Isolating and identifying microorganisms is very important
- 2. 1 Introduction: Isolating and identifying microorganisms is very important. There are many ways to determine the differences in microorganisms. They can be determined by their shape and staining properties, as well as the way they are grouped. They can also be determined by their reactions to different biochemical testing. Using these methods can be very important in discovering the type of organism present. Understanding the procedure in isolating and identifying bacteria can help in many different fields to include medical and biological research fields. The medical field can use identification to assure proper treatments are given to patients. Also “many of these tests are designed to identify Gram-negative organisms, since there is a larger percentage of pathogen” (Kerr & McHale, 2003) that are Gram-negative. The rise in antibiotic resistance makes this even more important. In biological research, soil and water microbiology play important parts in our everyday lives and in many discoveries. Some of these discoveries have led to new antibiotics in our constant battle to keep bacteria at bay within our bodies. Morphologies of bacteria play an important role in isolation and identification. The ability to determine different morphologies is the first step in isolation. This is followed by gram staining to determine if you have a gram positive or gram negative bacterium. The ability to determine these things help lead to biochemical tests which will further identify the bacteria present. Methods and Materials:
- 3. 2 Gram Positive Mannitol Fermentation Fermentation ofMannitol Staphylcoccus aureas s No Fermemtnation ofMannitol Staphylococcus epidermidis Streptococcus agalactiae Streptococcus salivarius Micrococcus luteus Bacillus subtilis Starch Agar Amalayse production Streptococcus salivarius Bacillus subtilis No Amylase Production Staphylococcus epidermidis Streptococcus agalactiae Micrococcus luteus Nitrate Reduction No Nitrate Reduction Streptococcus salivarius NO3 to NO2 Bacillus subtilis Urea Urease Production Staphylococcus Epidermidis No Urease Production Streptococcus agalactiae Micrococcus luteus TSI Glucose Fermentation only Micrococcus luteus GlucoseandLactosefermentation Streptococcus agalactiae Confirmation Glucose and Lactose Fermentation tubes
- 4. 3 Gram Negative Baird Parker Growth, brown colonies, no clearing Proteus vulgaris No growth Alcaligenes faecalis Enterobacter aerogenes Escherichia coli Psuedomonas aeruginosa Salmonella typhimurium Serratia marcescens Shigella flexneri Oxidative-Fermentation Metabolism Fermentation Enterobacter aerogenes Escherichia coli Salmonella typhimurium Serratia marcescens Shigella flexneri Oxidative Psuedomonas aeruginosa Non saccharolytic Alcaligenes faecalis Phenylalanine Slant Phenylalanine catabolism by phenylalanine deaminase Enterobacter aerogenes Confirmation Glucose fermentation and Lactose fermentation No catabolism Escherichia coli Salmonella typhimurium Serratia marcescens Shigella flexneri Sucrose fermentation Ferments Sucrose Escherichia coli Serratia marcescens No fermentation Salmonella typhimurium Shigella flexneri Citrate Citrate Utilizes Citrate Salmonella typhimurium Does not utilize citrate Shigella flexneri Does not utilize Citrate Escherichiacoli Utilizes Citrate Serratia marcescens
- 5. 4 Results: UNKNOWNS RESULTS FORM Student:Christine Kelly Unknown#: 3 Instructor: Dr. Paz Identification:Streptococcusagalactiae MORPHOLOGICAL CHARACTERISTICS Cell Shape &Arrangement:Coccusinchains ColonyGrowthon TSAYE: Small,creamandcircular Gram’s Reaction:Gram positive reaction Special Stains(optional): IsolationTemperature: 25C PHYSIOLOGICAL CHARACTERISTICS Media 1: Mannitol Fermentation Observations There was no change in the color of the tube and no gas present. Interpretation There was no fermentation of mannitol sugar. Staphylococcus aureas ruled out IncubationTemperature/Time 25 C / 48 hours Usedas a confirmationtest? No Media 2: Amylase Observations There was no growth on the plate and no clearing. Interpretation No Amylase was produced. Streptococcus salivarius and Bacillus subtilis ruled out IncubationTemperature/Time 25 C/ 48 hours Usedas a confirmationtest? No Media 3: Observations Interpretation
- 6. 5 Urea The tube remained yellow in color. There was no production of urease. Staphylococcus epidermidis ruled out IncubationTemperature/Time 25 C / 48 hours Usedas a confirmationtest? No Media 4: TSI Observations Both the slant and the bottom of the tube turned yellow. Interpretation Lactose and glucose were both fermented. Indicates Streptococcus agalactiae is the unknown. IncubationTemperature/Time 25 C / 48 Hours Usedas a confirmationtest? No Media 5: Glucose Fermentation Observations The tube changed from a blue to a more greenish color. Interpretation Glucose fermentation occurred. IncubationTemperature/Time 25 C/ 48 hours Usedas a confirmationtest? Yes
- 7. 6 UNKNOWNS RESULTS FORM Student: Christine Kelly Unknown#: 3 Instructor: Dr. Paz Identification:Enterobacteraerogenes MORPHOLOGICAL CHARACTERISTICS Cell Shape &Arrangement:bacilli,chainedtogether ColonyGrowthon TSAYE: small,circular,cream Gram’s Reaction:Gram Negative Special Stains(optional): IsolationTemperature:25C PHYSIOLOGICAL CHARACTERISTICS Media 1: Baird Parker Observations There was no growth observed on the plate Interpretation No coagulase, no tellurite reduction. Proteus vulgaris ruled out IncubationTemperature/Time 25 C / 48 Hours Usedas a confirmationtest? No Media 2: Oxidative Fermentation metabolism Observations Both tubes yellowed Interpretation Fermentationtookplace inboth aerobic and anaerobic tubes. Pseudomonas aeruginosa and Alcaligenes faecalis ruled out. IncubationTemperature/Time 25 C / 48 hours Usedas a confirmationtest? No Media 3:Phenylalanine Slant Observations The tube showed growth, and greening. Interpretation The presence of green mean Phenylalanine was catabolized by Phenylalanine deaminase. IncubationTemperature/Time 25 C / 48 hours
- 8. 7 Usedas a confirmationtest? Yes or No Indicates Enterobacter aerogenes as the gram negative bacteria Media4: Glucose Fermentation Observations The tube turned very yellow and had gas. Interpretation Glucose was fermented confirming Enterobacter aerogenes IncubationTemperature/Time 25 C / 48 hours Usedas a confirmationtest? Yes Media5: Lactose Fermentation Observations This tube was yellow and had the presence of gas Interpretation Lactose was fermented confirming Enterobacter aerogenes IncubationTemperature/Time 24 C / 48 hours Usedas a confirmationtest? Yes Discussion: Isolatingthe organismsthere wasaproblemgettingthe gramnegative organismtoseparate appropriately.There were noproblemsisolatingthe grampositive organism. Bothorganismswere viewedunderthe microscope.The grampositive organismwasobserved tobe coccus and inchains indicatingitwouldbe aStreptococcusspecies. The flow chart for the gram positive organismwasmade priorto the gram staining. The gram positive flowchartwaschosento gaina direct way to identificationwithoutusing manytests. There were no issueswiththe tests. Aftergram-stainingitwasknowntobe a Streptococcusspecies,sothe testsperformedasexpectedgiventhe resultof Streptococcusagalactiae.
- 9. 8 The gram negative bacteriawere hardertoisolate.Thismayhave beenbecause the morphologiesonboththe gram positive andgramnegative bacteriawere veryclose inappearance. Once separatedthe gram negative bacteriabecame muchmore cooperative. The testsproceededas expected.Againthe shortestpathtoidentificationwasused. The gramnegative flow chartstartedwith eliminatingone possibilitywiththe BairdParkerplate.The oxidativefermentationmetabolismwould have eliminatedtwoasthe bacteria.There wasno needtogo past the phenylalanineslantdue tothe positive resultof the bacteriumcatabolizingphenylalaninebyphenylalaninedeaminase. The catabolism of phenylalanine identifiedthe bacteriumas Enterobacteraerogenes. The knowledge gainedinperforming thesetestscanbe carriedon to manydifferentfields involvingmicrobiologytoincludethe medical professionaswellasresearchbasedprofessions. The understandingof gramstainingandbiochemicaltestinginisolationandidentificationare reallyrequired knowledge foranyone workinginthe microbiologyfield.
- 10. 9 Works Cited Kerr,T. J., & McHale, B. B. (2003). Applicationsin General Microbiology. Winston-Salem:Hunter TextbooksInc..