INTROD MICRO LAB
INTROD MICRO LAB MIBO 3510L
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This 7 page Class Notes was uploaded by Dr. Jayda Mayert on Saturday September 12, 2015. The Class Notes belongs to MIBO 3510L at University of Georgia taught by Dustman in Fall. Since its upload, it has received 70 views. For similar materials see /class/202390/mibo-3510l-university-of-georgia in Microbiology at University of Georgia.
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Date Created: 09/12/15
MBIO 3510 Lab Midterm Selective and Differential Media 1 Staph 110 S110 a Selective for Staphylococcus b Growth gtgt Staphylococcus present 2 TSI Tubes Triple Sugar Iron a Differential pH indicator and Fe indicates HzS prod b Slant Color change from red gtgtgt yellow lactose andor sucrose fermented to acids c Butt Color change from red gtgtgt yellow glucose fermented to acids d Black precipitate HzS productions 3 SIM Tubes Sulfur Indole Motility a Differential semisolid medium shows motility Fe shows HzS prod Kovac s shows indole production b Color change from Yellow gtgtgt Black H2S c Cloudy motile d Kovac s Reagent must be added after incubation to determine indole production i Amber gtgtgt no color change no indole production ii Amber gtgtgt red Indole 4 BP BairdParker Media a Differential AND Selective b Selective for Staphylococcus c Clearing of media around growth Coagulase activity d Black appearance tellurite reduction 5 MSA Mannitol Salt Agar a Differential AND Selective b Selective for Staphylococcus amphalotolerance c Differential for Mannitol Fermentation i Color change from Pink gtgtgt Yellow ferments mannitol to acids ii Color change from Pink gtgtgt hot pink no fermentation of mannitol to acids may be slightly alkaline 6 MS Mitis Salivarius Agar a Differential AND Selective b Differential bw two typical growth environments i Light Blue Streptococcus oral ii Dark Blue Enterococcus gut c Selective for StreptoEnterococcus 7 EMB Eosin Methylene Blue a Differential AND Selective b Selective for Gram Rods c Differential for lactose fermentation to acids i Transparent growth Doesn t ferment lactose or sucrose to acids ii Purpleblack growth Ferments lactose to acids iii Metallic Green E Coli ferments lactose and sucrose 8 MAC MacConkey Agar a Differential AND Selective b Selective for Gram 7 Rods c Differential for Lactose Fermentation to acids i Growth surrounded by pink Lactose fermentation to acids 9 SS SalmonellaShigella Agar a Differential AND Selective b Selective for Gram 7 Rods enteric c Differential for Lactose fermentation to acids and HzS production i No color change no lactose fermentation to acids ii Color change to pink lactose fermentation to acids iii Black present on growth HzS production 10 Starch Agar a Differential for enzyme amylase b Add iodine after incubation i Clear area around growth amylase presence 11 Skim Milk Plate a Differential for enzyme caseinase b Clear area around growth from cloudy caseinase presence 12 Blood Agar Plate a Differential for enzyme Hemolysin ability to break down hemoglobin b Alpha Hemolysis green color around growth c Beta Hemolysis clear area around growth d GammaHemolysis no break down of hemoglobin l3 DNAse Plate w Methyl Green a Differential Tests for enzyme DNase b Initial green color signifies acidity DNA is acidic c Color change to orangey green DNase activity present 14 Nitrate Reduction Broth a Differential for ability to reduce nitrate N03 to a terminal e39 acceptor b N0339 gtgtgt NOz39 gtgtgt N2 or NH4 c Steps i Culture for 48 hrs ii Add Dimethylalphanaphthylamine and Sulfanillic acid 1 Color Change to Red NO339 Reduced to N0239 2 No Color Change requires next step N0339 reduced all the way to N2 or NH4 OR Not reduced at all iii Add Zinc Powder forces Nitrate to Nitrite reduction 1 Color change to Red Species does not reduce nitrate 2 No Color change Organism reduced Nitrate to N2 or NH4 Oxygen Requirements 1 Obligate Aerobes a Require presence of O b Have enzymes that remove toxic byproducts c Growth found only at very top of the tube d Grows weakly in candle jar good in open and not at all in gas pak 2 Obligate Anaerobes a Oxygen is toxic b Do not have enzymes that remove toxic byproducts c Growth found at bottom of tube d Only will grow in the gas pak 3 Facultative Anaerobes a Can live with or without 02 b Prefer 02 but can also ferment sugars c Have enzymes to remove toxic byproducts d Growth found spread throughout tube with more concentrated towards top e Will grow in candle jar open air and in gas pak 4 Microaerophiles a Require Oz in LOW levels b Too much 02 is toxic c Limited quantities of enzymes to remove toxic byproducts d Growth found close to top of the tube e Will only grow in candle jar 5 Aerotolerant a Indifferent to Oxygen b Have enzymes to remove toxic byproducts c Even spread throughout tube White indicator in gas pak indicated NO 02 present Temperature Requirements 1 Psychrophile 0 to 20 C 2 Mesophile 15 to 45 C 3 Thermophile 45 to 70 C 4 Hyperthermophile 70 to 120 C 5 Thermoduric Thermotolerant a Cannot grow but can survive in extreme heat for periods of time b Mycolic acids and endospores can make an organism thermoduric pH Requirements 1 Acidophile pH 10 7 55 2 Neutrophile pH 55 7 80 3 Alkalophile pH 85 7115 4 Acidotolerant can persist in acidic conditions but does not grow Staphylococciiclusters of spheres Streptococciichains of spheres Diplococciitwo Tetradsifour Bacilli clustersirods Streptobacilliichains Spirochetesspirilla and vibrio comma Asexual fungal spores o SporangiosporesiRhizopus sacs holding spores o Conidiospores I Penicillium skeletal hand I Aspergillus dandelion 0 Yeast I Blastospores budding Growth on solid media Color white yellow red transparent Edge smooth lobed friedegg Size punctiform small large Height at raised convex Shape round spreading 0 Texture creamy dry slimy aky Chromophores chemical groups which possess a visible color and either a positive or negative charge 0 Basic dyesipositive charge 0 Acidic dyesinegative charge Negative stainsibackground stainediNigrosaIndia ink Gram Stain 0 Primary stainicrystal violent Water MordantiLugol s precipitates crystal violet Water Decolorizeri95 Ethanol dissolves LPS in Gram 7 and peptidoglycan Water Counterstainisafranin Water 0 Blot and oil immerse Gram Purple Positive Peptidoglycan Endospores Staining o ClostridiumiTriggers sporulation when oxygen present 0 Bacillusilack oxygen will sporulate 0 Has DPAistabilize DNA within endospores contain DNA repair enzymes 0 Steps I Primary Stainimalachite green 5 min I Water I Counterstainisafranin 2 minutes 00000 0000000 I Water 0 Greeniendospore redivegetative cell 0 Acid Fast Staining o M ycobacterium sp have waxy mycolic acids present in their cell walls I Steam heat softens it 0 Steps I Primary stainicarbol fuchsin 5 min breaks mycolic acids I Water I Decolorizeriacid alcohol 30 sec I Water I Counterstainimethylene blue 30 sec I Water 0 Fushciaiacid fast Streak plateiqualitative Spread and pour plateiquantitative Autotrophifix inorganic carbon C02 Heterotrophitakes up organic carbon Prototrophican synthesize shit itself Auxotrophibabyifastidious Direct motilityican see it physically Indirect motilityistabs in semisolid motility agar 0 Growth at top of tube doesn t count Oxygen o Obligate Aerobes I 02 necessary for growth I have enzymes which remove toxic byproducts o Obligate Anaerobes Gas Pak I O toxic to cells I do not have enzymes which remove toxic byproducts o Facultative Anaerobes I Prefer to use 02 but grow without it also I have enzymes which remove toxic byproducts I capable of fermenting sugars to alcohols or acids 0 Microaerophiles candle jar I Too much 02 is toxic need below atmospheric levels I have limited quantities of enzymes which remove toxic by products 0 Temperature 0 Psychrophile I Optimal growth at cool temperatures 0 7 20 C refrigerator spoilage o Mesophile I Optimal grth at warm temperatures 15 45 C pathogens 0 Thermophile I Optimal growth at hot temperatures 45 70 C compost heaps o Hyperthermophile Extreme thermophile I Optimal growth at hottest extremes 70 120 C hot springs thermal vents o Thermoduric thermotolerant I Cannot grow but can persist in extreme heat for a period of time without dying organisms survive pasteurization 0 Thermal Death Time TDT o TDT The shortest amount of time it takes to kill a given species that has been held at a constant temperature ie 70 C I Time is a variable I Subculture at 37 C to determine viability I Basis of pasteurization 0 Thermal Death Point TDP o TDP The lowest temperature it takes to kill a given species within 10 minutes I Temperature is a variable I Subculture at 37 C to determine viability I Used for food processing home canning methods 0 pH 0 Acidophiles pH 10 7 55 I Fermenting bacteria produce acids as byproducts of carbohydrate metabolism I Food spoilage wine to vinegar 0 Neutrophiles pH 55 7 80 I Most bacteria including pathogens 0 Alkalophiles pH 85 7 115 I Putrefying bacteria degrade proteins to amines and ammonia increasing pH 0 Acidotolerant Acidoduric I Capable of persisting in acidic conditions but not an increase in cell number 0 Osmotics o Halophiles I Require NaCl ions in order to grow I gt 15 NaCl 0 Osmotolerant I Can live in saline conditions and grow but do not reproduce I gt10 NaCl but ltl5 NaCl 0 Saccharophiles I Thrive in high sugar concentrations gt50 jams jellies I Often yeasts and molds 0 UV light 0 Photo reactivation small errors 0 Dark excision repair Experimental design owchart Dilutions Selective media7inhibits growth of a speci c set of microbes o S 1107Staphylococcus 110 Agar I Contains NaCl I Can have no growth and be salt tolerant 0 Differential mediaiused to discriminate between closely related organisms o TSliTriple sugar iron I Yellow buttiglucose fermented to produce H2S I Yellow slantilactose andor sucrose fermented I Gas I No yellowialkaline end product 0 SIMiSulfur indole motility I H2S production I Motility I Kovacs reagent 0 Amberiindole negative 0 Rediindole positive 0 Differentially selective media 0 BPiBaird Parker Media I Clear zoneicoagulase positive I No clearanceicoagulase negative I Blackitellurite reduction I Not for gram negatives o MSAiMannitol Salt Agar I Has NaCl I Neutral to alkalineiRed mannitol negative I Acidiciyellow Mannitol positive ferments mannitol acids I Growthisalt tolerant gram bacteria 0 MSiMitis Salivarius Agar I Blueistreptococci I Dark blueblackiEnterococci o EMBiEosin Methylene Blue Agar I Neutral to alkalineiopaque growth I Acidicipurpleblack growth I Highly acidicigreen metallic sheen pH3 I Ferments lactose to acidsipurple growth I Ferment sucrose and lactose to acidigreen E Coli o MacConkey Agar I Selects for Gram negative I Neutral to alkalineiopaque growth I Acidicipink growth lactose fermented to acids 0 Salmonella Shigella Agar I Same as before I H2Siblack o Enzymes o Exoenzymesiexcreted from cell I Skim Milk Agar 0 Clear mediaiorganism produces caseinase
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