{"id":1276,"date":"2013-04-03T13:54:43","date_gmt":"2013-04-03T17:54:43","guid":{"rendered":"http:\/\/www.ambrosiasymbiosis.org\/labprotocols\/extraction-of-fungi-from-beetles\/"},"modified":"2016-04-26T14:55:30","modified_gmt":"2016-04-26T18:55:30","slug":"extraction-of-fungi-from-beetles","status":"publish","type":"post","link":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/extraction-of-fungi-from-beetles\/","title":{"rendered":"Extraction of Fungi From Beetles"},"content":{"rendered":"<h1>Protocol<\/h1>\n<h2>Materials<\/h2>\n<ul>\n<li>1000ul and 100ul pipettes<\/li>\n<li>pipettes tips<\/li>\n<li>two pairs of fine hard forceps<\/li>\n<li>petri dishes for beetle dissection<\/li>\n<li>microscope in hood<\/li>\n<li>vials<\/li>\n<li>vial rack<\/li>\n<li>sterile 1X PBS<\/li>\n<li>alcohol-resistant marker<\/li>\n<\/ul>\n<h2>General notes<\/h2>\n<ol>\n<li><span style=\"line-height: 13px;\">Wash the beetle with a surfactant and\/or saline, to get most of the dirt off. You don&#8217;t need to get every last fungal spore off the surface (we will dilute those away). You also don&#8217;t need to use any toxic solutions, often used in older works. Some people use ethanol for surface sterilization; it probably helps in removing some contaminants, but there is also a high percentage of the mycangial symbiots that die. We have tested it&#8230;<\/span><\/li>\n<li>Don&#8217;t grind up the whole beetle &#8211; focus on the right body part. If the mycangium is in the head, use the head (most Xyleborini). If it&#8217;s in the prosternum (for example, Xyloterini, Corthylini) or pronotum (Platypodinae), use the prothorax. If it&#8217;s in the mesonotum (the <em>Xylosandrus<\/em>&#8211;<em>Anisandrus<\/em> clade of Xyleborini) it gets a bit trickier, but with a little practice you will learn how to excise that general part of the body out as well. The main point is to avoid most of the surface, the alimentary canal (particularly the hind gut which is full of yeasts) and the space under elytra, which also hosts many unwanted associates (including nematodes). Yes &#8211; the space under the elytra is dirty. When you want to study the microbial associates of people&#8217;s mouth, you also don&#8217;t grind up the whole person.<\/li>\n<li>Dilution plating! This is the ESSENTIAL\u00a0part of the process. Plate several dilutions of your inoculum, and use the lowest one where some Colony Forming Units end up growing in the end. The goal here is to dilute away all the non-specific associates which are likely present in lower abundances, and only capture the abundant symbionts. Those are typically present in thousands of cells, so you have a good chance of getting mostly those in the lowest dilution.<\/li>\n<\/ol>\n<h2>Dilution plating<\/h2>\n<ol>\n<li>Prepare two tubes per each sample, label them &#8220;0.1&#8221; and &#8220;0.01&#8221;, fill each with 500 ul of water or PBS.<\/li>\n<li>Suspend mycangium in the tube &#8220;0.1&#8221; and vortex.<\/li>\n<li>Plate\u00a0<b>50 ul<\/b>\u00a0of the suspension on media. Record the plate as &#8220;0.1 dilution&#8221; in [PLATES].[note] in the Isolations database.<\/li>\n<li>Transfer\u00a0<b>50 ul<\/b>\u00a0of the initial suspension to the second tube (&#8220;0.01&#8221;) and vortex.<\/li>\n<li>Plate\u00a0<b>50 ul<\/b>\u00a0of the second suspension on\u00a0<b>second<\/b>\u00a0media plate, and record that plate as &#8220;0.01 dilution&#8221;.<\/li>\n<li>Plate\u00a0<b>5 ul<\/b>\u00a0of the second suspension on a\u00a0<b>third<\/b>\u00a0plate, and record it as &#8220;0.001 dilution&#8221;.<\/li>\n<\/ol>\n<p>Quantititive extraction for fungus community characterization:<br \/>\nSee <a title=\"Calculating Colony Forming Units\" href=\"http:\/\/www.ambrosiasymbiosis.org\/labprotocols\/calculating-colony-forming-units\/\">Calculating colony-forming units<\/a><\/p>\n<p><!--\n\n\n<h3>Xyleborinus saxeseni<\/h3>\n\n\n1.Prepare the following vials for each beetle + one blank control:\n\n\n<blockquote>a. one vial with 1000 ul of water &amp; small drop of Tween 20 or Tween 80 (or both)\nb. four vials with glass beads and 500 ul of PBS (no Tween!)\nc. four vials with 500 ul PBS, labeled \u201c100x\u201d\nd. four vials with 500 ul PBS, labeled \u201c1000x\u201d\ne.\u00a0<b>do not dillute the blank control, plate directly from vial with beads<\/b><\/blockquote>\n\n\n2. Wash each beetle by vortexing 1 min in 1.0ml water + small drop of Tween (vial \u201ca\u201d)\n3. Divide each beetle to head, pronotum, elytra (both together), and abdomen.\n4. Crush each part with forceps and put in sterile tube with beads and PBS (vials \u201cb\u201d)\n5. Shake vials in bead beater for 30 sec\n6. Make 100x and 1000x serial dilutions:\n\n\n<blockquote>a. aliquot 5 uL from each vial \u201cb\u201d into the respective vial \u201cc\u201d (labeled 100x)\nb. shake or vortex vials \u201cc\u201d\nc. aliquot 50 ul from each vial \u201cc\u201d into the respective vial \u201cd\u201d (labeled 1000x)<\/blockquote>\n\n\n7. plate 100 ul of solution from vials \u201cc\u201d and \u201cd\u201d on the following media:\n\n\n<blockquote>a. PDA+antibacterials\nb. MEA + antibacterials (or YMEA+antibacterials)<\/blockquote>\n\n\n8. This will result in 16 plates from each beetle (four beetle parts, two dillutions, two types of media) plus two plates with blank control (two types of media)\n9. Record plates in a database (I will do that).\n--><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Protocol Materials 1000ul and 100ul pipettes pipettes tips two pairs of fine hard forceps petri dishes for beetle dissection microscope in hood vials vial rack sterile 1X PBS alcohol-resistant marker General notes Wash the beetle with a surfactant and\/or saline, to get most of the dirt off. You don&#8217;t need to get every last fungal [&hellip;]<\/p>\n","protected":false},"author":7,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_s2mail":"yes","footnotes":""},"categories":[5],"tags":[],"class_list":["post-1276","post","type-post","status-publish","format-standard","hentry","category-fungi"],"_links":{"self":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1276","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/users\/7"}],"replies":[{"embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/comments?post=1276"}],"version-history":[{"count":6,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1276\/revisions"}],"predecessor-version":[{"id":1902,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1276\/revisions\/1902"}],"wp:attachment":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/media?parent=1276"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/categories?post=1276"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/tags?post=1276"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}