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This 74 page Bundle was uploaded by Novecc13 on Monday October 12, 2015. The Bundle belongs to 2200 at Wayne State University taught by Penelope I Higgs in Summer 2015. Since its upload, it has received 37 views. For similar materials see Intro to Microbiology- Lab in Biology at Wayne State University.
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Date Created: 10/12/15
Purpose To differentiate between bacteria based on three tests sulfur reduction cysteine desulfurase indole production tryptophanase and motility Principle If a bacterium can produce the enzyme tryptohanase then it can use the amino acid tryptophan as a carbon and energy source as pyruvate One of the biproducts of this conversion is indole which is detected with Kovac39s reagent If the bacterium posseses the enzyme cysteine desulfurase sulfur containing amino acids will be broken down into pyruvate ammonia and hydrogen sulfide Iron in the medium reacts with hydrogen sulfide producing the characteristic black percipitate Motility is observed as growth away from the stab line but cannot be detected if hydrogen sulfide is formed The figure to the left represents the following SulfurlndoleMotility Results A Indole positive and hydrogen sulfide positive B Hydrogen sulfide positive C Indole positive and motility positive note fuzzy growth away from stab line D Negative Control Starch Hydrolysis Medium Created by ST Purpose To determine if a bacterium produces the enzyme amylase which breaks down starch to glucose Principle Starch Hydrolysis medium is a simple medium that contains beef extract soluble starch and agar Because starch is such a large molecule it cannot be directly transported into a bacterial cell A bacterium may secrete the extracellular enzyme alphaamylase to cleave the glucosidic linkage of starch releasing individual glucose molecules The presence of starch can be indicated by the development of a blue color therefore the absence of a blue color indicates starch hydrolysis The plate to the right illustrates the two typical reactions observed on a starch plate after the additon of iodine A Starch hydrolysis as indicated by a nonblue halo surrounding the growth arrow B Absense of starch hydrolysis notice there the blue color completely surrounds the colony Created by ST Phenol Red Fermentation Tubes A B e Possible phenol red tube results include A Formation of acid and gas bubble is indicated by arrow B Formation of acid C uninoculated control D alkaline byproducts and E no acid or gas formation Created by ST Purpose To determine if a bacterium can ferment a particular carbohydrate and determine the end products of that fermentation Principle Phenol Red Fermentation medium contains peptone phenol red a pH indicator and the carbohydrate to be tested Phenol red is yellow at a pH lt 68 and red at a pH of gt 74 therefore if a bacterium ferments a sugar to acid a yellow color will develop Additionally phenol red tubes contain an inverted Durham tube If a bacterium produces a gas during fermentation this tube will trap that gas a a bubble will be observed in the Durham tube Sometimes an orange color will develop in the tube this represents a pH gt 68 and should not be considered positive Also be catuious of tubes that inititally produce acid then turn red or pink as nitrogenous products are produced This type of reaction is characteristic of oxidative not fermentative metabolism Created by ST Spirit Blue Medium Created by ST Purpose To determine whether a bacterium produces a lipase that will hydrolyze a neutral fat to fatty acid and glycerol Principle The lipid tributyrin is emulsified into water using Tween 80 and ultrasonification The acid base indicator spirit blue is also present in the medium If tributyrin is hydrolyzed via a lipase the result will be fatty acid and glycerol The localization of fatty acid will cause the colony to take on a bluish hue The plate to the right illusrates possible reactions observed on Sprit Blue medium A Hydrolysis of lipid notice blue tint to colony B Absense of lipid hydrolyis Created by ST Nutrient Gelatin Results Created by ST Purpose To differentiate between organisms that produce the exoenzyme gelatinase and those that do not Principle Gelatin is a protein that is derived from collagen The gelatin molecule is to large to enter the cell as a whole so the exoenzyme gelatinase cleaves gelatin to polypeptides and then further degrades polypeptides to amino acids which are taken up and utilized by the cell When gelatin is converted to these smaller subunits it takes on a liquid form this is the basis for this test The tubes to the right depict a gelatinase negative A and gelatinase positive B and C reactions Created by ST Casein Hydrolysis Test Milk Agar Created by ST Purpose To determine if an organism can produce the exoenzyme casesase Principle Casease is an exoenzyme that is produced by some bacteria in order to degrade casein Casein is a large protein that is responsible for the white color of milk This test is conducted on milk agar which is a complex media containing casien peptone and beef extract If an organism can produce casein then there will be a zone of clearing around the bacterial growth The picture to the right illustrates two different casease positive organisms A and B and a casease negative organism C Created by ST SIM Tube Results Created by ST Purpose To differentiate between bacteria based on three tests sulfur reduction cysteine desulfurase indole production tryptophanase and motility Principle If a bacterium can produce the enzyme tryptohanase then it can use the amino acid tryptophan as a carbon and energy source as pyruvate One of the biproducts of this conversion is indole which is detected with Kovac39s reagent If the bacterium posseses the enzyme cysteine desulfurase sulfur containing amino acids will be broken down into pyruvate ammonia and hydrogen sulfide Iron in the medium reacts with hydrogen sulfide producing the characteristic black percipitate Motility is observed as growth away from the stab line but cannot be detected if hydrogen sulfide is formed The figure to the left represents the following SulfurlndoleMotility Results A Indole positive and hydrogen sulfide positive B Hydrogen sulfide positive C Indole positive and motility positive note fuzzy growth away from stab line D Negative Control Created by ST Urea Tube Results P03i tive Created by ST Purpose To differentiate between bacteria that produce the enzyme urease and those that do not Principle Urea may be hydrolyzed by some bacteria so that the ammonia that is liberated can be used as a nitrogen source Phenol red indicator is added to the broth so that when the pH reaches 84 due to ammonia production the tube will turn from an orange color to a pink color The tubes at the right represent a negative and a positive urea tube Created by ST The tubes above have been treated with dimethyl 1naptholamine and sulfanilic acid The tube on the left illustrated no change thus it was inconclusive The center tube also illustrated no change as expected The tube on the right indicates that the organism present reduced nitrate to nitrite In order to further determine positivenegative nitrate reduction zinc powder must be added to detect the presence of nitrate Created bv ST I A tube will turn a deep red color on the addition of zinc if nitrate is present The inconclusive tube did not turn red indicating that the organism present reduced nitrate to something besides nitrite positive The uninoculated control turned red indicating as expected that nitrate is still present in thetube Purpose Trypticasenitrate broth is used to detect an organisms ability to reduce nitrate to nitrite or a further reduced nitrogenous compound such as nitrous oxide or nitrogen gas Principle Nitrate may be reduce to multiple comounds by two processes Anaerobic respiration and denitrification In anaerobic respiration the bacterium uses nitrate as it39s terminal electron acceptor reducing nitrate to a variety of compounds while denitrification reduces nitrate solely to molecular nitrogen Sulfanilic acid and dimethyl 1naphthylamine are added to detect nitrite which will complex with these molecules forming a red color If no red color is observed there are two possibilities the nitrate has not been reduced or it has been reduced further than nitrite To differentiate between these two possibilities zinc powder is added which will complex with nitrate forming a red color Thus if the tube turns red after zinc nitrate has not been reduced and the result is negative If no red color is observed then the nitrate has been reduced further than nitrite and the result is positive Created by ST in l a I Iquot D 39l i 39 i quotl I I s f I I I i r quota I l i i i a I U i I I t L 39 I i m I I I I quot Gram Negative Cocci Gram Negative Rods Created by ST Gram Positive Rods Gram Stain Results Purpose To differentiate between Grampositive and Gramnegative organisms while simultaneously learning about the cellular morphology and arrangement Principle Grampositive cells have a thick peptidoglycan cell wall that is able to retain the crystal violetiodine complex that occurs during staining while Gramnegative cells have only a thin layer of peptidoglycan Thus Gram positive cells do not decolorize with ethanol and Gramnegative cells do decolorize This allows the Gramnegative cells to accept the counter stain safranin Grampositive cells will appear blue to purple while Gramnegative cells will appear pink to red Created by ST Colony Description Results Purpose To accurately describe common bacterial colonies The plate to the right shows colonies of Micrococcus Iuteus These colonies could be described in the following way Size 2mm diameter Shape Circular Margin Entire Elevation Convex Surface Smooth Density Opaque Pigment Yellow nondiffusible pigment preate Dy DI Simple Stain Results Purpose To recognize the three basic shapes of bacterial cells Principle In order to observe most bacterial cells using bright field microscopy the cells must be dark enough to see that is they must have contrast to the light To create contrast a simple stain can be used Simple stains use basic dyes which are positively charged These positive dyes interact with the slightly negatively charged bacterial cell wall thus lending the color of the dye to the cell wall The slide to the right shows a cocci that has been stained with crystal violet Created by ST Negative Stain Results Purpose To determine morphology arrangement and size of bacteria that may be affected by heat fixing Principle The negative stain uses the dye nigrosin which is an acidic dye By giving up a proton as an acid the chromophore of the dye becomes negatively charged Because the cell wall is also negatively charged only the background around the cells will become stained leaving the cells unstained Negatively Stained Bacillus A Vegetative Ce 3 Endospore Created bynggatlvely Stained COCCI AcidFast Stain Results Purpose To differentiate between acid fast and non acidfast bacteria Principle Some bacteria contain a waxy lipid mycolic acid in there cell wall This lipid makes the cells more durable and is commonly associated with pathogens Acid fast cell walls are so durable that the stain carbol fuschin must be driven into the cells with heat The cells are then decolorized with acidalcohol all other cells will decolorize with this strong r quot 39 solvent but acid fast bacteria will not Other cells are then counterstained with methylene blue To the right a mixed culture has been acidfast stained A Non Acidfast bacteria B Acidfast bacteria tii FI 139 I 3132 3 in Endospore Stain Results Purpose To differentiate between organisms that can produce endospores and those that cannot Also allows differentiation based on the position of the a endospore in the cell Principle Spores have a durable outer coating that is composed of the protein keratin This keratin coat resists staining so in order to stain a spore the primary stain malchite green must be heated to drive the Stain into the Spores Vegatative cells are 39 then decolorized with water and 05 safranin is used to counterstain Thus endospores are stained green while vegetative cells are stained red The stain to the right shows a bacterium capable of endospore production A Vegetative Cell B Endospore r 39l39 e H a Ediquot7quot I a LFIE39 Iiy A a gr w 139 en39ng a1 I quot44 Created by ST Streak Plate Results Purpose To obtain isolated colonies of a pure or mixed culture Principle By spreading a large amount of bacteria over the large surface area of a plate the amount of bacteria is diluted until individual cells are spread on the surface of the plate From these individual cells a single colony arises All of the cells in this colony are genetically iden caL The picutre to the right illustrates a properly performed streak plate Created by ST Motility Test Agar with TTC The culture on the left is nonmotile the cultures in the middle and right side are motile Created by ST Phenylethanol Agar selective for Grampositive organisms Mannitol Salt Agar a selective and differential medium It is selective for salttolerant staphylococci and differential for mannitol fermentation This medium is useful for the selection of pathogenic staphylococci such as Staphylococcus aureus Created by ST Eosin Methylene Blue Agar EMB This medium selects for Gramnegative bacteria and differentiates on the basis of lactose fermentation Lactose fermenters produce dark red growth or colonies see arrow This medim is useful for confirming the presence of coliform bacteria such as E coli E colialso produces a characteristic quotgreen sheenquot Visible better in streak plate on the right Created by ST Blood Agar plate BAP This medium is enriched because of the addition of sheep39s blood It is also differential for hemolysis a characteristic of some pathogens notably the pyogenic cocci Note the complete hemolysis quotbetaquot hemolysis in the lower left corner of the plate on the right Created by ST Production of a red pigment prodigiosin by Serratia marcescens From left to right slant culture grown at 25 C slant culture grownc tegge aqyylgroth culture grown at 25 C broth culture grown at 37 C c aer fat aerotol Growth in thioglycollate medium From left to right are uninoculated control strict aerobe aer facultative anaerobe fac and aerotolerant aerotol The pink color is from the oxygen indicator resazurin r T7 a mug im sn u Created by ST Thioglycollate results left top right Pseudomonas aeruginosa aerobic Staph yococcusaureus facultative Escherichia coli facultative and Clostridium butyricum obligate anaerobe Created by ST Growth of Clostridium in thioglycollate medium left and TSA shake right Note the effect of gas production on the agah Created by ST Anaerobe jar with GasPak system left and candle jar right The GasPak system generates hydrogen gas and carbon dioxide A palladium catalyst is used to combine hydrogen gas and oxyegn to create water thus removing oxygen from the atmosphere The candle jar is used to increase the level of carbon dioxide but is NOT a method of anaerobic culture Created by ST Hydrolysis of Casein milk protein by proteases Positive cultures indicated by arrows Hydrolysis of Gelatin using Frazier39s gelatin medium Plate was ooded with HClHgC12 necessary to precipitate the gelatin which is normally clear and soluble Strongly positive test indicated by arrow Organisms that can break down proteins almost always can digest gelatin Starch hydrolysis amylase test Plate was ooded with iodine which reacts with starch to produce a dark blueblack color Positive test indicated by arrow Not shQMHrIec qwi tFBlue Agar for Lipase Fermentation Sugar fermentation broth reactions Broth contains the pH indicator phenol red which turns yellow due to the production of acid The inverted Durham tube is used to collect gas produced as a result of fermentation Key to symbols neg no fermentation A acid production AG acid and gas AGR acid gas and reduction of the dye Some bacteria can use the dye as an electron acceptor and reduction makes the dye turn colorless Created by ST Key to symbols K alkaline pink A acid production yellow production of H28 gas Results are reported in the order of slantbuttHZS Tube on the far left was inoculated with a strain of Salmonella typhimurium which produces large amounts of H28 WALL1H wme mantra The tube on the left shows an acid slant acid butt with g note arrows but no H28 It would be scored AAG Acid production in both the butt and slant indicates fermentation of both glucose and lactose H Proteus vulgaris demonstrating fermentation of glucose but not lactose deamination of amino acids and H28 production Scored KA Created by ST Methyl RedVoges Proskauer Tests In the methyl red test dye is added to a 48hr culture If the methyl red indicator stays red upon hitting the culture the test is positive A positive test indicates a mixed acid fermentation pathway In the VogesProskauer test the reagents turn red within 15 60 minutes if the test is positive A positive VP test indicates the presenece of acetoin an intermediate in the butanediol fermentation pathway In the above pairs of tubes E coli is in the left tube MRVP and Enterobacter aerogenes is in the right tube MRlVP Created by ST In generala positive MR test is correlated with a negative VP test as shown here with E Q Created by ST Phenol red The tube on the far left which was inoculated with Escherichia coli produces acid and gas It would be read as AG The second tube from the left which was inoculated with Enterococcus faecalis produces acid but no gas It would be read as A The Alcaligenes faecalis degrades peptone and creates an alkaline byproduct It would be read as K The Micrococcus luteus provides no reaction and would be read as Q reated by ST Casease Test When casein is broken down into these component molecules it is no longer white If an organism can break down casein a clear halo will appear around the areas where the organism has grown The Bacillus megaterium on the left is negative for casease production The Bacillus subtilis on the rig rgrggtgpgitiye Gelatinase Test Gelatinase allows the organisms that produce it to break down gelatin into smaller polypeptides peptides and amino acids that can cross the cell membrane and be utilized by the organism When gelatin is broken down it can no longer solidify If an organism can break down gelatin the areas where the organism has grown will remain liquid even if the gelatin is refrigerated The Serratia marcescens on the left is positive for gelatinase production as evidenced by the liquidation of the media The Salmonella typhimurium on the right is negative as evidenced by the solidity of the media The gelatinase test can be used to differentiate between Staphylococcus aureus and Staphylococcus epidermidis It can also be used to differentiate Serratia marcescens Proteus vulgaris and Proteus mirabilis from other enterics Created by ST Lipase Test Lipase allows the organisms that produce it to break down lipids into smaller fragments Triglycerides are composed of glycerol and three fatty acids These get broken apart and may be converted into a variety of endproducts that can be used by the cell in energy production or other processes Tributyrin oil forms an opaque suspension in the agar When an organism produces lipase and breaks down the tributyrin a clear halo surrounds the areas where the lipaseproducing organism has grown The Salmonella typhimurium on the left is negative for lipase production The Pseudomonas aeruginosa on the right is positive for lipase production as evidenced by the clearing surrounding the growth Created by ST MEGATEHIUM Starch Hydrolysis Starch agar is a simple nutritive medium with starch added Since no color change occurs in the medium when organisms hydrolyze starch we add iodine to the plate after incubation Iodine turns blue purple or black depending on the concentration of iodine in the presence of starch A clearing around the bacterial growth indicates that the organism has hydrolyzed starch amusing 5 AG LEICTlAE si b39lwuamaa 339 Iodine has been added to this starch agar plate The zone of clearing surrounding Bacillus megaterium and Bacillus subtilis indicate that both were able to hydrolyze starch Iodine has been added to this starch agar plate The absence of any clearing indicates that neither Streptococcus agalactiae nor Staphylococcus epidermidis were able to Created by ST hydrolyze starch MR Methyl Red When methyl red is added to MRVP broth that has Pee When methyl red is added to MRVP Inoculated With Escherichia broth that has been inoculated with 09 39t Stays red39 quot3 393 a Enterobacter cloacae it turns pOSItIve result for the MR test yellow This is a negative MR result Created by ST When the VP reagents are added to When the VP reagents are added to MR39 MRVP broth that has been inoculated VP broth that has been iHOCUIated With with Escherichia coli the media turns Enterobacter Cloacae the media turns a copper coor This is a negative red This is a positive VP result Created bigsrult for the VP test When Simmons Citrate agar is inoculated with Salmonella typhimurium the medium turns royal blue This is a positive result for the citrate test Citrate Test When Simmons Citrate agar is inoculated with Escherichia coli the medium remains green This is a negative result for the citrate test Created by ST SIM Media Before the Kovac s reagent has been added Streptococcus pyogenes is negative for motility and sulfur reduction Serratia marcescens is positive for motility and negative for sulfur reduction Salmonella typhimurium is positive for motility and for sulfur reduction The tube on the far right has not beggtgaggtgated With the Kovac s reagent added Streptococcus pyogenes is negative for indole production Escherichia coli is positive for indole production Salmonella typhimurium is negative for indole production Proteus vulgars is positive for ing ggoogpgpction Urease Test Urea broth is formulated to test for rapid ureasepositive organisms The restrictive amount of nutrients coupled with the use of pH buffers prevent all but rapid urease positive organisms from producing enough ammonia to turn the phenol red pink Urease broth can be used to differentiate members of the genus Proteus as well as those of Morganela and Providencia but we don t use those in our lab from other enterics The Proteus mirabilis on the left is rapid urease positive as evidenced by the pink color of the media The Escherichia coli on the right is negativezreated by ST Triple Sugar Iron Agar Eterobacter cloacae exhibits fermenation of glucose and gas production but no sulfur reduction This would be read KAG Staphylococcus aureus exhibits acidic fermentation This would be read AA Salmonella typhimurium ferments glucose and reduces sulfur This would be read KA H28 Micrococcus luteus uses the amino acids and does not grow in the butt of the slant This would be ma aMST Bacillus megaterium fermented sugars but did not grow in the anaerobic environment of the butt This would be read ANC Enterobacter aerogenes fermented the sugars but turned to the amino acids This would be read KA Aacid production Kakaine reaction Ggas production H28sulfur reduction Results slantbutt Symbol Interpretation Redyellow KA Glucose fermentation only Peptone catabolized Yellowyellow AA Glucose and lactose andor sucrose fermentation Redred KK No fermentation Peptone catabolized Redno color change KNC No fermentation Peptone used aerobically Yellowyellow with bubbles AAG Glucose and lactose andor sucrose fermentation Gas produced Redyellow with bubbles KAG Glucose fermentation only Gas produced Redyellow with bubbles and black precipitate KAG H28 Glucose fermentation only Gas produced H28 produced Redyellow with black precipitate KIA H28 Glucose fermentation only H28 produced Yellowyellow with black precipitate AA H29 reategluQXseSalnd lactose andor sucrose fermentation H28 produced No changeno change NCNC No fermentation DNase Test DNase agar is a differential medium that tests the ability of an organism to produce an exoenzyme called deoxyribonuclease or DNase that hydrolyzes DNA DNase agar contains nutrients for the bacteria DNA and methyl green as an indicator Methyl green is a cation which binds to the negativelycharged DNA Deoxyribonuclease allows the organisms that produce it to break down DNA into smaller fragments When the DNA is broken down it no longer binds to the methyl green and a clear halo will appear around the areas where the DNaseproducing organism has grown SNEQSSQEVW Q The Staphylococus aureus on the left is negative for DNase production the Serratia marcescens on the right is positive for DNase production as evidenced by the ar fiif cleagrlng around the growth Phenylalanine Deaminase When 10 ferric Chloride is added to When 10 ferric chloride is added to phenylalanine deaminase medium Phenylalanine deaminase medium inoculated with Proteus mirabilis the inocu39ated Witn StaphyOCOCCUS aUFeUS presence of phenylpyruvic acid causes the no color change occurs This is a negative media to turn dark green This is a positive resurf resut Created by ST Blood Agar Blood agar contains general nutrients and 5 sheep blood It is useful for cultivating fastidious organisms and for determining the hemolytic capabilities of an organism Some bacteria produce exoenzymes that lyse red blood cells and degrade hemoglobin these are called hemolysins Bacteria can produce different types of hemolysins Betahemolysin breaks down the red blood cells and hemoglobin completely This leaves a clear zone around the bacterial growth Such results are referred to as Bhemolysis beta hemolysis Alphahemolysin partially breaks down the red blood cells and leaves a greenish color behind This is referred to as dhemolysis alpha hemolysis The greenish color is caused by the presence of biliverdin which is a byproduct of the breakdown of hemoglobin lfthe organism does not produce hemolysins and does not break down the blood cells no clearing will occur This is called yhemolysis gamma hemolysis The hemolysins produced by streptococci perform better in an anaerobic environment Because of this it is standard procedure to streak a blood plate and then stab the loop into the agar to provide an area of I r r I I The Strepooccus aglaCtiae 0n the left eXhibits v Thls Streptococcus pyogenes exh39b39ts 6 Cr igfgelyais SI39ITl39ile the Staphylococcus epidermidis on the right hemolySIs is dhemolytic SHUHEUS Mannitol Salt Agar The Staphylococcus aureus ferments mannitol and turns the medium yellow The Serratia marcescens does not grow because of the high salt content hi i l SNEQESQHEW HGALAET ME smmaamaa s Streptococcus agalactiae does not grow on MSA because of the high salt content Staphylococcus epidermidis grows but Created by ST does not ferment mannitol Phenyethyl Alcohol Agar Phenylethyl alcohol agar PEA is a selective medium used to cultivate Gram positive organisms The active ingredient phenylethyl alcohol inhibits or markedly reduces growth of Gram negative organisms by interfering with DNA synthesis PEA also prevents Proteus species from swarming across the surface of the agar Staphylococcus aureus a Gram positive organism grows on this PEA plate while Serratia mamesoelss a Gram negative organism does not MacConkey Agar MacConkey Agar MAC is a selective and differential medium designed to isolate and differentiate enterics based on their ability to ferment lactose Bile salts and crystal violet inhibit the growth of Gram positive organisms Lactose provides a source of fermentable carbohydrate allowing for differentiation Neutral red is a pH indicator that turns red at a pH below 68 and is colorless at any pH greater than 68 Organisms that ferment lactose and thereby produce an acidic environment will appear pink because of the neutral red turning red Bile salts may also precipitate out of the media surrounding the growth of fermenters because of the change in pH Nonfermenters will produce normallycolored or colorless colonies ll E to a 3 o g a E E t KebS eIa Pneumon a ferment Salmonella typhimurium does not ferment lactose and Pmduces Pmk COIOn39eS lactose and produces colorless colonies 0 MAC M 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