Introduction - Central New Mexico Community College | CNM



Unit 13: Identification of Unknown BacteriaBy Karen Bentz, Patricia Wilber and Heather Fitzgerald, and Andrea Peterson, 2018Creative Commons Attribution-NonCommercial 4.0 International License.IntroductionThe purpose of this project is to demonstrate basic understanding of microbiological techniques and media you have learned this term, mastery of logical processing and fact-based decision-making, and flexibility, resilience and creativity in the face of unexpected outcomes.You will be separating and identifying two bacteria, one a Gram(+) and one a Gram(-), that are mixed together in a broth. Each student will do their own unknown identification of the two species that are in the broth that they choose. You will have several days to determine the identity of the two species by using lab techniques and biochemical tests you have learned and fact-based decision-making. Your instructor will provide a format for the lab report that you will write and turn in for this unknown identification process. Your lab work and written lab report will count as the final exam for this course. (Formats do vary a bit by instructor, so follow the rubric provided by your instructor, only.)Tips for SuccessCarefully consider the media and techniques you will use before you do them. Do not wing it.Use good aseptic technique to ensure your cultures remain pure.Take detailed notes and photos of your procedures and results. These will make it easier to write a detailed lab report.You will usually perform two tests and/or inoculate two media per lab session. Generally, you will choose one test and/or media for your Gram(+) identification, and one test and/or media for your Gram(-) identification. Once again, take your time and choose wisely. Once you check out your media from your instructor, you cannot exchange it. You may consult your lab manual, other students, or other resources regarding interpretation of your results. You may not ask your instructor or other instructors to interpret results for you.Materials and Media (per student)For Day 1 inoculation: Students will choose THREE of the following four plates: MacConkeyTSA BloodChocolate AgarCNA blood Gram stain materialsAdditional media/tests as needed to identify both bacterial species. No student will use all of the media/tests available, but the possible media/ tests will include: Catalase, Oxidase, T-Soy, MacConkey, Chocolate Agar, Blood Agar, MR-VP, Lysine, TSI, Lactose, Sucrose, SIM, PA, Urease, Coagulase, Novobiocin, Bacitracin, Optochin.Bacterial CulturesYour instructor will mix 0.05 ml (50?l) of one Gram(-) species into a 5ml broth containing one Gram(+) species. Thus, you will have two unknown species mixed together in your unknown tube; one Gram(-) and one Gram(+). The Gram(-) will be one of these: Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Citrobacter freundii, Klebsiella pneumoniae, Serratia marcescens, and Shigella flexneri.The Gram(+) will be one of these: Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, Staphylococcus saprophyticus, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Enterococcus faecalisProcedureDay 1Pick up one of the broth tubes of unknowns. Each numbered tube contains one Gram(+) and one Gram(-) bacterial species that have been mixed together. Record the tube number on your instructor’s sign-out sheet, and also on your notes. Choose THREE of these four plates: MacConkey, TSA Blood, Chocolate Agar, CNA Record WHY you chose each plate.Label your three plates with your full name and unknown number, as well as the standard label information. STIR your mixed broth thoroughly with your sterilized loop. The Bacillus species grow on the top and the Streptococcus are on the bottom of the tube, so if you have one of those, you will want them to be mixed in really well with your sterilized loop before streaking!Inoculate your plates using the inoculation pattern of your choice. YOU NEED A GOOD REASON FOR EACH CHOICE. Record your reason on your data table.Put your plates in the proper rack/candle jar for incubation. Return your unknown tube to the original test tube rack.Take careful notes of your procedures today, as these will be included in your lab report.You should BEGIN WRITING your report now! And continue to write as you go.Day 2Check your plates for growth, isolation of colonies, and metabolic results. Record your observations from each plate (colony appearances, lactose fermentation, hemolysis, etc. if you can tell) on the data collection sheet. IDENTIFY YOUR GRAM POSITIVE COLONIES:Hint: On Chocolate or TSA blood agar, Gram(+) colonies are often smaller (but not always) than Gram(-) colonies. Gram(-) colonies are often slimier than Gram(+) colonies. If you do not have isolated Gram(+) or Gram(-) material WHAT SHOULD YOU DO?? Don’t panic. Science is like that e up with some ideas and discuss with your instructor.You could use your original tube, as it was saved. That might not be the best idea for your situation, though.Your instructor may require you to Gram stain to identify the colonies you think are Gram(+). Record cell shape. DO NOT worry about arrangement.Estimate and record the size of your cells. Record all other information about your Gram(+) you can gain from the plates you chose, which may include hemolysis, color, size, basic description.You may wish to perform a spot test. If so, request the materials from your instructor and follow their instructions. RECORD exactly which colonies and which plate used. Record your results.You may also choose a medium upon which to grow more of your Gram(+) to aid in further identification of your Gram(+). Record the name of the new test and EXACTLY which colonies and plate you used to perform this test.PUT YOUR FULL NAME, Sample # and G+ on the label.Incubate in the proper location. IDENTIFY YOUR GRAM NEGATIVE COLONIES:Your instructor may require you to Gram stain the colonies you think are Gram(-) on your TSA blood and/or your chocolate agar plates. DO NOT GRAM STAIN OFF THE MACCONKEY PLATE. THE RESULTS ARE JUST MESSY. If so, record cell shape and size in your data sheet.Record all other information about your Gram(-) you can gain from the plates you chose, which may include hemolysis, color, size, basic pare to your colleagues.Choose a next test for your Gram(-) that will help you identify the Gram(-) species. PUT YOUR FULL NAME, Sample # and “G-” on the label.Place the new test in the proper place for incubation.Sign for your new tests/media on your instructor’s sign-out sheet at the front of the lab. Your instructor will then give you any media/materials required. Once you have signed for and received your medium/materials, it/they cannot be returned, so choose wisely. If you are choosing one of the antibiotic discs, you will also need to specify which medium to use it with. Reagents and Gram staining materials will be out in the lab for you to use, but you will need to sign the instructor sheet for these as well. Some instructors may require you to ask them for each media or reagent instead of using a sign out sheet.If you are performing a spot test, complete the test and record your results. Your instructor may wish to see you perform the test. Write down details of your observations.You may keep two of your three Day 1 plates for future inoculations. These two plates will be stored in the refrigerator, and you can use them any time during this identification project as a source of bacteria. Place the two media you wish to save in the designated rack and discard your third plate. You should continue writing your Unknown Lab Report TODAY. Use the instructions provided by your professor (the instructions ARE NOT the same from instructor to instructor) to set up the format for your paper. At the end of today’s lab you should have enough information to write the introduction, the Day 1 and Day 2 materials and methods section of your lab report, as well as the Day 1 results and interpretation sections. DO NOT wait until the end to start writing your paper. If you write it as you go, you will not be rushing at the end to finish your paper. Day 3 Collect your media and record your detailed observations on the data collection sheet. You may need to add reagents to some media before observing results. The reagents are available as part of the original test but you may still need to get them from your instructor. Your instructor may wish to see the results or watch you perform the test.If you are still working on isolating your Gram(+) or your Gram(-)on Day 3, refer to the Day 2 instructions and consult with your instructor. Your instructor may provide you with a pure culture plate at this time. Or they may not. You may lose points.Choose two new media and/or tests as needed to further identify your two unknowns. Sign for this media at the front of the lab. On the label put PUT YOUR FULL NAME, Sample # and “G+” or “G-“ as appropriate.Inoculate your two new media and place them in the appropriate rack for incubation. If you are performing a test, complete the test and record your results. You may keep only two of your old media. These may be the two you saved on Day 2, or two from today or a combination. Discard all other old media.If you have identified your bacteria, you do not need to run any more tests.Continue writing your lab report, adding the details of today’s inoculations and results.Day 4Last day to inoculate. No one needs to do all of the available tests or media inoculations. In most cases, a relatively small number of carefully selected tests will be enough to identify your organism. Choose two new media and/or tests if needed. If you have identified both of your bacteria, STOP! You do not need to run any new tests. On the label PUT YOUR FULL NAME, Sample # and “G+” or “G-“ as appropriate.Inoculate your media and place it in the proper rack for incubation. If you are performing a test, complete the test and record your results. Discard all old media including your “saved” media.Continue/finish writing your lab report. Day 5: Last day to observe results. Collect your media and record results. Discard all media when finished. Finish your report.662656-200025Unknown Bacteria Identification Project00Unknown Bacteria Identification ProjectResults and InterpretationThe inoculations performed on your three media on Day 1 will hopefully produce obvious results you will see clearly on Day 2. These results and perhaps some spot tests should be used as the basis for your decision making process on Day 2. Once you have narrowed the possible choices to a few organisms, the additional tests performed should only be ones that help you narrow your choices down still further. Tests where all of your possible organisms would give the same result (for instance, all positive or all negative) are of no value. If you did not get isolation of one of your species, consult the information above, come up with a plan and consult with your instructor.Take detailed notes on the results of your tests. Interpret the results of all of the media and/or tests that you conduct. Explain in detail what the results indicate about the cell wall structure and metabolism of your unknown. Also discuss the evidence for or against your bacteria being a possible pathogen. Always record EXACTLY where you got your samples for any test, and the dates of your inoculations and observations.Your Unknown Bacteria tube number: ________________Final Unknown Project Data Collection TableDay 1 InoculationsDate_________________Plates inoculated from Unknown tube #___________ Plate chosenWhy did you pick that plate?Inoculation techniqueWhy did you choose that technique?Day 2 Results and Interpretation of your plates. Date ____________________Plate chosenDescribe the color, texture and size of each type of colony.For each colony type on each plate, describe what you learned about the species from the colony (cell wall, metabolism, pathogenicity, etc.)Insert photographs of your results here:Days 2/3 Next Tests results and interpretation (add/ subtract or modify rows as needed) DayMedia Inoculated or Test performed and date performedExactly where did you get the bacteria for this test?Results: Describe IN DETAIL what the results looked like and date observed. Interpretation: What does each result indicate about organism’s size, shape, cell wall, metabolism, pathogenicity, etc.2Date:Test: Gram stain?Date:2Date:Test: Catalase?Date:2Date:Test:Date:2/3Date:Test:Date:2/3Date:Test:Date:Insert photographs of your results as needed:Days 3/4Final Unknown Project Data Collection Table (continued)DayMedia Inoculated or Test performed and date performedExactly where did you get the bacteria for this test?Results: Describe IN DETAIL what the results looked like and date observed. Interpretation: What does each result indicate about organism’s size, shape, cell wall, metabolism, pathogenicity, etc.3/4Date:Test:3/4Date:Test:?Date:Test:Insert photographs of your results as neededDays 4/5Final Unknown Project Data Collection Table (continued)DayMedia Inoculated or Test performed and date performedExactly where did you get the bacteria for this test?Results: Describe IN DETAIL what the results looked like and date observed. Interpretation: What does each result indicate about organism’s size, shape, cell wall, metabolism, pathogenicity, etc.4/5Date:Test:4/5Date:Test:4/5Date:Test:Insert photographs of your results as neededBio 2192 Gram(-) Organisms and Expected ResultsMedia/TestCitrobacter freundii (Cf)BSL 2Escherichiacoli (Ec)BSL 1Klebsiella pneumoniae (Kp)BSL 2Proteusvulgaris (Pv)BSL 2Pseudomonas aeruginosa (Pa)BSL 2Serratia marcescens (Sema) BSL 1Shigella flexneri (Sf) BSL 2Gram Stain(-) pink(-) pink(-) pink(-) pink(-) pink(-) pink(-) pinkCell ShapebacillusbacillusbacillusbacillusbacillusbacillusbacillusO2Catalase(+) bubbles(+) bubbles(+) bubbles(+) bubbles(+) bubbles(+) bubbles(+) bubblesOxidase(-)(-)(-)(-)(+) purple(-)(-)BloodTSA with bloodgammagamma or betagammagrayish Grayish, iridescent silver; or betaalpha may change to beta over timegammaCNA with bloodNo growthNo growthNo growthNo growthNo growthNo growthNo growthCarbohydrate TestsMacConkey’sgrowth + lac (fuchsia)growth + lac (fuchsia)growth + lac (fuchsia)growth, (-lac)growth, (-lac)growth, (-lac)growth, (-lac)Lactose Durham acid, gasAG yellow, gasAGyellow, gasAGyellow, gas-, - red, no gas-, - red, no gas- , - red, no gas-, - red, no gasSucrose Durham acid/gasAG yellow, gas-, - red, no gasAGyellow, gasA-yellow, no gas-, - red, no gasAG yellow, gas-, - red, no gasMR/VP+ (red) / -+ ( red) / -+ ( red) / -+ (red) / -- / -+ (red) or - / + (red ring)+ (red) / -TSI Slant/ButtA/A yellow/blackA/Ayellow/yellowA/A yellow/yellowA/A yellow/blackK/K no growth (red/red)A/A or K/A(yellow/yellow)(red/yellow)K/A(red/yellow)TSI gas(+)(+)(+)(+)(-)(-)(-)TSI H2S(+) black(-)(-)(+) black(-)(-)(-)Protein TestsLysine(-) (+) purple(+) purple(-)(-)(-) OR (+) purple(-)SIM sulfur(+) black(-)(-)(+) black(-)(-)(-)SIM indole(-)(+) red ring(-)(+) red ring(-)(-)(-)SIM motility(+) black(+) cloudy(-)(+) black(-)(-)(-)Phenylalanine(-)(-)(-)(+) green(-)(-)(-)Urease(-) or weak fuchsia (-)(+) fuchsia (+) fuchsia(-)(-)(-)AntibioticsNovobiocin (NB) > 16 mm = Sresistantresistant (11 mm)resistantresistantresistantresistant (10 mm)resistantBacitracin (A) >10 mm = SresistantresistantresistantresistantresistantresistantresistantOptochin (P) > 14 mm = SresistantresistantresistantresistantresistantresistantresistantBio 2192 Gram(+) Organisms and Expected ResultsMedia/TestBacillus megaterium (Bm) BSL 1Bacillus subtilis (Bs)BSL 1Enterococcus faecalis (Ef)BSL 2Staphylococcus aureus (Sa)BSL2Staphylococcus saprophyticus (Ss) BSL 1Streptococcus oralis (So)BSL 2Streptococcus pneumoniae (Spn) BSL 2Streptococcus pyogenes (Spy) BSL 2Gram Stain(+) purple(+) purple(+) purple(+) purple(+) purple(+) purple(+) purple(+) purpleCell Shapebacillusbacilluscoccuscoccuscoccuscoccus (can look bacillus off Choc.)coccuscoccusO2Catalase(+) bubbles(+) bubbles(-)(+) bubbles(+) bubbles(-)(-)(-)Oxidase(-)(-)(-)(-)(-)(-)(-)(-)BloodTSA with bloodgrayish coloniesgrayish coloniesgammabetagammaalphaalphabetaCNA with bloodNo growthNo growthgammabeta gammaalphaalphabetaCarbohydrate TestsMacConkey’sno growthno growthno growthno growthno growthno growthno growthno growthLactose Durham (Acid, Gas)-, - red, no gas-, - red, no gas- , - red, no gasAGyellow, gas- , - red, no gas-, - red, no gas-, - red, no gasAG yellow, gasSucrose Durham (Acid, Gas)-, - red, no gasAG yellow, gasAG yellow, gas A –yellow, no gas A-yellow, no gasA-yellow, no gas-,- red, no gasAGyellow, gasMR/VP- / - - / + (red ring)+ (red) /+(red ring)+ (red) /+ (red ring)+ (red) / - + (red) / -- / -no growth+ (red) / -TSI Slant/ButtA/K yellow/orange-redA/A yellow/yellowA/Ayellow/yellowresults inconsistent/ do not use this testresults inconsistent/ do not use this testresults inconsistent/ do not use this testresults inconsistent/ do not use this testresults inconsistent/ do not use this testTSI gas(-)(-)(-)TSI H2S(-)(-)(-)Protein TestsLysine(-)(-)(-)(-)(-)(-)(-) no growth(-)SIM sulfur(-)(-)(-)(-)(-)(-)(-)(-)SIM indole(-)(-)(-)(-)(-)(-)(-)(-)SIM motility(-)(-)(-)(-)(-)(-)(-)(-)Phenylalanine (lt +) pale green(-)(-)(-)(-)(-) (-) no growth(-)Urease(lt +) fuchsia slant(-)(-)(lt +) fuchsia slant(+) fuchsia(-)(-)(-)AntibioticsNovobiocin (NB) > 16 mm = Ssensitive (20 mm)sensitive (25 mm)sensitive (16 mm)sensitive (26 mm)resistantsensitive (23 mm)Sensitive(18 - 35 mm)sensitive (16 mm)Bacitracin (A) >10 mm = Sresistantresistantresistantresistantresistantresistantresistantsensitive (14 mm)Optochin (P) > 14 mm = Sresistantresistantresistantresistantresistantresistantsensitive (22 mm)resistantPRE-Lab Questions TO BE COMPLETED BEFORE YOU BEGIN YOUR UNKNOWN PROJECTName_______________What grows on a MacConkey’s plate? ____________________Give two reasons why this could be a good plate to use for one of your three plates.What grows on a CNA with blood plate? ____________________Give two reasons why this could be a good plate to use for one of your three plates.What grows on a TSA with blood plate? _________________________Give two reasons why this could be a good plate to use for one of your three plates. What grows on a Chocolate Agar plate?_________________________Give two reasons why this could be a good plate to use for one of your three plates.What inoculation technique is best to produce isolated colonies on plate media?Should you use a streak isolation inoculation on selective media like the MacConkey’s and CNA-blood? DEFEND YOUR ANSWER.If you have two distinct colonies on a plate, how will you determine and then keep track of which is G(+) and which is G(-)?Determine G(+) vs. G(-)Keep track of which colony is G(+) or G(-)List three things you can learn about your isolated colonies from your day two Gram stain / microscope work? For the following questions use the Gram (+) and Gram (-) Tables on the previous pages. If you could choose one media/ test to differentiate between E. coli, Citrobacter and Klebsiella, which one would you choose? Why would you choose this particular test? Which one media/ test would you use to differentiate between Proteus, Pseudomonas and Shigella?Why would you choose this particular test? Which antibiotic disc test would you use to differentiate between Streptococcus oralis and Streptococcus pneumonia? What plate medium would you use to put this disc on?Use the Gram(+) and Gram(-) tables to identify the following unknown organisms. TestResults for Organism “A”Results for Organism “B”MacConkeyPink coloniesClear coloniesTSIYellow tube, bubbles in mediaRed slant, yellow buttMR-VPMR redMR redSIMNo red ring, no black color no fuzzinessNo red ring, no black color, no fuzzinessOxidaseNo colorNo colorUreaPinkYellowish PhenylalanineYellow after reagent addedYellow after reagent addedSucroseYellow, 25% gas in Durham tubeRed2316480101600046234351016000Correctly written scientific name:A.B.Results for Unknown “C”Results for Unknown “D”Cell ShapeCocciCocciCatalaseBubblesNo bubblesA discGrowth up to discGrowth up to discP discGrowth up to disc22 mm zone of inhibitionNovobiocinGrowth up to disc30 mm zone of inhibitionHemolysisNo color change in mediaMedia is an olive green color4036695323850017030702286000Correctly written scientific name:C.D. ................
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