{"id":1111,"date":"2018-08-13T21:41:18","date_gmt":"2018-08-13T21:41:18","guid":{"rendered":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/?p=1111"},"modified":"2018-08-13T21:41:18","modified_gmt":"2018-08-13T21:41:18","slug":"microbiology-lab-mediatests-notes","status":"publish","type":"post","link":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/microiology\/microbiology-lab-mediatests-notes\/","title":{"rendered":"Microbiology Lab Media\/Test Notes"},"content":{"rendered":"<p><strong>Microbiology Lab Notes (media and tests) By \u00a92018 Shirley S. Chung, Green River Community College<\/strong><\/p>\n<p><a href=\"http:\/\/www.integrativewellnessandmovement.com\/iwmstudentresources\/MicroLabProcNotes_stainsSEC.pdf\" target=\"_blank\" rel=\"noopener\">Download these notes.<\/a><\/p>\n<p><strong>Thioglycollate Broth<\/strong>:<\/p>\n<ul>\n<li>Test aerotolerance of bacteria.<\/li>\n<li>Turns pink in presence of oxygen via Resazurin indicator. Alternatively, methylene blue indicator (colorless in an anaerobic environment and greenish-blue in the presence of oxygen).<\/li>\n<li><strong>Uniform growth<\/strong> = facultative anaerobic bacteria.<\/li>\n<li><strong>Bubbles<\/strong> = gas-producing bacteria.<\/li>\n<li><strong>Bottom growth<\/strong> = anaerobic bacteria.<\/li>\n<li>Contains sodium thioglycollate, thioglycollic acid, L-cystine, methylene blue, and 0.05% agar.\u00a0 The sodium thioglycollate, thioglycollic acid, and L-cystine reduce the oxygen to water.<\/li>\n<\/ul>\n<p><a href=\"http:\/\/www.austincc.edu\/microbugz\/fluid_thioglycollate_medium.php\" target=\"_blank\" rel=\"noopener\">http:\/\/www.austincc.edu\/microbugz\/fluid_thioglycollate_medium.php<\/a><\/p>\n<p><strong>GasPak<\/strong>:<\/p>\n<ul>\n<li>For incubation of anaerobic cultures in a nonreducing medium.<\/li>\n<li>Generates water and CO2.<\/li>\n<li>Methylene blue strip indicator is blue in presence of oxygen, and colorless in absence of oxygen.<\/li>\n<\/ul>\n<p><strong>Selective, Phenylethyl alcohol agar<\/strong>:<\/p>\n<ul>\n<li>For isolation of most G+<\/li>\n<li>Partially inhibitory to G- (may form, but stunted growth, suboptimal).<\/li>\n<li>Inhibits E. coli, selects for S. aureus.<\/li>\n<\/ul>\n<p><strong>Selective, Crystal violet agar<\/strong>:<\/p>\n<ul>\n<li>Selective for most G-<\/li>\n<li>Inhibitory to most G+<\/li>\n<\/ul>\n<p><strong>Selective, 7.5% NaCl agar<\/strong>:<\/p>\n<ul>\n<li>For halophilic<\/li>\n<li>Inhibitory to most other non-halophilic organisms.<\/li>\n<li>*Most useful in detection of genus Staphylococcus.<\/li>\n<\/ul>\n<p><strong>Differential\/Selective, MacConkey agar<\/strong>:<\/p>\n<ul>\n<li>Has bile salts and crystal violet, lactose, neutral red.<\/li>\n<li>Inhibit G+<\/li>\n<li>Selective for G-<\/li>\n<li>Contains Lactose<\/li>\n<li>Contains pH neutral red which differentiates RED-Lactose-fermenting colonies; translucent-non-fermenting colonies.<\/li>\n<li>Differentiate between enteric bacteria.<\/li>\n<li><strong>Coliform bacili<\/strong>: lactose fermenters, make acid, <strong>RED<\/strong> color on their surface. coli is a super fermenter and there will be <strong>pink<\/strong> zone surrounding growth.<\/li>\n<li><strong>Dysentery, typhoid, paratyphoid<\/strong>: non-lactose fermenters; TAN appearance or transparent.<\/li>\n<\/ul>\n<p><strong>Differential\/Selective, Mannitol salt agar (MSA)<\/strong>:<\/p>\n<ul>\n<li>High salt concentration, 7.5% NaCl<\/li>\n<li>Select for staphylococci, inhibit most other non-halophilic bacteria.<\/li>\n<li>Contains mannitol (carbohydrate) for differential (some staphylococci can ferment).<\/li>\n<li>Phenol red indicator detect acid from mannitol-fermenting staphylococci.<\/li>\n<li><strong>Yellow<\/strong> zone around growth is <strong>positive<\/strong> for mannitol fermentation; no color change is <strong>negative<\/strong>.<\/li>\n<\/ul>\n<p><strong>Differential\/Selective, Eosin-methylene blue agar (Levine)<\/strong>:<\/p>\n<ul>\n<li>Has lactose and dyes eosin and methylene blue.<\/li>\n<li>Partly inhibitory to G+<\/li>\n<li>Promote G-<\/li>\n<li>Differentiate between enteric lactose fermenters and nonfermenters.<\/li>\n<li>Can identify between E. coli (<strong>blue-black w\/metallic green<\/strong> sheen due to lots of acid).<\/li>\n<li>Can identify E. aerogenes (thick mucoid, <strong>pink<\/strong> colonies).<\/li>\n<li>Enteric bacteria that do <strong>NOT ferment lactose<\/strong>&#8212;<strong>colorless<\/strong> colonies, transparent, and appear to take on purple color of medium.<\/li>\n<\/ul>\n<p><strong>Enriched Media, Blood agar<\/strong>:<\/p>\n<ul>\n<li>For cultivation of fastidious organisms (e.g. Streptococcus).<\/li>\n<li>Demonstrate hemolytic properties.<\/li>\n<li><strong>Gamma<\/strong>: no lysis of RBC. No change in medium.<\/li>\n<li><strong>Alpha<\/strong>: incomplete lysis of RBC; reduction of Hb to methemoblogin results in greenish halo around growth.<\/li>\n<li><strong>Beta<\/strong>: lysis of RBC; results in clear zone.<\/li>\n<\/ul>\n<p><strong>Starch Hydrolysis<\/strong>:<\/p>\n<ul>\n<li>Extracellular enzyme amylase to hydrolyze starch down to maltose (maltase cat.) then glucose.<\/li>\n<li>Starch agar.<\/li>\n<li>Flood with iodine to test: blue-black = presence of starch and <strong>NEG<\/strong> for starch hydrolysis; clear zone (exoenzymes present) = <strong>POS<\/strong> for starch hydrolysis.<\/li>\n<\/ul>\n<p><strong>Lipid Hydrolysis<\/strong>:<\/p>\n<ul>\n<li>Tributyrin agar.<\/li>\n<li>After inoculation, CLEAR zone <strong>POS<\/strong> for lipid hydrolysis (lipase).<\/li>\n<\/ul>\n<p><strong>Casein Hydrolysis<\/strong>:<\/p>\n<ul>\n<li>Milk agar to test for protein hydrolysis.<\/li>\n<li>Clear zone = <strong>POS<\/strong>.<\/li>\n<\/ul>\n<p><strong>Gelatin Hydrolysis<\/strong>:<\/p>\n<ul>\n<li>Test for liquifaction via gelatinase to hydrolyze protein to amino acids.<\/li>\n<li>Gel deep tubes get inoculated.<\/li>\n<li>After incubation, put in fridge for 30min. Cultures that remain liquified produce gelatinase and rapid gelatin hydrolysis (<strong>POS<\/strong>).<\/li>\n<li>If solid, then re-incubate cultures for 5 more days. Put in fridge for 30min. If liquify, then they are <strong>POS<\/strong> for SLOW gelatin hydrolysis. If solid, then <strong>NEGATIVE<\/strong>.<\/li>\n<\/ul>\n<p><strong>Carbohydrate Fermentation<\/strong>:<\/p>\n<ul>\n<li>*Facultative anaerobes are usu. the fermenters.<\/li>\n<li>Need broth and Durham tube.<\/li>\n<li>Observe w\/in 48 hrs.<\/li>\n<li>Add phenol red: red turns yellow = <strong>POS<\/strong> (no color change of indicator = <strong>NEG<\/strong>).<\/li>\n<li>Gas = <strong>POS<\/strong>.<\/li>\n<li>Beware that neg result does NOT mean no growth.<\/li>\n<\/ul>\n<p><strong>Hydrogen Sulfide:<\/strong><\/p>\n<ul>\n<li>2 fermenting pathways to produce H2S (g).<\/li>\n<li>Stab inoculation.<\/li>\n<li>Black ferrous sulfide = <strong>POS<\/strong>.<\/li>\n<li>Also indicate <strong>motility<\/strong>.<\/li>\n<\/ul>\n<p><strong>Urease<\/strong>:<\/p>\n<ul>\n<li>Useful to i.d. Proteus vulgaris (produces urease). Other organisms also can produce urease.<\/li>\n<li>Inoculate urea broth containing indicator phenol red.<\/li>\n<li>Deep pink = <strong>POS<\/strong> urease presence. No deep pink = <strong>NEG<\/strong>.<\/li>\n<\/ul>\n<p><strong>Nitrate Reduction Test:<\/strong><\/p>\n<ul>\n<li>Reduction of nitrates by some aerobic\/facultative anaerobic organisms occur in absence of oxygen. Use inorganic subtrates NO3 or SO4. Some can further reduce Nitrite to ammonia.<\/li>\n<li>Solution A=sulfanilic acid. Solution B is alpha-naphthylamine.<\/li>\n<li>Solution A+B = <strong>cherry red<\/strong> = <strong>POS<\/strong> for reducing nitrates to nitrites.<\/li>\n<li><strong>No red<\/strong> gives 2 possibilities: end products were reduced even further down to ammonia; or no reduction took place.<\/li>\n<li>Add <strong>zinc<\/strong>. No color change = no nitrates = <strong>POS<\/strong>. Red color change = <strong>NEG<\/strong> = yes nitrates are present.<\/li>\n<\/ul>\n<p><strong>IMViC (Indole, Methyl red, Voges-Proskauer, Citrate utilization)<\/strong>:<\/p>\n<ul>\n<li>Identification of enteric bacteria.<\/li>\n<li><strong>Indole production<\/strong>.<br \/>\nSome bacteria hydrolyze Trp to produce organic compound indole. Use SIM agar containing Trp. After incubation, add Kovac&#8217;s reagent.<\/p>\n<p>Turn CHERRY RED = <strong>POS<\/strong> for Trp hydrolization. No cherry red = <strong>NEG<\/strong>.<\/li>\n<li><strong>Methyl red<\/strong>.<br \/>\nDetermine if organism can ferment glucose via high-acid end-products. Differentiate between glucose-enterics (especially valuable to separate E. coli [low pH] and E. aerogenes [converts to nonacidic end products]).<\/p>\n<p>Low pH (&lt;4.4) Methy red indicator turns RED = <strong>POS<\/strong>. pH &gt; 6.2 turns YELLOW = <strong>NEG<\/strong>.<\/li>\n<li><strong>Voges-Proskauer<\/strong>.<br \/>\nDetermine if organism produces nonacidic\/neutral end-products from organic acids from glucose metabolism&#8230;Glucose fermentation. Characteristic of E. aerogenes.<\/li>\n<\/ul>\n<p>Barritt&#8217;s reagents A+B. Wait 15 min. Rose color = <strong>POS<\/strong> (for glucose fermentation). No pink rose color = <strong>NEG<\/strong>.<\/p>\n<ul>\n<li><strong>Citrate Utilization<\/strong>.<br \/>\nDifferentiate enteric organisms ability to use citrate as sole source of carbon.<br \/>\nGrowth, <strong>blue<\/strong> medium = <strong>POS<\/strong> for citrate. <strong>Green<\/strong>, no growth = <strong>NEG<\/strong> for citrate.<\/li>\n<\/ul>\n<p><strong>Catalase<\/strong>:<\/p>\n<ul>\n<li>Aerobic respiration, hydrogen peroxide and superoxides are produced. Capable of producing catalase or superoxide dismutase.<\/li>\n<li>Strict anaerobes don&#8217;t produce these enzymes.<\/li>\n<li>Differentiate catalase-positive Staphylococci and catalase-negative Streptococci and members of Enterobacteria.<\/li>\n<li>Inoculate on TSA slant\/plate.<\/li>\n<li>Add H2O2 (hydrogen peroxide). <strong>Bubbles<\/strong> = <strong>POS<\/strong> for catalase. No bubbles = <strong>NEG<\/strong> = strict anaerobe.<\/li>\n<\/ul>\n<p><strong>Oxidase<\/strong>:<\/p>\n<ul>\n<li>Aerobic bacteria and some facultative exhibit oxidase activity.<\/li>\n<li>Differentiate between Neisseria and Pseudomonas (both oxidase-positive) and Enterobacteria (oxidase-negative).<\/li>\n<li>Test reagent alpha-aminodimethylaniline.<\/li>\n<li>Pink then maroon then dark purple = <strong>POS<\/strong> for cytochrome oxidase production.<\/li>\n<li>No color change or light pink = <strong>NEG<\/strong>.<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<p><strong>Microbiology Lab Procedure Notes By \u00a92018 Shirley S. Chung, Green River Community College<\/strong><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Microbiology Lab Notes (media and tests) By \u00a92018 Shirley S. Chung, Green River Community College Download these notes. Thioglycollate Broth: Test aerotolerance of bacteria. Turns pink in presence of oxygen via Resazurin indicator. Alternatively, methylene blue indicator (colorless in an anaerobic environment and greenish-blue in the presence of oxygen). Uniform growth = facultative anaerobic bacteria. [&hellip;]<\/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-1111","post","type-post","status-publish","format-standard","hentry","category-microiology"],"_links":{"self":[{"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/posts\/1111","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=1111"}],"version-history":[{"count":1,"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/posts\/1111\/revisions"}],"predecessor-version":[{"id":1112,"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/posts\/1111\/revisions\/1112"}],"wp:attachment":[{"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/media?parent=1111"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/categories?post=1111"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/integrativewellnessandmovement.com\/iwmbasicscience\/wp-json\/wp\/v2\/tags?post=1111"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}