Introduction - Central New Mexico Community College | CNM



Unit 12: Susceptibility of Bacteria to Antibiotics and Bacterial Identification and Streak IsolationBy Karen Bentz, Patricia G. Wilber and Heather Fitzgerald and Andrea Peterson, 2022Creative Commons Attribution-NonCommercial 4.0 International License.IntroductionThe term antibiotic generally refers to a chemical that inhibits the growth of bacteria (which is why they do not work on viruses). The first widely used antibiotic, penicillin, was isolated from a fungus in the 1920s. During WWII (in the 1940s) it was mass produced and used to treat wound infections in injured soldiers. Since that time, a variety of antibiotics have been developed to treat bacterial infections. Unfortunately for us, some populations of bacteria develop resistance, usually in less than one year after introduction, thus reducing the usefulness of the antibiotic as a treatment. The response of bacteria to an antibiotic can be termed sensitive if bacterial growth is inhibited by the antibiotic. If bacterial growth is unaffected by the presence of the antibiotic, the bacteria is considered resistant. A common method to test the response of bacteria to an antibiotic is to spread bacteria on a media plate using a lawn pattern, and then to place a paper disc containing a specified amount of antibiotic on top of the bacteria. The inoculated plate containing the disc is then incubated, and growth patterns of the bacteria are observed.Figure 12-1. Lawn of bacteria showing sensitivity and resistance to various antibiotic discsAccessed 8/9/2015 from GNU Free DocumentationNovobiocin is an antibiotic produced by the soil bacterium Streptomyces niveus. Novobiocin is a very effective inhibitor of the bacterial enzyme DNA gyrase, thus it inhibits DNA replication, resulting in death of susceptible bacteria. Clinically, novobiocin is used to separate novobiocin-resistant Staphylococcus saprophyticus, a common urinary tract pathogen, from novobiocin -sensitive skin flora such as Staphylococcus aureus and Staphylococcus epidermidis. Novobiocin is also effective against MRSA (methicillin resistant Staph. aureus), many Gram(-) species and some streptococcal species. The antibiotic Bacitracin is produced by Bacillus subtilis and it disrupts peptidoglycan synthesis in the cell walls of bacteria. Bacitracin is used as a topical (skin) preparation only, as it is highly toxic if taken internally. Its widespread use in triple antibiotic ointments such as Neosporin has contributed to the emergence of antibiotic resistant bacteria such as MRSA. Clinically, bacitracin is used to distinguish between β-hemolytic streptococci (such as Streptococcus pyogenes), which are sensitive to bacitracin, and various other Staphylococcal and Streptococcal species which are resistant to bacitracin. Optochin is a chemical (ethylhydrocupreine hydrochloride) that interferes with ATP production in Streptococcus pneumonia. This helps distinguish S. pneumonia from other α-hemolytic streptococci. It entered medical use around 1914. Identification of Bacteria: Bacterial species are characterized by their unique physical and metabolic traits, and these traits are used in medical laboratories to identify causes of infection and aid in determining treatments. DAY 1: Inoculation and Start Unknown IdentificationNovobiocin / P Disk Video has this on a CNA blood plate but we will also use the TSA with Blood. created by Corrie Andries.Materials Five sterile cotton swabs/pairTweezersAntibiotic discs: Novobiocin (NB) discs, Bacitracin (A) discs, Optochin (P) discs1 T-Soy per personMedia (per group of 4) Five TSA-blood plates Five CNA-blood platesBacteria Cultures Bacillus subtilis in brothStreptococcus pyogenes (Spy) in brothStreptococcus pneumoniae (Spn) in brothProteus vulgaris (Pv) in brothUnknown Bacteria “A” in a brothUnknown Bacteria “B” in a brothProceduresNovobiocin, Optochin and Bacitracin Antibiotic Sensitivity Test Work in a group of four.One pair of the group of four use the five TSA blood plates: Label the bottom of four of your TSA-blood agar plates with your name, date, and the name of the bacteria that you’ll be testing for antibiotic sensitivity. Save the 5th plate for the unknown. See Figure 12-2.The other pair in the group of four use the five CNA blood plates: Label the bottom of four of your CNA blood plates with your name, date, and the name of the bacteria that you’ll be testing for antibiotic sensitivity. Save the fifth plate for the unknown. See Figure 12-2.Create a lawn of bacteria on each blood plate. See Unit 3 for a review of the technique. Leave a bit of the edge of the plate unswabbed so you can see the original plate color. Figure 12-2. Use of the three antibiotic discs, Novobiocin (NB), Optochin (P) and Bacitracin (A) on blood plate (TSA or CNA) lawns. Figure created by Karen BentzSterilize the end of a pair of tweezers by holding it in the micro incinerator for a few seconds. Let tweezers cool before using.Use the sterile tweezers to carefully grab one of the novobiocin (NB) discs from the container.Carefully place the NB disc on your bacterial lawn.Press the disc gently, but firmly onto the plate. Do not break the agar below it. You want the disk to stick when you invert the plate, but not be embedded in the agar.Re-sterilize and cool the tweezers. Get an Optochin (P) disc and repeat steps 5 – 9 above.Get a Bacitracin (A) disc and repeat steps 5 – 9 above. Repeat steps 5 – 9 for your other bacterial lawn plates. Place your team’s eight inoculated plates upside down in a candle jar for incubation. Return the tweezers to the canister.Note: Using blood agar can show you bacterial hemolysis as well as antibiotic sensitivity, if the organism grows on the plate; not everything will grow on a CNA. The antibiotic discs can also be tested on bacterial lawns made on T-soy agar.Characterizing and Identifying Unknown BacteriaYour group of four will start your unknown bacterial identification today by using either Unknown bacteria “A” or Unknown bacteria “B” and running an antibiotic sensitivity test on your unknown.Novobiocin, Optochin and Bacitracin Antibiotic Sensitivity Test1. Choose either Unknown Bacteria A or Unknown Bacteria B to identify.2. One pair of your group of four will label the bottom of a TSA blood plate with your name, date, and the Unknown letter. The other pair will label a CNA blood plate.3. Use a swab to make a lawn of your unknown bacteria on a blood plate. Leave a bit of an edge of the plate unswabbed so you can see the original plate color after incubation.4. Using sterile, cooled tweezers carefully place a novobiocin, optochin, and bacitracin disc on the plate, keeping the discs far apart on the plate. 5. Sterilize the tweezers between discs and when finished.6. Place your inoculated plate upside down in the candle jar for incubation.Streak Isolation of Unknown BacteriaEach member of the group will perform a streak isolation on their own T-soy using the group’s Unknown bacteria.III. DAY 2: Results and InterpretationAntibiotic Disc Results and InterpretationIntroductionIf the species grew on the plate, antibiotic resistance can be assessed. If the species did not grow, antibiotic resistance cannot be observed. For those that grew, observe the growth. Inhibition of bacterial growth by the antibiotic produces a clearing (a circular region without growth whose diameter can be measured) around the disc, called the zone of inhibition. If the zone of inhibition is big enough, the bacteria are considered to be sensitive to the antibiotic, and that antibiotic might be an effective medication. If the lawn of bacteria on the plate grew up to or close to the disc, the bacteria are said to be resistant to that particular antibiotic and would not be an effective medication.Use a metric ruler to measure the diameter of the zone of inhibition (in mm) around each antibiotic disc. Then compare your measurement to the table provided to determine if the bacteria are sensitive or resistant to the antibiotic. See Figure 12-3 below. Figure 12-3. How to measure the zone of inhibition using a metric ruler. Figure by Karen Bentz and Patty WilberProcedureUse a metric ruler to measure the diameter of the zone of inhibition in mm for all of the antibiotic discs tested. Record your measurements in Tables 12-1 and 12-2. Compare your measurements with the metric measurements in Table 12-3 to determine if your bacteria are sensitive or resistant to the antibiotics, and record your results in Tables 12-1 and 12-2.Table 12-1. Results of the TSA-blood plateName of Bacteria TestedAntibiotic TestedMeasurement of the diameter of inhibition (mm)Is the bacteria sensitive to or resistant to the antibiotic tested?Novobiocin (NB)Optochin (P)Bacitracin (A)Novobiocin (NB)Optochin (P)Bacitracin (A)Novobiocin (NB)Optochin (P)Bacitracin (A)Novobiocin (NB)Optochin (P)Bacitracin (A)Unknown Bacteria ____Novobiocin (NB)Optochin (P)Bacitracin (A)Table 12-2. Results of the CNA-blood plateName of Bacteria TestedAntibiotic TestedMeasurement of the diameter of inhibition (mm)Write NA if there was no growth.Is the bacteria sensitive to or resistant to the antibiotic tested?Write NA if there was no growthNovobiocin (NB)Optochin (P)Bacitracin (A)Novobiocin (NB)Optochin (P)Bacitracin (A)Novobiocin (NB)Optochin (P)Bacitracin (A)Novobiocin (NB)Optochin (P)Bacitracin (A)Unknown Bacteria ____Novobiocin (NB)Optochin (P)Bacitracin (A)Table 12-3. Required Zones of Inhibition for Three AntibioticsAntibioticRequired Diameter in mm for Bacterial SensitivityNovobiocinGreater than 16 mmOptochinGreater than 14 mm BacitracinGreater than 10 mmNow look at the media under your bacteria and record your bacteria’s ability to grow and hemolyze blood in Table 12-4. If there is no growth, you cannot assess hemolysis. Record no growth and do not assign a hemolysis type.Table 12-4. Results on the two blood platesBacteriaTSA with blood:Growth/no growthIf growth: alpha, beta or gamma CNA with blood:Growth/no growthIf growth: alpha, beta or gamma Unknown Bacteria ____Which antibiotic was most effective for each species tested, including the unknown. Record your results in Table 12-5.Table 12-5. Give this table your own title_____________________________________SpeciesMost Effective AntibioticBacillus subtilisStreptococcus pyogenesStreptococcus pneumoniaeProteus vulgarisUnknown Bacteria ____Insert Photos of Results Here:Bacterial Identification Analyze your Streak Isolation.You will be performing a streak isolation for the midterm. Score yourself between 0 and 1 for each of the three criteria (a-c) below.Do you have the correct pattern? ________Does your pattern show dilution? _________Do you have 8-10 isolated colonies? _________If not, why not? Circle all that apply.Bad pattern. Too much bacteria. No sterilizing between streak. Too much overlap.Have your instructor assess the grade you gave yourself.If you did not get a 3/3, determine what you can do better to get 3/3 on the midterm’s SI.Record your colony results in Table 12-6.Insert a photo of your streak isolation here.Four ideas to help fix your SI pattern if it did not work:Have you mastered the basic pattern? If no, look at the pattern in U3 and practice the pattern on a piece of paper to develop muscle memory.Are you using too much bacteria? If yes, USE LESS! Touch your loop to a solid colony if using a plate; do not scoop it. Tap the excess liquid off your loop if using a broth.Did you sterilize your loop between sections? If no, just remember to use that incinerator!Are you overlapping too much? If yes, practice the pattern with (see 1) less overlap between sections.Catalase test of your Unknown BacteriaPerform a catalase test of your Unknown off your streak isolation plate. Record your results in Table 12-6.Gram Stain of Unknown BacteriaPerform a Gram stain of the Unknown. Refer to Unit 5 for the technique if need be. Use your assigned microscope to view your cells at a total magnification of 1000X. Be sure to use oil. Record your results in Table 12-6.Table 12-6. Record the results for your unknown.TestResultsGram stain:Cell colorCell sizeCell shapeCatalase result:Hemolysis Type:Zone of Inhibition (mm)Sensitive or Resistant?Novobiocin:Bacitracin:Optochin:Create a Dichotomous Key to Identify Your Unknown BacteriaYour Unknown Bacteria is one of the following four bacteria: Bacillus subtilis Streptococcus pyogenes Streptococcus pneumoniae Proteus vulgaris Using the information you have obtained (cell structure, presence of catalase, ability to hemolyze blood, and antibiotic sensitivity/resistance, growth on CNA-blood), and the U13 G+/G- key, make a dichotomous key that identifies each of the four bacteria listed above. You may not need to use all the information you have.Dichotomous Key:Use your dichotomous key, and Table 12-6 to identify your Unknown bacteria. Write the name of your bacteria here: Analyze Results to Determine PathogenicitySometimes some evidence suggests pathogenicity and other evidence suggests a non-pathogen, but if there are any test results that indicate pathogenicity, you should consider the organism a possible pathogen.Based on your results, do you think your Unknown bacterium is a pathogen? (yes or no). List all of the evidence that suggests your Unknown bacteria might be pathogen.Post-Lab QuestionsName ________________________When might an antibiotic sensitivity test be ordered by a medical professional?What unit of measure do we use for a zone of inhibition?Given the picture shown here, what steps do you need to go through to decide which of these antibiotics would be best for use on a patient?What was the purpose of the catalase test that you did for the Unknown Identification?Explain how each of the following contributes to bacterial pathogenicity.HemolysinAntibiotic resistance Explain what you learned about your Unknown from your CNA-blood plate.If you wanted to find out what kind of hemolysis a Gram(-) organism can perform, which medium would you use and why? ................
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