{"id":237,"date":"2016-05-14T23:46:01","date_gmt":"2016-05-14T23:46:01","guid":{"rendered":"http:\/\/blogs.oregonstate.edu\/schilkelab\/?page_id=237"},"modified":"2016-05-14T23:53:18","modified_gmt":"2016-05-14T23:53:18","slug":"nisin-activity-ps-microspheres","status":"publish","type":"page","link":"https:\/\/dev.blogs.oregonstate.edu\/schilkelab\/safetysops-2\/lab-procedures\/nisin-activity-ps-microspheres\/","title":{"rendered":"Nisin Activity"},"content":{"rendered":"<div id=\"wikitext\">\n<p>Protocol from Matt Ryder 2007-2008 bioactivity assay (Ryder UHC thesis).<\/p>\n<h2 class=\"vspace\">Nisin Activity on PS Microspheres<\/h2>\n<h2>General Schedule<\/h2>\n<h5>Day Before: Date __________<\/h5>\n<ul>\n<li>Make MRS Broth (change bi-weekly):<\/li>\n<\/ul>\n<div class=\"indent\">Vol __________ Date __________<\/div>\n<ul>\n<li>Make overnight <em>Pediococcus<\/em> culture (16 hr incubation):<\/li>\n<\/ul>\n<div class=\"indent\">Time\/Date Start __________ Finish __________<\/div>\n<div class=\"indent\">Vol __________<\/div>\n<div class=\"indent\">OD<sub>600<\/sub> __________<\/div>\n<div class=\"indent\">rpm __________<\/div>\n<ul>\n<li>Incubate microsphres with F108 (16 hr min):<\/li>\n<\/ul>\n<div class=\"indent\">Start __________ Finish __________<\/div>\n<div class=\"vspace\"><\/div>\n<h5>Day of Test: Date __________<\/h5>\n<ul>\n<li>Dilute 100x the overnight <em>Pediococcus<\/em> culture: OD<sub>600<\/sub> __________<\/li>\n<li>Make MRS agar<\/li>\n<\/ul>\n<div class=\"indent\">Total number samples: __________ MRS Gel Vol: __________ ml<\/div>\n<ul>\n<li>Make MRS agar tubes<\/li>\n<\/ul>\n<div class=\"indent\">Total number of tubes: __________<\/div>\n<ul>\n<li>Incubate microspheres with nisin (1 hr) Date __________<\/li>\n<\/ul>\n<p class=\"vspace\">Perform Colony Count Assay<\/p>\n<div class=\"indent\">Performed Number __________ Date __________<\/div>\n<div class=\"indent\">Day 1: __________<\/div>\n<div class=\"indent\">Day 7: __________<\/div>\n<div class=\"indent\">Day 14: __________<\/div>\n<div class=\"indent\">Day 21: __________<\/div>\n<div class=\"indent\">Day 28: __________<\/div>\n<div class=\"indent\">*Day 35: __________<\/div>\n<div class=\"vspace\"><\/div>\n<h2>Inventory Check<\/h2>\n<ul>\n<li>PS microsphers, (Seradyn, Part No: 81002497100290, 1.15 \u00b5m, 10% solid. In refrigerator.)<\/li>\n<li>Pluronic F-108 Solution (5% in refrigerator Lab 300.)<\/li>\n<\/ul>\n<div class=\"indent\">if diluted note here: __________<\/div>\n<ul>\n<li>South African nisin solution (-80\u00b0<sup>C<\/sup> freezer)<\/li>\n<li>0.01 M phosphate buffer. 0.2 \u00b5m filtered<\/li>\n<\/ul>\n<div class=\"indent\"><em>Phosphate buffer is NOT PBS<\/em><\/div>\n<ul>\n<li>Screw-top test tubes<\/li>\n<li><em>Pediococcus<\/em> Stock Solution (FBB 612, In -80\u00b0<sup>C<\/sup> freezer)<\/li>\n<li>Lactobacilli MRS Broth, (EMD, Product No: 1.10661.0500)<\/li>\n<li>Agar, Granulated (Difco, Product No: 214530)<\/li>\n<li>Petri Dishes, 100 mm in diameter.<\/li>\n<\/ul>\n<div class=\"indent\">Number of dishes: __________<\/div>\n<ul>\n<li>Serum nedded: __________ ml<\/li>\n<\/ul>\n<div class=\"vspace\"><\/div>\n<h2>Preparation<\/h2>\n<ul>\n<li>Make sure incubator is on 37\u00b0<sup>C<\/sup>.<\/li>\n<li>Make sure steam valve for autoclave is open 1 hr before use.<\/li>\n<li>Wash hands carefully with soap and 70% alcohol. Put on gloves.<\/li>\n<li><ins><strong>MRS Broth:<\/strong><\/ins><\/li>\n<\/ul>\n<div class=\"indent\">Add <strong>52.2g<\/strong> of MRS to every <strong>1 liter<\/strong> of RO water. Heat to dissolve MRS powder, then take to the autoclave at 121\u00b0<sup>C<\/sup> for 20 min sterilizing and 10 min drying. Run on liquid setting.<\/div>\n<div class=\"indent\">Vol made: __________ L<\/div>\n<ul>\n<li><ins><strong>Overnight <em>Pediococcus<\/em> Culture:<\/strong><\/ins><\/li>\n<\/ul>\n<div class=\"indent\">Take <em>Pediococcus<\/em> glycerol stock from -80\u00b0<sup>C<\/sup> freezer. Dip a loop of <em>Pediodoccus<\/em> in a 50ml Falcon test tube contianing 10ml of MRS broth. Incubate at 37\u00b0<sup>C<\/sup> for 16hrs. Store at 4\u00b0&#8217;^C&#8217; immediately after use.<\/div>\n<div class=\"indent\">Date\/Time Start: __________ Finish: __________ OD<sub>600<\/sub>: __________<\/div>\n<ul>\n<li><ins><strong>100x Dilution of <em>Pediococcus<\/em>:<\/strong><\/ins><\/li>\n<\/ul>\n<div class=\"indent\">Dilute <em>Peidococcus<\/em> for neccessary total volume.<\/div>\n<div class=\"indent\">Stock Vol: __________ Diluent Vol: _________<\/div>\n<div class=\"indent\">Vol made: __________ Date: __________<\/div>\n<ul>\n<li><ins><strong>MRS Agar Solution<\/strong><\/ins>:<\/li>\n<\/ul>\n<div class=\"indent\">Add <strong>15g<\/strong> agar to every <strong>1 liter<\/strong> of MRS broth. Heat to dissolve powder, then take to autoclave at 121\u00b0<sup>C<\/sup> for 20 min sterilizing and 10 min drying. Run on liquid setting.<\/div>\n<ul>\n<li><ins><strong>Glassware:<\/strong><\/ins><\/li>\n<\/ul>\n<div class=\"indent\">Autoclave necessary number of screw-top glass test tubes and flasks with agar solution.<\/div>\n<ul>\n<li><ins><strong>MRS Agar tubes:<\/strong><\/ins><\/li>\n<\/ul>\n<div class=\"indent\">Pipette 12ml of MRS agar solution into each autoclaved screw-top tube. Tubes may be stored at 4\u00b0&#8217;^C&#8217; for 2 weeks.<\/div>\n<div class=\"indent\">Date\/Time made: __________<\/div>\n<div class=\"vspace\"><\/div>\n<h2>Preparation of Pluronic F-108 Coated PS Microspheres<\/h2>\n<ol>\n<li>Mark 40ml Oakridge tubes to organize for later testing, according to formulation. Add F-108 to beads and incubate overnight \u2014 10% total volume final solution of 5% F108. Date\/Time: __________<\/li>\n<li>After incubation, vortex the tubes for 30 seconds to well-mix the microsphere solution.<\/li>\n<li>Spin down microspheres at max rpm for 15 min in swing arm centrifuge (if not available, use centrifuge in Gleeson 003).<\/li>\n<li>Remove supernatant and fill tubes with 0.01M PBS (possibly with NaCl?) up to 30ml.<\/li>\n<li>Vortex tubes to disperse microspheres again.<\/li>\n<li>Repeat steps 3-5 twice more.<\/li>\n<li>Spin down microspheres at max rpm for 15 min, remove supernatant.<\/li>\n<li>Fill tubes with 0.01M PBS up to 30ml.<\/li>\n<\/ol>\n<div class=\"vspace\"><\/div>\n<h2>Preparation of nisin loaded Microspheres<\/h2>\n<ol>\n<li>Add nisin and other constituents according to experiment, except serum.\n<ul>\n<li>List ingredients and quantities thereof to prevent forgotten material<\/li>\n<li>Constituents added: _________________________________________________<\/li>\n<\/ul>\n<\/li>\n<li>Mount samples on rotator. Incubate for 1 hr at room temperature.<\/li>\n<\/ol>\n<p class=\"vspace\"><ins><em>1st Washing<\/em><\/ins><\/p>\n<ol>\n<li>Spin down microspheres at max rpm for 15 min.<\/li>\n<li>Remove supernatant.<\/li>\n<li>Fill tube to 30ml with 0.01M PBS.<\/li>\n<li>Repeat steps 1-3 twice more.<\/li>\n<li>Let nisin coated microspheres stand for 1 hr.<\/li>\n<li>Make sure MRS agar solution tubes are standing by in the 40\u00b0<sup>C<\/sup> water bath. <em>This takes a long time. Start far in advance (approximately 4-5 hours).<\/em><\/li>\n<li>Clean incubator with 70% alcohol before incubation.<\/li>\n<\/ol>\n<p class=\"vspace\"><ins><em>2nd Washing<\/em><\/ins><\/p>\n<ol>\n<li>Spin down microspheres at max rpm for 15 min.<\/li>\n<li>Remove supernatant.<\/li>\n<li>Fill tubes to 30ml with 0.01M PBS.<\/li>\n<li>Repeat steps 1-3 twice more.<\/li>\n<li>Remove supernatant from the tubes of the previous step. Add buffer and\/or serum according to formulation.<\/li>\n<li>Vortex (1 min) and sonicate (5 min) samples to disperse beads. Put samples on rotator and incubate at 37\u00b0<sup>C<\/sup> until designated test day.<\/li>\n<\/ol>\n<div class=\"indent\">Date\/Time: __________<\/div>\n<div class=\"vspace\"><\/div>\n<h2>Assay of Microsphere Acitivty<\/h2>\n<h3>Plate count Method<\/h3>\n<ol>\n<li>Spin down incubated beads and remove supernatants. Wash, as in previous procedure, microsphere samples three times. Final nisin concentration in worst case: _________<\/li>\n<li>Add 100x dilute <em>Pediococcus<\/em> culture up to 1.5ml and disperse the 1ml (perhaps 1.5ml) dead sample by vortexing.<\/li>\n<li>Add another 1.5ml 100x dilute <em>Pediococcus<\/em> culture and sonicate for 3 min.<\/li>\n<li>Pipette solution into 15ml Falcon plastic tubes.<\/li>\n<li>Add 7ml of 100x <em>Pediococcus<\/em> culture to reach a 10ml volume.<\/li>\n<li>Rotate tubes at 37\u00b0<sup>C<\/sup> for 4 hours.<\/li>\n<li>Take out 4-hr incubated tube; disperse contents by vortexing.<\/li>\n<li>Add 1.0ml of of the 4-hr culture to 9ml of MRS broth to achieve 1000x dilution.<\/li>\n<li>Add 0.5ml of well-mixed 4-hr, 100x diluted samples at the center of Petri dish.<\/li>\n<li>Obtain a MRS agar tube from water bath and pour on top of the microsphere-containing solution.<\/li>\n<li>Plate the solution using either the spin or pour plate method (see <a class=\"wikilink\" href=\"http:\/\/blogs.oregonstate.edu\/schilkelab\/safetysops-2\/lab-procedures\/plating\/\">Plating<\/a>).<\/li>\n<li>Count the colonies on the plates. Record data in Excel spreadsheet. <em>(Determine a useful range of colonies?)<\/em><\/li>\n<\/ol>\n<p class=\"vspace\">Total 100x dilute <em>Pediococcus<\/em> solution needed = 150ml<br \/>\nTotal MRs broth needed = 500ml<br \/>\nPlates Needed = 21<br \/>\nMRS Agar needed = 400ml (15ml\/plate)<br \/>\n<em>Experimental treatments and controls: List explicitly so they are consistent and not forgotten<\/em><\/p>\n<p class=\"vspace\"><a class=\"createlinktext\" href=\"https:\/\/secure.engr.oregonstate.edu\/wiki\/schilke\/index.php\/Profiles\/Auxierj?action=edit\" rel=\"nofollow\">auxierj<\/a><a class=\"createlink\" href=\"https:\/\/secure.engr.oregonstate.edu\/wiki\/schilke\/index.php\/Profiles\/Auxierj?action=edit\" rel=\"nofollow\">?<\/a><\/p>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>Protocol from Matt Ryder 2007-2008 bioactivity assay (Ryder UHC thesis). Nisin Activity on PS Microspheres General Schedule Day Before: Date __________ Make MRS Broth (change bi-weekly): Vol __________ Date __________ Make overnight Pediococcus culture (16 hr incubation): Time\/Date Start __________ Finish __________ Vol __________ OD600 __________ rpm __________ Incubate microsphres with F108 (16 hr min): [&hellip;]<\/p>\n","protected":false},"author":6147,"featured_media":0,"parent":117,"menu_order":4,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-237","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/dev.blogs.oregonstate.edu\/schilkelab\/wp-json\/wp\/v2\/pages\/237","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/dev.blogs.oregonstate.edu\/schilkelab\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/dev.blogs.oregonstate.edu\/schilkelab\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/dev.blogs.oregonstate.edu\/schilkelab\/wp-json\/wp\/v2\/users\/6147"}],"replies":[{"embeddable":true,"href":"https:\/\/dev.blogs.oregonstate.edu\/schilkelab\/wp-json\/wp\/v2\/comments?post=237"}],"version-history":[{"count":3,"href":"https:\/\/dev.blogs.oregonstate.edu\/schilkelab\/wp-json\/wp\/v2\/pages\/237\/revisions"}],"predecessor-version":[{"id":241,"href":"https:\/\/dev.blogs.oregonstate.edu\/schilkelab\/wp-json\/wp\/v2\/pages\/237\/revisions\/241"}],"up":[{"embeddable":true,"href":"https:\/\/dev.blogs.oregonstate.edu\/schilkelab\/wp-json\/wp\/v2\/pages\/117"}],"wp:attachment":[{"href":"https:\/\/dev.blogs.oregonstate.edu\/schilkelab\/wp-json\/wp\/v2\/media?parent=237"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}