Unit 11



Unit 11: Microbial Blood Tests and Streak Isolation By Patricia G. Wilber, Karen Bentz, Heather Fitzgerald, and Andrea Peterson, 2022.Creative Commons Attribution-NonCommercial 4.0 International License.IntroductionIn this unit you will use media plates that contain whole sheep’s blood (TSA-blood, CNA-blood), lysed RBCs (Chocolate) and conduct coagulase and catalase tests. The Chocolate agar contains lysed sheep’s blood and is a supportive medium. This is the only medium that supports the growth of fastidious (picky) species such as Aggregatibacter sp. and is a good plate to use to observe colony morphology.The TSA-blood plate contains whole sheep blood and is non-selective; meaning all types of non-fastidious bacteria can grow on the media. It will differentiate between the bacteria that grow based on their ability to hemolyze blood. Alpha hemolysis is a partial digestion of the hemoglobin, producing an olive color in the media and the colonies. Beta hemolysis is a complete digestion of the blood cells in the media under the bacteria, producing a clear area in the media. No digestion of blood is termed gamma hemolysis.A CNA-blood plate is both selective and differential. It selects for Gram(+), coccus-shaped bacteria and selects against everything else. CNA-blood is also differential. It will differentiate between Gram(+) cocci based on their ability to hemolyze blood, just like a TSA-blood plate (alpha, beta or gamma). See Unit 7 for complete details on media ingredients.Figure 11-1. Photographs of Alpha (α), Gamma (γ) and Beta (β) hemolysis Left: Accessed 7/29/2015 from , but licensed for use by the American Society for Microbiology, Creative Commons Attribution – Noncommercial – NoDerivatives 4.0 International licenseRight: photo by Karen BentzThe Latex Coagulase test identifies the presence of the enzyme coagulase in bacteria. Coagulase allows a bacterium to clot the blood plasma in the host organism, thus creating a faux capsule around the bacteria. The faux capsule makes it difficult for the phagocytic host white blood cells to detect and eat the bacteria. Production of coagulase by a bacterium is therefore considered an indicator of pathogenicity. In a medical diagnostics lab (like Tri core here in Albuquerque), the latex coagulase test is used primarily to confirm identification of Staphylococcus aureus, after other tests have strongly suggested that the organism in question is, in fact, S. aureus. Figure 11-2. The results of a coagulase test. A positive reaction is on the left (the organism CAN coagulate plasma resulting red clumps in the test) and a negative reaction (no ability to coagulate plasma; no clumping of any kind) is on the right.Photo by Andrea PetersonThe latex coagulase test does not allow differentiation between S. aureus and S. saprophyticus, Therefore, it is important to follow a decision-making process prior to applying the latex coagulase test.The Catalase test allows us to determine if the organism produces the enzyme catalase. An important thing to remember about the catalase test is that it does not distinguish Gram(+) from Gram(-). The catalase test is used diagnostically to distinguish between Gram(+) cocci such as Staphylococcus species (catalase +) and Streptococcus species (catalase -). This test also gives us information about oxygen utilization. (See Unit 8)The blood cells in CNA-blood and TSA-blood plates also contain catalase. Thus, it is best to avoid doing the catalase test off a CNA or a TSA-blood plate. This prevents you from obtaining a false positive result. You can use your chocolate plate for the catalase test without worry because the catalase enzyme was denatured when the red blood cells were lysed.DAY 1: InoculationsMaterials:1 Chocolate agar plate for each student, for Streak Isolation1 Chocolate agar plate for two students1 TSA-blood plate for two students2 CNA-blood plate for two studentsinoculating loopsincinerator (on)Sharpiescandle jarscultures:BacteriaCell WallHemolysisStaphylococcus aureus (Sa)Gram(+)BetaStreptococcus oralis (So) Gram(+)AlphaEscherichia coliGram(-)Beta or GammaBacillus subtilis (Bs) Gram(+)Grayish coloniesEnterococcus faecalis (Ef)Gram(+)GammaAggregatibacter aphrophilus (Aa)Gram(-)Can’t tell; won’t grow on blood plates Procedures:Streak isolation on Chocolate AgarEach person will perform this procedure. Properly label the bottom of your Chocolate plate.Perform a streak isolation using Sa or Ef ONLY! Remember that you are taking your sample from a plate, so ONLY TAKE A TINY bit.Place your SI plate in a rack for incubation.Figure 11-3. The Streak Isolation procedureFigure by Patricia G. WilberTSA- Blood plateWork with a partner.Use a Sharpie and draw lines on the bottom of your plate dividing it into thirds. Also label each third with the initials of one of the bacteria as shown below.Using a sterilized loop, inoculate the plates with the three bacterial species as shown in Figure 11-4 below.Re-sterilize your loop after each species inoculated.Re-sterilize and return the loop to the metal canister at the end.Place the inoculated plate (agar side up, like normal) in the candle jar for A-blood plateWork with a partner.Use a Sharpie and draw lines on the bottom of your plate dividing it into thirds. Also label each third with the initials of one of the bacteria.Using a sterilized loop, inoculate the plates with the six different bacterial species as shown in Figure 11-4 below.Re-sterilize your loop after each species inoculated.Re-sterilize and return the loop to the metal canister at the end.Place the inoculated plate (agar side up, like normal) in the candle jar for incubation.Chocolate AgarWork with a partner.Use a Sharpie and draw lines on the bottom of your plate dividing it into thirds. Also label each third with the initials of one of the bacteria.Using a sterilized loop, inoculate the plates with the three bacterial species shown in Figure 11-4 below.Re-sterilize your loop after each species inoculated.Re-sterilize and return the loop to the metal canister at the end.Place the inoculated plate (agar side up, like normal) in the rack for incubation.Figure 11-4. Inoculate your TSA-blood, CNA-blood and Chocolate agar plates with the species and pattern shown. Figure by Patty WilberDay 2: Media Interpretation, Coagulase and Catalase TestsLatex Coagulase Test created by Corrie Andries and Karen BentzStreak Isolation Results and InterpretationObserve your streak isolation, photograph it and place the photo in the space below.Did your streak isolation grow? If it grew, great!If it did not grow, why might that be?Did you achieve isolation on your plate? (8-10 isolated colonies in streak area 3 or 4).If so, YAY!If not, how can improve/ modify your technique to get better results?Look at your isolated colonies. Notice the size, appearance and color of the colonies.TSA-Blood Plate and CNA-blood plate Hemolysis Results and InterpretationObserve your TSA-blood plate and your CNA-blood that were both inoculated with Ec, Ef and Bs. Photograph the results and insert the photos below. Fill in Table 11-1.Table 11-1. Summary of Bacterial Growth on TSA-blood vs. CNA-bloodBacterial speciesGrowth?(yes/no)CNA TSA Effect of Bacteria on Blood on plate(s) with growthType of Hemolysis(alpha, beta, gamma)Escherichia coliEnterococcus faecalisBacillus subtilisWhich of these bacterial species are possible pathogens? Defend your answer.Of the three species above, why did only Ef grow on the CNA?If you had a mixed culture containing one Gram(+) and one Gram(-) and you needed both of them to grow on one single plate, which plate(s) would work for this?TSA-bloodChocolateMacConkey’sCNA-BloodCNA-blood Plate vs. Chocolate Agar: Growth and HemolysisPhotograph your CNA-blood and your Chocolate Agar plates that were inoculated with Sa, Hh and So. Insert them here. Fill in Table 11-2.Table 11-2. Summary of Bacterial Growth on Chocolate vs. CNA-bloodBacterial speciesGrowth?(yes/no)CNA Choc Effect of Bacteria on Blood on the CNA-blood Plate, if the species grewType of Hemolysis(alpha, beta, gamma)Staphylococcus aureusStreptococcus oralisAggregatibacter aphrophilus Which of these bacterial species are Gram(+) cocci? Defend your answer.Which of these species is fastidious and will only grow on the chocolate? Work with people at your table to Gram Stain all the species that grew on the two CNA blood plates to verify they were Gram(+) cocci. Each person should do one Gram Stain. It is good practice for the midterm, too. Fill in Table 11-3. Table 11-3. Gram stain results for the species that grew on the CNA-blood plateSpecies that grew on CNA bloodGram stain results:Gram + or Gram -?Cell shapeCell size(0.2mm/# of cells across)List the species that did not grow on the CNA. Explain why they did not grow.Latex Coagulase Test and InterpretationThe latex coagulase test is used to confirm the presence of Staphylococcus aureus. A positive test will show red clumps, a negative test will not form clumps. Materials:your four inoculated plates coagulase test kit Procedure:Only Gram(+), coccus-shaped, beta hemolytic, catalase positive species should be considered as candidates for the coagulase test. Look at the results on your plates, and put a star by the culture(s) that has/have the first three features.List that species below and BASED on your RESULTS, explain how you knew. Species name:How you knew it was coccus:How you knew it was Gram(+):How you knew it was Beta hemolytic:Catalase test that/ those species off the CHOCOLATE AGAR (to avoid a false positive)If the result is catalase positive, you may have found the species to test with the coagulase test.Check with your instructor to verify the species you are planning to Coagulase test is the right one.Get a coagulase test card with one circle on it. Shake the reagent bottle.Put ONE drop of the test reagent (it is red) on the card.Use a toothpick to pick up a good amount of the bacteria you are testing FROM THE CHOCOLATE PLATE, and put it on the test card next to the drop of reagent.Stir the bacteria and reagent together in slow circles until you see clumps. Insert a photograph of your results here:Did the results of the coagulase test confirm your bacteria was Staphylococcus aureus?Based on your results, what enzyme does your organism possess?What does this enzyme allow the bacteria to do if it is in the bloodstream of a human?Is this bacterial species a possible pathogen? Defend your answer.Catalase Test Materialsyour inoculated TSA-blood, CNA-blood and Chocolate agar plates hydrogen peroxide bottles (2 per table)ProcedureTest the three media for the presence of catalase.Put a drop of hydrogen peroxide directly on the media, away from the bacteria.Watch for a little bit as the bubbling, if it occurs is a often little slow to develop.Record your result in Table 11-4.Repeat for the other two plate media.Test the six bacterial species for the presence of catalase from any plate. Put a drop of hydrogen peroxide directly on the bacteria growing on the plate.If the species is catalase positive, bubbling will be immediate-much much faster than bubbling that occurs on the plate alone.Record your result in Table 11-4.Repeat for the other bacterial species.Table 11-4. Catalase results for media and bacteriaMedia/Bacteria TestedGram(+) or Gram(-)Catalase + or - TSA-blood plateNACNA-blood plateNAChocolate plateNAEscherichia coliStaphylococcus aureusStreptococcus oralisBacillus subtilisAggregatibacter aphrophilus Enterococcus faecalisIf an organism bubbles when hydrogen peroxide is added, what enzyme does it produce?Explain why the blood plates were catalase positive, but not the chocolate plate.Based on your results, which plate is the safer choice when conducting a catalase test?ChocolateCNA-bloodTSA-bloodDoes the catalase test distinguish Gram(+) from Gram(-)? Explain your answer. Staphylococcus species should be catalase positive and Streptococcus species should be catalase negative. Do your results support this? (yes or no).Does producing catalase make an organism pathogenic? If an organism is catalase positive, which aerotolerance categories could it be in? Circle all that apply.Obligate aerobeObligate anaerobeAerotolerant anaerobeFacultative anaerobemicroaerophilePost-lab QuestionsName ________________List the species grew on TSA-blood but not on CNA-blood plate.Based on these results, what do you know about the bacteria that grew only on the TSA-blood and not on the CNA-blood?For those species that only grew on the TSA-blood plate, what media could you grow these species on to figure out if they are Gram(-)?What stain could you use to determine if the above bacteria are Gram(-)?Chocolate agar contains RBCs that were lysed when heated in an autoclave. How does this aid bacterial growth?What did the heating process do to the enzyme catalase?Based on your results, determine which enzymes each bacterial species has and whether or not there is evidence for pathogenicity.Bacteria Catalase + or Catalase - Hemolysis(alpha, beta, gamma) CoagulaseEvidence for pathogenicityEnterococcus faecalisNAStaphylococcus aureusStreptococcus oralisNABacillus subtilisNAEscherichia coliNAAggregatibacter aphrophilus NANA ................
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