Daily beetle colony maintenance
\nI.\tNew females<\/p>\n
1.\tCheck black tubes for emergent females. Set aside emergent females. Check collection dates on black tubes. Discard any sticks older than 45 days. Clean emptied tubes with bleach, dry completely, and store in zip lock bags.<\/p>\n
2.\tThaw frozen sticks for newly emerged females. Seal stick ends with wax (paraffin or parafilm). Place in new sterile petri dishes.<\/p>\n
3.\tAdd emergent females, 1 per stick, per petri dish. Label petri dish with date. Place in Starter Box<\/p>\n
II.\tStarter Box<\/p>\n
1.\tCheck starter box for excavating beetles. Label dishes with excavating females with the \u201cbored \u2013 in\u201d date and unique ID number. Move dish to Working Colony and enter ID and \u201cbored \u2013 in\u201d date in spreadsheet. <\/p>\n
2.\tDiscard any dishes with dead females, signs of mites, or females that have failed to bore in for longer than 5 days.<\/p>\n
III.\tWorking colony<\/p>\n
1.\tCheck Working Colony spreadsheet for ripe galleries. Harvest galleries that have reached the \u201cripe date\u201d. Date will depend on current demand for experiments (ask James). If eggs are needed, this will be 4 \u2013 6 days after \u201cbored \u2013 in date\u201d. If pre-pupae are needed, this will be 14 \u2013 16 days after \u201cbored \u2013 in\u201d date.<\/p>\n
i.\tHarvesting<\/p>\n
1.\tPlace sterile #40 Whitman filter paper in sterile petri dish and moisten with 500 ul sterile PBS. <\/p>\n
2.\tUse sterile pipet tips to transfer eggs\/pre-pupae to moistened filter paper, being as fast and sanitary as possible. Add no more than 12 eggs or 6 pre-pupae per filter paper. Wear gloves and clean gloves with EtOH between every stick.<\/p>\n
3.\tIf gallery is in good shape and may produce more eggs\/pre-pupae, carefully reclose gallery, taking care not to crush beetles. Use small strips of parafilm to bind the ends of the stick back together. Record \u201copened\u201d date on petri dish and spreadsheet. Return petri dish to working colony. Check 2-3 days later.<\/p>\n
4.\tDiscard spent galleries and remove from spreadsheet.<\/p>\n
ii.\tSpecimen treatments<\/p>\n
1.\tIf current experimental protocol requires axenic larvae\/pre-pupae, apply chemical treatment per protocol to eggs\/pre-pupae on filter paper (ask James for protocol). <\/p>\n
2.\tTransfer cleaned eggs\/pre-pupae to fresh sterile filter paper in new sterile petri dish. Label dish with \u201charvested\u201d date and chemical treatment, then seal petri dish with parafilm, and move it to the small incubator (27\u00b0 C).
\n\u2003
\nWeekly beetle colony maintenance<\/p>\n
Monday: Clean Starter Box and re-supply stocks. <\/p>\n
1.\tRemove all petri dishes<\/p>\n
2.\tPour perlite into autoclave bag using large funnel and autoclave. Leave
\nperlite in closed autoclaved bag for use next week.<\/p>\n
3.\tWipe down container with bleach solution. Dry well.<\/p>\n
4.\tReplace with autoclaved and cooled perlite from previous week.<\/p>\n
5.\tCheck and replenish stocks of sterile filter papers, sterile tubes of PBS, media, etc. <\/p>\n
Tuesday: Collect fresh galleries.<\/p>\n
1.\tCollect as many occupied X. compactus galleries as possible. Effort will have to be adjusted to match need\/loss. Only collect galleries with female visible at entrance.<\/p>\n
2.\tCut twigs to fit in black tubes. Seal cut twig ends with wax.<\/p>\n
3.\tPlace 4-6 galleries per clean black tube. Put tubes in walk-in hot freezer.
\nWednesday: Clean Working Colony. <\/p>\n
6.\tRemove all petri dishes\/ tubes<\/p>\n
7.\tPour perlite into autoclave bag and autoclave.<\/p>\n
8.\tWipe down container with bleach solution. Dry well.<\/p>\n
9.\tReplace autoclaved perlite, petri dishes, and tubes.
\nThursday: Collect fresh sticks.<\/p>\n
1.\tFind sweet gum stems 2-6 mm in diameter. Cut sticks ~5 cm length, with no nodes!<\/p>\n
2.\tCollect enough sticks to replace what was used in previous week.<\/p>\n
3.\tStore in zip lock bag in freezer<\/p>\n","protected":false},"excerpt":{"rendered":"
Daily beetle colony maintenance I. New females 1. Check black tubes for emergent females. Set aside emergent females. Check collection dates on black tubes. Discard any sticks older than 45 days. Clean emptied tubes with bleach, dry completely, and store in zip lock bags. 2. Thaw frozen sticks for newly emerged females. Seal stick ends […]<\/p>\n","protected":false},"author":16,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_s2mail":"","footnotes":""},"categories":[6],"tags":[],"class_list":["post-1908","post","type-post","status-publish","format-standard","hentry","category-beetles"],"_links":{"self":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1908","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\/16"}],"replies":[{"embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/comments?post=1908"}],"version-history":[{"count":1,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1908\/revisions"}],"predecessor-version":[{"id":1909,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1908\/revisions\/1909"}],"wp:attachment":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/media?parent=1908"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/categories?post=1908"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/tags?post=1908"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}