Introductory Microbiology Laboratory
Introductory Microbiology Laboratory MICRB 202
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MICRB 202 Introductory Mcrobiology Lab 1 Unit IX Identification of a Gram Negative Intestinal Pathogen Unit IX Identi cation of a GramNegative Intestinal Pathogen Activities 201 Isolation amp Phenetic ID of Enteric Pathogens EX 26 p 1 221 Molecular ID of Enteric Pathogens EX 27 p 6 251 Enterotube II Multiple Test System EX 28 p 12 Introduction The enteric pathogens of prime medical concern are the salmonella and shigella They cause enteric fevers food poisoning and bacillary dysentery Salmonella typhz39 which causes typhoid fever is by far the most signi cant pathogen of the salmonella group In addition to the typhoid organism there are 10 other distinct salmonella species and over 2200 serotypes The shigella which are the prime causes of human dysentery comprise four species and many serotypes Serotypes within genera are organisms of similar biochemical characteristics that can most easily be differentiated by serological typing testing of the unknown bacterium for reactivity to different antibodies speci c to each serotype Routine testing for the presence of these pathogens is a function of public health laboratories at various governmental levels The isolation of these pathogenic enterics from feces is complicated by the fact that the colon contains a diverse population of bacteria Species of such genera as Escherichia Proteus Enterobacter Pseudomonas and Clostridium exist in large numbers hence it is necessary to use media that are differential and selective to favor the growth of the pathogens In this laboratory unit we will isolate putative intestinal pathogens and other enteric bacteria on differential and selective media and proceed to con rm the isolate to be a pathogenic species using two different identi cation ID procedures Identi cation of bacteria has traditionally relied on phenetic characters such as morphological features and metabolic or physiological potential The later characters can be assessed rapidly using miniaturized multipletest kit systems such as the Enterotube II designed for identifying species of the P 39 iaceae 39 39 39 39 1 provide alternate approaches to the identi cation of unknown bacteria Here we will PCR amplify the 16SrRNA gene from our putative isolates and compare its 16SrDNA restriction fragment length polymorphism RFLP pattern to a set of known patterns for identi cation 201 Isolation amp Phenetic ID of Enteric Pathogens EX 26 Identi cation of unknown enteric bacteria to genus will follow a stande separation outline procedure used to demonstrate the presence of salmonella or shigella in a patient s blood urine or feces Figure 261 Note that lactose fermentation separates the salmonella and shigella from most of the other Enterobacteriaceae Final differentiation of these two enteric pathogens from Proteus relies on motility hydrogen sul de production and urea hydrolysis Differentiation of the positive lactose fermenters on the left side of the separation outline is used for the identi cation of enteric coliform bacteria some genera of which can include pathogen strains like Escherichia call 0157 MICRB 202 Introductory Microbiology Lab Unit IX Identification of a Gram Negative Intestinal Pathogen Lactose lactose 39 L i Lactose ndole nd39oleH W Glucose Glucoser I v v Pseudomonas Citrate Citrate I Uea quot Urea Motile Nonmoiile Aloegenes H2sr Citrobacrer 39 Escherichia K bsie lla Enterobacter shigella 39 Urea Urea r Proteus PrOvidencia Salmonsa MorganaIa Figure 261 Separation outline for genus level identi cation of the Enterobacteriaceae In this experiment you will be given a mixed culture containing a coliform and Proteus and salmonella or shigella The pathogens will be of the less dangerous types but their presence will naturally demand utmost caution in handling Your problem will be to isolate the pathogen from the mixed culture and make genus identi cation There are ve steps that are used to prove the presence of these pathogens in a stool sample 1 enrichment 2 isolation 3 fermentation tests 4 nal physiological tests Figure 262 and 5 serotyping We will not perform the latter Enrichment from stool There are two enrichment media that are most frequently used to inhibit the nonpathogens and favor the growth of pathogenic enterics They are selenite F and gram negative GN broths While most salmonella grow unrestricted in these two media some of the shigella are inhibited to some extent in selenite F broth thus for shigella isolation GN broth is preferred In many cases stool samples are plated directly on isolation media In actual practice 1 to 5 grams of feces are placed in 10 ml of enrichment broth In addition plates of various kinds of selective media are inoculated directly The broths are usually incubated for 4 to 6 hours Because we are not using stool samples in this exercise the enrichment procedure is omitted Instead you will streak the agar isolation media directly from the unknown broth culture and broth cultures of other unknowns you would like to work with Isolation of coliform and noncoliform enterics There are several excellent selective differential media that have been developed for the isolation of coliform and noncoliform enteric pathogens Various inhibiting agents such as brilliant green bismuth sul te sodium desoxycholate MICRB 202 Introductory Mcrobiology Lab 3 Unit IX Identification of a Gram Negative Intestinal Pathogen and sodium citrate are included in them For Salmonella typhz39 bismuth sul te agar or Salmonella Shigella SS agar appears to be the best media Colonies of S tpr on these media appear black due to the reduction of sul te to sul de Other widely used media are ENDO agar MacConkey agar Hektoen Enteric HE agar and Xylose Lysine Desoxycholate XLD agar These media may contain bile salts andor sodium desoxycholate to inhibit grampositive bacteria Some like SS agar contain citrate to inhibit coliforms and other nonenterics All of them contain lactose and a dye so that if an organism is a lactose fermenter putative coliform its colony will take on a color characteristic of the dye present Recall the dark colonies with greenmetallic sheen grown on ENDO agar are likely to be coliform enteric bacteria Once colonies are isolated we will proceed with fermentation tests Fermentation tests Recall the fermentation characteristic that separates the SS pathogens from the coliforms is their ability to ferment lactose Once we have isolated colonies on differential media that look like coliforms and those that look like salmonella or shigella the next step is to determine whether these isolates can ferment lactose All media for this purpose contain at least two sugars glucose and lactose Some contain a third sugar sucrose They also contain phenol red to indicate when fermentation occurs the indicator turns yellow under acid conditions created by sugar fermentation Russell s Double Sugar RDS agar is one such media Final Mu 39 39 39 39 and Iquot 39 39 39 39 Tests MotilityHzSindole production and urease activity will be accessed for each isolate using SIM medium and urea broth respectively Also citrate and tryptophan utilization will be determined to differentiate between different lactosefermenting coliform bacteria Figure 261 Any salmonella will be a nonlactosefermenter that exhibits motility produces hydrogen sul de yet cannot produce urease Any Shigella will be a nonlactose fermenter with urease activity but is nonmotile and cannot produce hydrogen sul de Figure 261 You will also be making a gramstained slide to perform a purity check on your isolates We will also inoculate Entrotube II multitest kits during the third week of this unit see Exercise 28 below Exercise 26 Isolation amp Penetic ID Materials unknown broth cultures one a mixture of a coliform proteus and a salmonella or shigella Petri plates of different selective media ENDO and SS agars Slants of Russell Double Sugar RDS agar 1 tube of SIM medium for each SS pathogen 1 tube of urea broth for each SS pathogen or coliform Gram Stain Kits 1 stab tube of Simon Citrate agar for each coliform Kovacs reagent and chloroform for Indole testing ie tryptophan utilization 5ml pipettes MICRB 202 Introductory Microbiology Lab Unit IX Identification of a GramNegative Intestinal Pathogen SELECTIVE MED UM such as MacConkey HE or XLD agar ENHICHMENT 37 C 18 24 hr red slants ll Kligler iron agar slants have a black precipitate H25 is produced NO GLUCOSE GLUCOSE Slants of ROS or Kligler iron agar FERMENTATION ONLY AND LACTOSE are streaked and stabbed from typical Salmonella and Shigella colonies Tubes of urea broth and SIM medium are inoculated rom each u that exhibits glucose fermentation SIM medium is stabbed to 23 at depth of medium 0th media are incubated at 37 C for 18 to 24 hours GRAM STAINED SLIDE is made to see if culture is pure Serotyping is generally necessary UFIEA SIM BROTH MEDIUM Figure 262 Protocol for SS pathogen isolation We will add steps to this for coliform isolation MICRB 202 Introductory Mcrobiology Lab 5 Unit IX Identification of a Gram Negative Intestinal Pathogen Procedure 1 Fore each unknown label plates of each media type with your name and unknown number or code 2 With a loop streak each plate with your unknown in a manner that will produce good isolation Incubate the plates at 37 C for 24 to 48 hours ltlt EX 26 iDay 2 gtgt For each unknown and media inoculate three slants using three different colonies on the selective media that look like either salmonella or shigella or like a coliform The reason for using three slants is that you may have difficulty distinguishing Proteus from the SS pathogens By inoculating three tubes from different colonies you will be increasing your chances of success 3 For each unknown label the three slants with your name and the number of your unknown 4 Look for isolated colonies that look like salmonella or shigella organisms and those that look like coliforms With a straight wire inoculate the three agar slants from separate SSappearing colonies Use the streakstab technique When streaking the surface of the slant before stabbing move the wire over the entire surface for good coverage V39 Repeat step 3 for any putative coliform isolates 0 Incubate the slants at 37 C for 18 to 24 hours Longer incubation time may cause alkaline reversion Even refrigeration beyond this time may cause reversion Alkaline reversion is a condition in which the medium turns yellow during the first part of the incubation period and then changes to red later due to increased alkalinity by ammonia produced as proteins degrade ltltEX267Day3 gt 7 Examine the slants from the previous period to separate possible coliforms and possible SS pathogens for further testing Figure 262 Select those tubes that have a yellow butt with a red slant as possible SS pathogens These tubes contain organisms that ferment only glucose nonlactose fermenters Slants that are completely yellow are able to ferment lactose as well as glucose and considered coliforms Tubes that are completely red are either nonfermenters or examples of alkaline reversion Ignore the later tubes 8 With a loop inoculate one tube of urea broth from each coliform and each possible SS pathogen 9 With a straight wire stab one tube of SIM medium from each coliform and each possible SS pathogen Stab in the center to twothirds of depth of medium 10 With a straight wire stab the Simmon s Citrate agar for each coliform using the technique in 9 ll Incubate all ofthese tubes at 37 C for 18 to 24 hours 12 Make gramstained slides from the same slants and confirm the presence of gramnegative rods MICRB 202 Introductory Mcrobiology Lab 6 Unit IX Identification of a Gram Negative Intestinal Pathogen ltlt Ex 26 iDay 4 gtgt 13 Examine the tubes of SIM medium checking for motility and HzS production If you see cloudiness spreading from the point of inoculation the organism is motile A black precipitate will be evidence of HzS production 15 Test for indole production by pipetting 2 ml of chloroform into each SIM tube and then adding 2 ml of Kovacs reagent A pink to deep red color will form in the chloroform layer if indole is produced Salmonella are negative Some Shigella may be positive Citrobacter and Escherichia are positive 16 Examine the urea broth tubes If the medium has changed from yellow to red color the organism is ureasepositive 17 Examine the Simmon Citrate tubes If they have turned from green to blue they are positive for citrate utilization 18 Summarize all of your results in a table and use the identi cation outline Figure 261 to determine the genera of your unknowns 7 record 19 Address all questions and discussion points presented at the end of Exercise 28 221 Molecular ID of Enteric Pathogens Ex 27 Another approach to identify bacteria is based on the Restriction Fragment Length Polymorphism RFLP of 16SrRNA genes 16SrDNA its results in a type of DNA ngerprint The secondary structure of the 16SrRNA Figure 271 is highly conserved and functions well as a molecular chronometer For this approach DNA is extracted from a bacterial culture and the 16SrRNA gene is ampli ed via the Polymerase Chain Reaction PCR of partial or entire PCR ampli cation of the 16srRNA gene is possible with more information gained by having more of the molecule Restriction endonucleases are then used to cleave the gene into fragments of different sizes Tetrameric restriction endonucleases are preferred because 4 nucleotide target sequences permit a greater number of cuts and are more sensitive to variation among highly conserved rRNA gene sequences than restriction enzymes with a greater number of target nucleotides Typically 2 to 3 enzymes are used in separate digests of the same sample to obtain adequate information for identi cation at the species level Patterns are compared with known patterns based on the computerized database or determined empirically MICRB 202 Introductory Microbiology Lab 7 Unit IX Identification of a GramNegative Intestinal Pathogen Figure 271 Secondary structure of the 1542 base pair 16S rRNA of Escherichia 001139 For our project we will identify an unknown bacterium from a pure culture using the 16S rDNA RFLP strategy outlined above We will begin by extracting the bacterial genomic DNA to use as template for PCR ampli cation of the 16S rRNA gene As PCR primers we will use an oligonucleotide sequence equal that beginning at position 68 on the sense strand forward primer 68F 5 TNANACATGCAAGTCGAKCG3 and the complement sequence of the sense strand ending at position 1406R reverse primer 1404R39 5 ACGGGCGGTGTGTRC3 Figure 272 Therefore our PCR product will be 1338 base pairs or about 200 base pairs short ofthe complete 16S rRNA gene We will select two tetrameric restriction enzymes for digesting the PCR product after its precipitation and separate the fragments on a 3 agarose gel To identify your unknown RFLPs will be compared with a selection of results from known species provided by your instructor 68 1406 5 l i sense strand J 3 16SrRNA l 3 39 5 Figure 272 Orientation of primers from position 68 forward 68F and from position 1406 in reverse 1406R relative to the sense strand DNA Dark arrows represent primers next to their complement target arrow point is the 3 end of primer where DNA elongation will proceed MICRB 202 Introductory Mcrobiology Lab 8 Unit IX Identification of a Gram Negative Intestinal Pathogen Exercise 27 The exercise is divided into four separate protocols 1 extraction of genomic DNA from a bacterial culture 2 PCR ampli cation of the 16SrRNA gene 3 Restriction enzyme digest of amplicons and 4 agarose gel electrophoresis Extraction of Genomic DNA from Bacteria Materials TSB cultures of bacterial unknowns Lysozyme Buffer 50mgml Lysozyme in 10 mM Tris 1 mM EDTA at pH 80 GITC Buffer 5M guanidinium isothiocyanate 100 mM EDTA 05 sarkocyl detergent 75 N NH4Ac ammonium acetate ChloroformPentanol 241 ratio Isopropanol 70 EtOH Eppendorf tubes Sterile DIwater Micropipetters and appropriate sterile tips Procedure We will need to share the microcentrifuge at each step 1 Place 15 ml of bacterial culture in a well labelled eppendorf tube Spin down the bacteria in the microcentrifuge for 1 min at 13000 rpm Always orient your eppendmf tube in the microcentifuge with the hingeside outwards this will be the side wh ere your pellet forms Pour off the supernatant into the red wasteautoclave bag Use the 10100 microliter pipetter to remove the remainder of the supernatant 2 Add 100 microliters of Lysozyme Buffer to the bacterial pellet and use the broader tip of the toothpick to mix the suspension by twirling the toothpick between your thumb and fore nger Place the suspension in the heating block at 37 C for 30 min 3 Add 50 microliters of GITC Buffer to the eppendorf tube and vortex the suspension Place the eppendorf tube on ice for 10 min 4 Add 250 microliters of icecold 75 N NH4Ac ammonium acetate to the eppendorf tube and mix Return the tube to ice for 10 min 5 Add 500 microliters of ChloroformPentanol 241 ratio to the eppendorf and vortex the tube 6 Spin the eppendorf tube for 5 min at 13000 rpm If the supernatant is still visibly turbid then spin for another 5 min interval 7 Carefully remove the upper phase from the eppendorf tube and place this in a new labelled eppendorf tube MICRB 202 Introductory Mcrobiology Lab 9 Unit IX Identification of a Gram Negative Intestinal Pathogen 8 Add 540 microliters of icecold Isopropanol to the upper phase and mix Spin the eppendorf tube for 10 min at 10000 rpm 9 Carefully pour off the supernatant into the waste bag Add 1 ml of 70 icecold EtOH ethanol to the eppendorftube and mix Spin the tube for 10 min at 13000 rpm 10 Carefully pour off the supernatant and use a 10 100 microliter pipetter to remove the remaining residual 70 EtOH at the bottom of the tube Be careful to pipet from the side opposite the DNA pellet ie opposite the tube hinge which may or may not be visible Place the tube upside down on KimWipe and leave this on your bench for 20 min 11 Resuspend the Genomic DNA by slowly washing the side of the eppendorf tube with 50 microliters of TE Buffer 10 mM Tris 1 mM EDTA at pH 80 Have faith that your DNA is there because you probably won t see it This DNA suspension is will be used as template for ampli cation of the 16SrRNA gene via PCR polymerase chain reaction PCR Ampli cation of the 16SrRNA Gene ltlt Ex 27 Day 2 gtgt Materials PCR tubes Genomic DNA Reaction Master Mix see below Procedure IMPORTANT NOTE Because you are amplifying bacterial DNA there is a need for extremely sterile technique both lab space and handling Wear gloves use only sterile pipetter keep eppendorf lids closed as much as possible and NEVER use a pipetter tip more than once for any solution transfer 1 Label a sterile PCR tube for each genomic DNA sample in your group 2 Add 48 micrliters of PCR Master Mix to the tube this represents the following 39 microliters of sterile deionized H20 5 microliters of 10X PCR Buffer 1 microliters of 10 mM dNTP mix of 4 nucleotides 1 microliters of 68F forward primer N100 ng 1 microliters of 1406R reverse primer N100 ng 05 microliters of Tth Polymerase 3 Add 2 microliters of template genomic DNA With a new pipette tip on your 10100 microliter pipetter slowly pipett the 50 microliters of PCR reaction cocktail up and down to thoroughly mix reagents 4 Place it in the PCR machine technically caller a thermocycler 5 The temperature program used preset for you is as follows MICRB 202 Introductory Mcrobiology Lab 10 Unit IX Identification of a Gram Negative Intestinal Pathogen a 94 C for 1 min denatures double stranded DNA template b 60 C for 1 min cooling permits the selective annealing of primers with target sequences of the template DNA c 72 C 4 min activates the Tth Polymerase to polymerize proceeding from the 3 end of the annealed primers a DNA strand complimentary to the template strand 4 d Return to step a Repeat the cycle 30 times after which the temperature is maintain at 4 C Next time we meet we will digest the ampli ed PCR product with a selection of tetrameric restriction endonucleases and separate the digested fragments by size via agarose gel electrophoresis Restriction Enzyme Digestion of PCR Amplicon ltlt Ex 27 Day 3 gtgt This period we will continue with the molecular identi cation of our unknown bacterium by use of molecular techniques speci cally the restriction fragment length polymorphism RFLP pattern of the PCR ampli ed 16S rRNA our amplicon First digest your amplicon with tetrameric restriction endonucleases ie restriction enzymes with 4bp recognition sites Materials PCR tubes Sterile DIwater PCR reaction for amplicons DNA Selected restriction enzymes and their respective buffers 10x Glycerol Loading Buffer Procedure 1 Label a 2 new 05 microliter eppendorf tube one for each of 2 restriction enzymes used details will be provided Add in order the following to each tube 12 microliters of sterile deO 2 microliters of 10X restriction enzyme buffer 4 microliters of precipitated PCR product 2 microliters of the appropriate restriction enzyme 2 Mix the contents of the tubes by slowly pipetting up and down and place the tubes into the 37 C heating bloack or thermocycler for 30 min Periodically give the tubes a mix and return them to the heating block 3 After the reactions have been incubated remove them from the heating block and add 2 microliters of 10x GlycerolLoading Buffer GLB to each tube 4 An undigested sample of PCR product will need to be prepared for running on the agarose gel along side of the digests To a new eppendorf tube add the following MICRB 202 Introductory Mcrobiology Lab 11 Unit IX Identification of a Gram Negative Intestinal Pathogen 2 microliters PCR product 7 microliters sterile deO 1 microliters of 10X GLB Agarose Gel Electrophoresis to Determine RFLP pattern ltlt ltlt EX 27 Day 3 gtgtgt Materials 3 agarose gel with Ethidium Bromide EtBr staining solution incorporated 05x TAE electrophoresis buffer Horizontal electrophoresis apparatus and power supply UV light Transluminator and photodocumentation equipment Marker DNA Procedure 1 You will share a 3 agarose gel and horizontal gel apparatus with other groups Therefore all samples and standard marker DNA need to be loaded within a reasonable time frame to avoid diffusive loss from the wells 2 Draw your entire sample into the tip of a pipetter but do not create an air space between the tip point and the sample within the tip If you have an air bubble there it will dislodge your sample out of the well as you attempt to load 3 Keep your pipetter tilted at an angle so you can view what is going on during loading sample into the gel well Lower the pipette tip to just inside the side of the next empty well 4 Slowly expel the tip contents It will sink to the bottom of the well due to the density of the sucroseloading buffer Make sure not to introduce any air bubbles as the last bit of sample leaves the pipette tip I usually sacri ce the last microliter of sample to avoid bubbles and sample loss Certainly never push the pipettes plunger to its second stop when loading gels 5 When all samples have been loaded close the cover and insert the electrodes matching blackwith black and redwithred Turn on the voltage We will run at 50 Volts for 1 hour in 05X Tris Acetate EDTA TAE Buffer We have already added Ethidium Bromide 1 ugml to the gel during its casting 6 After electrophoresis remove the gel and photodocument while transilluminating with UV light Each student will receive a picture of their gel It will look similar to that in Figure 273 7 Compare your results to the known database provided by the instructor RB 202 Introductory Microbiology Lab Unit IX Identi cation of a GramNegative Intestinal Pathogen Figure 273 Restriction fragment length polymorphism for a 1338 bp PCR for the E coli loSrRNA es 2 3 4 and 5 are restriction enzymes digests using HaeIII HpaII CfoI and RsaI respectively Lanes 1 and 6 are molecular size markers 251 Enterotube II Multiple Test System Ex 28 The Enterotube II miniaturized multitest system is supplied by BectonDickinson of Cockeysville Maryland for rapid identi cation of Enterobacteriaceae It incorporates twelve different conventional media and een biochemical tests into a single readyto use tube that can be simultaneously inoculated in a moment39s time with a minimum of equipment If you have an unknown negative rod or coccobacillus that appears to be one of the Enterobacteriaceae you may wish to this system on it Before applying this test however make certain that your unknown is oxidase negative since very few of the Enterobacteriaceae are oxidasepositive Each of the 12 compartments of an Enterotube 11 contains a different agarbased medium Compartments that require aerobic conditions have openings for access to air Those compartments that require anaerobic conditions have layers of paraf n wax over the media Extending through all compartments of the entire tube is an inoculating wire To inoculate the media one simply picks up some organisms on the end of the wire and pulls the wire through each of the chambers in a single rotating action Figure 281 A er incubation the indole and VogesProskauer tests must be performed and the reactions in all compartments are noted Figure 302 Positive reactions are given numerical values which are totaledto arrive at a vedigit code Figure 301 Identi cation ofthe unknown is achieved by consulting a booklet the Enterotube II Interpretation Guide which lists these numerical codes for the Enterobacteriaceae MICRB 202 Introductory Microbiology Lab Unit IX Identification of a GramNegative Intestinal Pathogen prevent damaging media do not heatsterilize the 1 Remove organisms trom a wellisolated colony To inoculating wxre inoculate each compartment twice by pulling wire all the way out and then forcing it back through all the chambers again Wire should be rotated as it passes through H28 compartment At this point break off the wire 3 Withdraw the wire a second time until tip is in the at a notch in the wire Discard broken and After replacing the caps at both ends strip off the blue plastic tape that covers holes on eight chambers Holes allow for aerobic growth After incubation at 35 37 C for 24 hours record results with or on side of tube and perform lndole and VogesProskauer tests Record results before doing two tests 6 Enclrcle numbers of positive tests on tabulatth form Lab Report and total up the numbers of each bracketed series to determine 5dlglt code number See Enterotube II Interpretation Guide Figure 281 Enterotube II inoculation and analysis protocol MICRB 202 Introductory Mcrobiology Lab 14 Unit IX Identification of a Gram Negative Intestinal Pathogen Exercise 28 Enterotube 11 Materials Culture plates of 2 unknowns Enterotube II for each of two unknowns Kovacs reagent for indole test VP reagents 10 KOH with 03 creatine solution 5 alphanaphthol in absolute ethyl alcohol disposable Pasteur pipettes testtube rack Procedure Figure 281 1 Write your initials or unknown number on the white paper label on the side of the tube above VP 2 Unscrew both caps from the Enterotube II 3 Without heatsterilizing the exposed inoculating wire insert it into a wellisolated colony 4 Inoculate each chamber by rst twisting the wire and then withdrawing it through all 12 compartments Rotate the wire as you pull it through Again without sterilizing reinsert the wire and with a turning motion force it back through all 12 compartments 6 Withdraw the wire again until the tip is in the H2Sindole compartment At this point break the wire by bending and discard the handle end of the wire A notch in the wire makes this relatively easy Leaving the wire in the last three chambers excludes oxygen from the three paraffin covered chambers GLUGAS LYS ORN The fermentation of glucose and the decarboxylation of lysine and omithine occur anaerobically The small portion of wire protruding into the H2Sindole compartment will not interfere with these two tests 7 Replace the caps at both ends 8 To expose some small holes in the sides of eight of the compartments strip off the blue plastic strip If no blue plastic strip is present the punch holes using the sterile wire These holes provide aerobic conditions for the following compartments ADON LAC ARAB SORB VP DULPA UREA and CIT 9 Incubate at 300 to 370 C for 18 to 24 hours with the Enterotube II lying on its at surface When incubating several tubes together allow space between them to allow for air circulation MICRB 202 Introductory Mcrobiology Lab 15 Unit IX Identification of a Gram Negative Intestinal Pathogen ltlt EX 28 Day 2 gtgt 10 Record results as positive or negative Figure 282 for all reactions except the VP and Indole 11 Add Kovac s reagent for indole production record result 12 Add reagents for VP reaction record results 13 Interpret the results as indicated by the instructor This may involve computation of code numbers and comparison to a known database or by more conventional mean by comparing each test result to tabular data for various Enterobacteriaceae 14 Address all questions and discussion points presented at the end of Exercise 28 UNINOCULATED REACTED SYMBOL COLOR C TYPE OF REACTION Glucose GLU The end products of bacterial fermentation of glucose are either acid or acid and gas The shift in pH due to the production of acid is indicated by a color change from red alkaline to yellow acidic Any degree of yellow should be interpreted as a positive reaction orange should be considered nega ive GLUGAS Gas Production GAS Complete separation of the wax overlay from the surface of the glucose medium occurs when gas is produce The amount of separation between the medium and overlay will vary with the strain of bacteria OLOR 39 a Lysine Decarboxylase Bacterial decarboxylation of lysine which results in the formation of the alkaline end product cadaverine is indicated b a change in the color of the indi cator from pale yellow acidic to purple alkaline Any degree of purple should be interprete as a positive reaction The medium remains yellow if decarboxylation of lysine does not occur acidic yellow nature of the medium is converted to purple as alkalinity occurs Any degree of purple should be interpreted as a positive reaction The medium remains yellow if decarboxyla tion of ornithine does not occur H28 Production Hydrogen sulfide liberated by bacteria that reduce sulfurcontaining compounds such as peptones and sodium thiosulfate reacts with the iron salts in the medium to form a black precipitate of ferric sulfide usually along the line of inoculation Some Proteus and Providencia strains may pro duce a diffuse Drown coloration in this medium which should D HZSlN not be confused with true HES production ORN I lndole Formation The production of indole from the metab olism of tryptophan by the bacterial enzyme tryptophanase is detected by the development of a pink to red color after the addition of Kovaos39 reagent Figure 282 Description of Enterotube II reaction compartment tests and appearance of negative and positive results for each test MICRB 202 Introductory Mcrobiology Lab Unit IX Identification of a Gram Negative Intestinal Pathogen UNINOCULATED REACTED SYMBOL COLOR 0 on TYPE OF REACTION 7 ADON Adonitol Bacterial fermentation oi adonitol which results in the formation of acidic end products is indicate by a change in color of the indicator present in the med39um from red alkaline to yellow acidic Any sign of yellow should be inter re e as a positive reaction orange should be considere negative Lactose Bacterial fermentation of lactose which results in the formation of acidic and products is indicated by a c ange in color of the indicator present in the medium from red alkaline to yellow acidic Any sign of yellow should be interpreted as a positive reaction orange should be considered negative ARAB Arabinose Bacterial fermentation of arabinose which results in the formation of acidic end products is indicated by a change in color from red alkaline to yellow acidic Any sign of yellow should be interpreted as a positive reaction orange should be considered nega ive SORB Sorbitol Bacterial fermentation of sorbitol which results in the formation of acidic end pro ucts is indicated by a change in color from red alkaline to yellow acidic Any sign of yellow should be interpreted as a positive reaction orange should be considered negative VP Voges Proskauer Acetylmethylcarbinol acetoln is an inter mediate in the production oi buty ene glycol from glucose fer mentation The presence of acetoin is indicated by the develop ment of a red color within 20 minutes Most positive reactions are evident within 10 minutes DULPA Dulcitol Bacterial fermentation of dulcitol which results in the formation of acidic en ts is indicate y a change in color oi the indicator present in the medium from green alkaline to yellow or pale yellow acidic Phenylalanine Deamiuase This test detects the formation oi pyruvic acid from the deamination of phenylalanine The pyruvic acid formed reacts with a ferric salt in the medium to produce a characteristic black to smoky gray color UREA Urea The production of urease by some bacteria hydrolyzes urea in this medium to produce ammonia which causes a shift in pH from yellow acidic to reddish purple alkaline This test is strongly positive for Proteus in 6 hours and weakly positive tor Klebsiella and some Enterabacter species in 24 hours Citrate Organisms that are able to utilize the citrate in this medium as their sole source of carbon pro uce alkaline metabo lites which change the color of the indicator irom green acidic to deep blue alkaline Any degree of blue should be considered positive Figure 282 Continued MICRB 202 Introductory Mcrobiology Lab 17 Unit IX Identification of a Gram Negative Intestinal Pathogen Results Assignment and Discussion 1 Create a standard curve of log marker band size versus migration distance and provide the equation for this curve and the r2 value for the linear regression Use this equation to determine the size of each band in your digested sample 2 Use the undigested sample to con rm the presence of target PCR product 1340 bp and any nonspeci c PCR products Also use the information from the undigested sample to determine any undigested or extra nonspeci c DNA bands in your 2 digested samples Report any aberrant results and discuss possible sources 3 Use your RFLP data and the RFLP data for known bacterial species provided by the instructor to propose the identity of your unknown 4 How does this approach compare with phenetic approaches used in Exercises 26 amp 28 Which would you use under what circumstances and why 5 How might the phenetic and molecular phyletic approaches compliment each other in learning about a newly isolated bacterium for which there is no known species
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