Microbiology 4000 Final Lab Study Guide
Microbiology 4000 Final Lab Study Guide 4000
Popular in Basic and Practical Microbiology
Popular in Microbiology
This 0 page Study Guide was uploaded by Victoria Gonzalez on Friday December 4, 2015. The Study Guide belongs to 4000 at Ohio State University taught by Madhura Pradhan,Tammy Bullwinkle in Summer 2015. Since its upload, it has received 239 views. For similar materials see Basic and Practical Microbiology in Microbiology at Ohio State University.
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Date Created: 12/04/15
Microbiology 4000 Lab Final Study Guide Victoria Gonzalez 1 Microscopes a b Resolving powerResolution ability to distinguish two closely spaced points as separate Parfocality ability of microscope to remain focused as it switches from objective to objective Working distance space needed between the specimen and the lens in order for the specimen to be in focus With increasing magni cation the lens size decreases looks darker False motility i Brownian motion organism appears to be vibrating in place due to random collision of particles in suspension ii Current ow everything on the slide moves in same direction Microscope used in lab compound bright eld microscope i Specimens appear dark against a bright background ii Close iris diaphragm when looking at unstained slides iii Immersion oil used only with 100X lens 1 Without oil immersion most light is refracted and lost because lens is so small 2 Resolving power is increased Dark eld microscope i Bright images against a dark background ii Easier to view unstained objects with light shined at an angle iii Used to view living viable unstained organisms in order to determine their morphology and motility Phase contrast microscope i Useful for determining microbial motility and morphology ii Ability to see certain cellular structures iii Should NOT be used to view specimens that have been stained Fluorescence microscopes i Chemicals called uorochromes are used to stain specimens ii lmmuno uorescence microscopy uorochromes attach to antibodies antibodies attach to the bacteria j Electron microscopes i Useful for viewing ne structures ii Cannot be used to determine motility iii Specimens must be stained iv Time consuming and expensive techniques v Transmission electron microscope TEM 1 Specimen is placed in the middle of the microscope 2 Image is an internal view of the specimen vi Scanning electron microscope SEM 1 Specimen is placed at the bottom of the microscope 2 Used to see the surfaces of objects 3 Image produced is 3D vii TEM a vs SEM b BEENgm E3 151 llch39aMillal Citinglungs I Par39m5san reacnrz39d for lepwu tgn tr LE 5 5w lal l ll quot1391 l I turn 2pm 2 Dilution and enumeration a Enumerating microbes used to determine microbial cell concentrations i Viable count method allows observer to quantify the number of organisms in a volume of culture 1 Countable plate has between 30 and 300 colonies b Calculating dilutions i Individual dilutionvol sample addedsample vol diluent ii Total dilution individual dilution x individual dilution iii When multiplying powers add the exponents c Calculating original concentration i Original concentrationcfuvolume plated x total dilution ii If two plates made from different dilutions in a series have colony counts between 30 and 300 the original concentrations calculated can be averaged 3 Microbial diversity a Fungi types i Hyphae majority 1 Produce spores small resistant structures ii Yeast less common 1 Reproduce asexually through budding b Bacteria reproduce through binary ssion Yeast Bacteria Large in size Smaller in size Budding is visible No budding Nucleusinternal structures No visible internal structures 4 Techniques for generating isolated colonies a Streak plate technique three phase streak b Spread plate technique spreading cells from a diluted liquid culture i This technique also allows enumeration of bacteria determining the concentration of bacteria in a given volume of sample ii This technique can also be used to produce a lawn from undiluted pure liquid sample iii Spread plates are used for 1 Testing pure cultures to various antiseptics disinfectants or antibiotics 2 Identifying bacterial subspecies by determining their susceptibilities to speci c bacteriophages 5 Simple and differential stains a b Stains make bacteria more visible under bright eld illumination Biological dyes used to stain bacteria are salts i Basic dye colour is carried on the positive ion ii Acidic dye colour is carried on the negative ion iii Bacteria have an overall negative ionic charge so they have a strong af nity for basic dyes Simple stain one basic dye is applied all cells stain i Used to determine size cellular morphology and arrangement ii Crystal violet used Differential stain uses multiple biological dyes i Used to determine cellular morphology and arrangements ii Used to differentiate between two types of bacteria Gram stain a type of differential stain i 1 Primary stain crystal violet stains purple ii 2 Mordant enhances the uptake and retention of the primary stain gram s iodine iii 3 Decolorizing step acetone alcohol 1 Differential step 2 In gram negative bacteria acetone dissolves outer membrane and decolorizes thin peptidoglycan layer 3 In gram positive bacteria acetone does not decolorize because peptidoglycan layer is thick iv 4 Secondary stain safranin stains pink 1 Absorbed by 6 because they have been decolorized f Acid fast stain a type of differential stain i For cells with mycolic acid waxy layer no gram stain ii Primary stain carbolfuschin iii Two mordants turgitol and phenol iv Decolorizing agent solution of HCI and alcohol v Counterstain methylene blue vi Nonacidfast cells lose carbolfuschin and stain blue vii Acidfast cells stain pink from carbolfuschin g Capsule stain i Capsules distinct shape and slime layers irregular shape protect bacteria from invaders facilitate attachment impede phagocytosis contribute to virulence 1 Capsules composed of polysaccharides ii To visualize capsules 1 pH indicator dye Congo red 2 Staining reagent Maneval s solution iii Capsule remains unstained background is dyed 1 Negative stain structure being observed remains colorless h Endospore staining i Method one simple stain with basic dye 1 Crystal violet is used 2 Vegetative cells take up the stain 3 Endospores remain unstained 4 Negative stain ii Method two phase contrast microscope 1 Endospores and vegetative cells have different densities 2 Endospores appear as bright objects 3 Vegetative cells appear dark 6 Morphology a Coccus spherical b Bacillus rodshaped c Vibrio comma shape d Spirillum wavelike shape e Spirochete corkscrew shape 7 Cellular arrangements a Single b Pairs diplo c Chains strepto d Clusters staphylo e Groups of four tetra f Packets of eight sarcinae 8 Colony morphologies a Color b Texture c Type of marginedge regular undulating lobed lamentous d Type of elevation at raised convex e Optical characteristicsconsistency dull shiny opaque transparent translucent 9 Bacteria classi cation Auto uses inorganic carbon carbon dioxide Hetero uses organic compounds as carbon sources Photo uses light as energy Chemo uses inorganic components for energy 10 Media classi cation a Chemically de ned i Complexunde ned component amounts are not known ii De ned component amounts are known in precise amounts b Nutritionally de ned i Minimal supplies only nutrients microbe can t make ii Rich supplies a wide variety of nutrients c Functionally de ned i General purpose grows a wide variety of microbes ii Selective can only grow some microbes iii Differential grows a variety of microbes that are visually distinguishable iv Selectivedifferential selects for limited organisms and enables observers to distinguish between the types grown 11 Bacteria that live in the presence of oxygen produce cytochrome oxidase and catalase obligate anaerobes do not a Test for cytochrome oxidase production i Smear culture on lter paper with toothpick ii Add a drop of oxidase reagent TMPD iii Dark purple colour within 30 seconds reagent has been oxidized and the organism is oxidasepositive b Test for catalase production i Transfer culture to microscope slide with inoculation loop ii Add one drop of hydrogen peroxide iii Bubbles produced positive for catalase production 006m 12 13 14 15 U9 1 Catalase catalyzes H202 l H20 02 2 02 accounts for bubbles Growth curve represents growth of a bacterial population Generation time time it takes for population to double Stages i Lag phase cells do not dividedivide slowly while adapting to their surroundings and synthesizing necessary materials ii Log phage grow exponentially iii Stationary phase essential nutrients are depleted and growth plateaus iv Death phase occurs if a renewed source of nutrient doesn t become available Spectrophotometer measures the amount of light of a speci ed wavelength that passes through a sample of a bacterial culture Protein utilization in bacteria In order to use protein as energy cells must produce peptidases i Exoenzymes peptidases active outside the cells 1 Gelatinase hydrolyses gelatin ii Endoenzymes intracellular peptidases 1 Tryptophanase tryptone broth 2 Desulfhydrase KIA Anaerobic conditions GasPac anaerobic system Contains inorganic carbonate activated carbon ascorbic acid and water ii Reduces the amount of free oxygen making anaerobic conditions in the jar iii Indicator strip turns from blue to white indicating no oxygen Enterotube II and API 20E Both i Costly methods ii Quick iii Can only identify organisms in the database Enterotube ll i The same inoculum is added at once ii The Computer Coding and Identi cation System provides an identi cation of the organism iii Dif cult to add necessary chemicals API 20E i Dehydrated form in 20 separate cupules are inoculated individually 16 ii A sevendigit code is generated and identi ed with the API Index for Identi cation Osmosis diffusion of solvent through semipermeable membrane a b 17 18 19 20 U9 Osmotic pressure amount of pressure needed to stop the net ow of water across semipermeable membrane Plasmolysis cell s membrane collapses due to loss of water from cytoplasm Most bacterial habitats are hypotonic water ows into the cell i Can cause cell to lyse Autoclave kills endospores by moist heat Microbes and food Constructive processes result in desirable products i Often involve fermentation ii Ex bread cheese yogurt soy sauce tofu vinegar sauerkraut beer and wine Destructive processes produce spoilage i Can breakdown food products into less desirable forms ii Can consist of the growth of organisms that cause food poisoning Meat was spread on a selectivedifferential medium eosinmethylene blue EMB agar Yogurt is a product of fermentation of milk i Requires two bacteria 1 Streptococcus thermophies 2 Lactobacilus bugaricus ii These bacteria catabolize lactose into lactic acid Mouthwashes on bacterial growth Measured through lter disk method i Use lter disks saturated with an agent ii Zone of inhibition is measured to estimate the relative effectiveness of the agents Normal oral microbiota simulated by i Streptococcus salivarius ii Staphylococcus epidermidis Antibiotics Bactericidal antibiotic kills bacterium Bacteriostatic antibiotic inhibits bacterium growth Etest rapid way to measure minimum inhibitory concentration MIC i Plastic strip with concentration gradient is laid on a spread plate ii MIC can be read by observing where the growth intersects with the strip if in between two use higher concentration d KirbeyBauer method tests microbial sensitivity to antibiotics through the use of lter disks containing antibiotics i Zone of inhibition can be measured and compared ii To determine if organism is killed or inhibited a subculture must be made with a sample from within the zone of inhibition 1 If there is growth the antibiotic was bacteriostatic 2 If there is no growth antibiotic was bactericidal iii Interpretation 1 Susceptible organism is susceptible to antibiotic biggest zone of inhibition 2 Intermediate antibiotic may be useful under certain circumstances 3 Resistant organism causing the infection is resistant to antibiotic not an appropriate choice smallest zone of inhibition 21 Transformed bacteria a Bacteria was transformed giving it a new phenotype antibiotic resistant b During transformation ces take up new plasmids and express the genes i Plasmid A 1 Gene encodes enzyme betaIactamase ampicillin resistant 2 Gene that synthesizes a protein involved in bioluminescence found in jelly sh ii Plasmid B 1 Gene responsible for kanamycin resistance 10 22 23 up Um h Symbiosis Mutualism bene ts both organisms i Ex soybeanalfalfapeas and bacteria in nodules 1 Bacteria xes nitrogen into a useable form for the plant 2 Plant nodules provide protection plant provides bacteria with cabohydrates Mouth and nose microbiota i Normal microbiota can be mutualistic 1 E coli make vitamin K for us and bene ts from nutritious environment in our GI tract ii Nose and mouth harbor large ora of microorganisms most are harmless few may become pathogenic under certain conditions ELISA Test antigen binds to well surfaces Test serum is added and antibodyantigen complexes form with the positive test serum Cell wells are washed to remove unbound antibodies Conjugated secondary antibodies are added and bind to antibodies that remain in wells ampli ed response Unbound conjugated second antibodies are washed away Substrate is added to well green colour change is noticed in wells that contain conjugated secondary antibodies 11 1 MM1 minimal media a MM1 without glucose minimal media b MM1 without sulfur minimal media 2 TSA complex rich general purpose 3 BHI brainheart infusion agar complex very rich general purpose a Contains many factors required by fastidious organisms 4 PIA pseudomonas isolation agar a Only pseudomonas grows selective b Contains a broad spectrum antimicrobial agent that inhibits the growth of a wide variety of organisms 5 Starch agar differential a Starch is a carbohydrate used for energy b Bacteria that use starch for energy require the exoenzyme aamylase c Appearance of growth does not determine ability to utilize starch d To test i Agar must be ooded with iodine which turns the starch black ii If an organism produces aamylase it will degrade the starch and a clear area will appear 6 MacConkey agar selectivedifferential a Selective for enterics due to bile salts and crystal violet i Gram negative enterics grow ii Gram positive and other gram negatives do not grow b Differential for colforms due to neutral red pH indicator i Bright pink coliform enterics ferment lactose ii Yellowcolorless noncoliform enterics 7 MRVP a Used for identi cation of enterics b Used to determine metabolic pathway used to ferment glucose c MR test i Uses a pH indicator methyl red to determine amounts of acid produced from glucose fermentation 1 When a lot of acid is present pH 43 or below indicator turns red uses mixedacid pathway 2 When above pH 43 medium remains yelloworange 12 d VP test i Determine if high amounts of neutral products are produced via butanediol pathway 1 Pink is positive result 8 Simmons citrate agar a Used to determine if citric acid citrate has been utilized b pH indicator bromothymol blue c If citric acid is utilized less acid raises the pH and medium turns blue d Citrate negative green 9 KIA kliger iron agar a KIA is made up of i Carbohydrates lactose and glucose ii Peptides and proteins peptone and beef iii pH indicator phenol red turns yellow below pH 63 b Letters i K basic peptones used turns red ii A acid carbohydrates used turns yellow c Results i KA top is red bottom is yellow 1 Bacteria used glucose not lactose ii AA all turned yellow 1 Bacteria used glucose and lactose iii Kno change top is red bottom is colorless 1 Bacteria cannot use glucose or lactose bacteria can use peptones only aerobically iv KK all red 1 Bacteria cannot use glucose or lactose bacteria can use peptone aerobically and anaerobically v Cloudy red no growth vi Black precipitate used sulfur H25 10 Glucose lactose or mannitol broths with Durham tubes a pH indicator phenol red b No reaction broth remains clear and red organism cannot utilize carbohydrate and did not grow anaerobically c Growth no acid the broth is turbid and red organism grows using alternative source peptones d Acid production broth turns yellow organism used carbohydrate 13 11 12 13 14 15 Acid and gas production broth turns yellow and gas bubble appears in Durham tube organism used carbohydrate and produced gas Nitrate broth a Positive test nitrate was used turns red i N03 l N02 b Negative test no colour change and zinc is then added i Red organism did not use nitrate ii No colour change check Durham tube 1 Bubble denitri cation 2 No bubble nitrate was used N03 N02 Gelatin strips a To utilize gelatin microorganisms must produce gelatinase b Blue colour on gelatin strip organism used gelatin c No blue colour negative for gelatinase production Tryptone broth a Tryptone broth contains tryptophan to use tryptophan bacterium must produce tryptophanase b Tryptophanase activity can be con rmed by the presence of indole c Kovac s reagent reacts with indole i lndole positivetryptophanase positive red on top of broth ii lndole negative yellow ring on top of broth PDA potato dextrose agar a Rich and unde ned b Encourages growth of fungi c Yeasts appear starshaped inside the agar appear large and white on the surface of the agar Eosinmethylene blue EMB agar selectivedifferential medium a Selective i Growth gram negative ii No growth gram positive b Differential I Yellow nonlactose fermenter ii Black ferment lactose through butanediol pathway iii Green metallic ferment lactose through mixedacid pathway coliform in feces
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