li>\n<li>Heat the slide for 5 minutes in steam over a boiling water bath<\/li>\n<li>Rinse with &#8220;<span class=\"moreinfo\" title=\"70% alcohol with a small amount of Hydrochloric Acid\">Acid Alcohol<\/span>&#8220;<\/li>\n<li>Stain for a minute with Methylene blue.<\/li>\n<\/ul>\n<p>At the end, anything &#8220;Acid-Fast&#8221; (having a &#8220;waxy&#8221; outer layer) will show up red, anything else will be blue.<\/p>\n<p>My objection is the messy and time-consuming steam-bath.<\/p>\n<p>Today&#8217;s lab included one &#8220;unknown&#8221; which we suspected to be a Mycobacterium, so while one of us was going through the tedious 19th-century-style procedure, I decided to try something.<\/p>\n<p>The steambath heat is just intended to (I believe) slightly &#8220;melt&#8221; the waxy layer of the cell and otherwise help &#8220;drive&#8221; the dye into it.  So, instead of dealing with the time to make a water bath, heat until it steams, and then wait for the slide to sit there and hope the bubbling bath doesn&#8217;t splatter the slide with crud, I just stuck the flooded slide in the lab microwave and cooked it for 20 seconds.<\/p>\n<p>It worked.  Quite well, actually (other than letting the slide dry out, leaving some crystals on the slide) &#8211; the bright red mycobacterial cells showed up nicely.  I&#8217;m annoyed that my &#8216;stick the camera up to the eyepiece&#8217; technique came out slightly out of focus (I may see if I can enhance it later &#8211; if so, I&#8217;ll post it.).  Somebody commented that it looked as good as a &#8220;textbook&#8221; example, which was nice for my ego&#8230;<\/p>\n<p>Unfortunately, I guess I&#8217;m far from the first person to think of this.  I don&#8217;t know if anyone&#8217;s done this exactly the same way, but <a title=\"A method for acid-fast staining with microwave heat\" href=\"http:\/\/library.med.utah.edu\/WebPath\/HISTHTML\/MANUALS\/AFB.PDF\">This procedure<\/a> describes directly heating the Carbolfuschin in the microwave and soaking the slides directly in it.  There is also apparently an old Lancet[2] article which I don&#8217;t currently have access to &#8211; I&#8217;ll have to check it out later.<\/p>\n<p>Next thing to do is try the endospore stain this way.  Behold, the miracles of applying last century&#8217;s technologies to the problems of century-before-last!<\/p>\n<p>[1] Ehrlich P. Zur Fa?rbung der Tuberkelbakterien. Aus dem Verein fu?r innere Medizin zu Berlin. Deutsche Med Wochenschr 1882; 8:269?270<br \/>\n[2] Hafiz, S., R. C. Spencer, M. Lee, H. Gooch, and B. I. Duerden. 1984 . Rapid Ziehl-Neelsen staining by use of microwave oven. Lancet ii:1046.<\/p>\n","protected":false},"excerpt":{"rendered":"<p>The &#8220;standard&#8221; acid-fast differential staining process for the &#8220;High G+C Gram-Positive&#8221; bacteria[1] as we learned it is pretty archaic. It goes something like this: Smear and heat-fix the slide flood the slide with Carbolfuschin Heat the slide for 5 minutes in steam over a boiling water bath Rinse with &#8220;Acid Alcohol&#8220; Stain for a minute &hellip; <a href=\"https:\/\/www.bigroom.org\/wordpress\/?p=35\" class=\"more-link\">Continue reading <span class=\"screen-reader-text\">Making the great leap out of the 19th Century&#8230;&#8221;Acid-Fast&#8221; staining<\/span><\/a><\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[13,15,6,5,8],"tags":[],"class_list":["post-35","post","type-post","status-publish","format-standard","hentry","category-me-me-me","category-methods","category-microbiology","category-nerdity","category-why-does-it-work"],"_links":{"self":[{"href":"https:\/\/www.bigroom.org\/wordpress\/index.php?rest_route=\/wp\/v2\/posts\/35","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.bigroom.org\/wordpress\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.bigroom.org\/wordpress\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.bigroom.org\/wordpress\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.bigroom.org\/wordpress\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=35"}],"version-history":[{"count":0,"href":"https:\/\/www.bigroom.org\/wordpress\/index.php?rest_route=\/wp\/v2\/posts\/35\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.bigroom.org\/wordpress\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=35"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.bigroom.org\/wordpress\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=35"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.bigroom.org\/wordpress\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=35"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}