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Formal Lab report 5

Many microorganisms grow abundantly both inside and on the surface of the normal adult body. The microorganisms that establish more or less permanent residence without producing diseases are known as normal microbiota. Organisms that may be present for a few days or months are called transient microbiota. At one time, bacteria and fungi were thought to be plants and thus the term flora was used. You are provided with directions for the isolation and identification of the gram positive bacteria associated with the human skin. Gram-positive, catalase-positive, moderately halophilic bacteria are among the normal microflora of the human skin. These bacteria tend to be resistant to drying and to relatively high salt concentrations. In this lab, you will isolate and identify one strain of bacteria from your own skin. Once identified, you will characterize the bacteria by determining growth curves under two different conditions (different media, temperatures, aeration, etc.). You will also characterize the bacteria in terms of its sensitivity to various antibiotics and antimicrobial agents. INTRODUCTION Introduction: A short, concise statement (approximately 200 – 300 words) explaining what the experiment is designed to show and providing some background. ·         Begin with a short review of background material – specifically information that helps explain WHY you are performing this experiment. ·         State the problem that you are trying to solve. What questions are you trying to answer? What hypotheses are you testing? What are you predicting based on your hypothesis?  [15 points] MATERIALS & METHODS Preparation of Soil Microcosms (Part I) Equipment □        Spectrophotometer □        Antibiotic disk dispenser □        Shaker   Supplies □        Sterile swabs □        Hydrogen peroxide □        Coagulase plasma □        Various antibiotic disks   Media and Reagents □        Sterile saline □        Mannitol-salt agar plates (MSA) □        Mueller-Hinton agar plates (MH) □        Glucose fermentation tubes □        Trehalose fermentation tubes □        Xylose fermentation tubes □        Small tubes of ½ NB, TSB, BHI □        Disinfectants (H2O2, Listerine, Cepacol, lab disinfectant, etc.)     Isolation and Identification of Skin Bacteria 1.       From Johnson & Case. 2010. Laboratory Experiments in Microbiology, Exercise 45 [Bacteria of the Skin] and Appendix H.3 Follow procedures in this exercise to isolate and identify the one organism from your skin. 1.       Wet a swab with sterile water and swab any surface of your skin. 2.       Swab ½ of a MSA plate. Using a sterile loop, streak back and forth into the swabbed area a few times, then streak away from the inoculum. 3.       Incubate the plate, inverted at 35oC for 24 to 48 hours. 4.       Examine the colonies. Record the appearance of the colonies and any mannitol fermentation. Gram stain the colonies and test for catalase production. Perform the catalase test by suspension of the desired colony on a slide with a toothpick and adding a drop of H2O2. Discard the toothpick in the biohazard container. 5.       Subculture a catalase-positive, gram positive coccus on another mannitol salt agar plate. Transfer the bacteria to a slant, incubate, and refrigerate it for later use. 6.       Using the key, proceed to identify your isolate. a.       To test for coagulase, place a loopful of rehydrated cooagulase plasma on a clean slide. Add a loopful of water and make a heavy suspension of the bacteria to be tested. Observe for the clumping of the bacterial cells (clumping = coagulase positive) b.       Inoculate the appropriate fermentation tubes. Incubate the tubes at 35oC for 24 to 48 hours.   Key to gram positive cocci commonly found on skin Gram-positive cocci ↓ Catalase positive ↓ I.                     Cells arranged in tetrad; glucose not fermented Micrococcus A.      Colonies have yellow pigment M. luteus B.       Colonies have red pigment M. roseus II.                   Cells arranged in grape-like clusters; glucose fermented Staphylococcus A.      Acid produced from mannitol                                                                1.      Coagulase-positive S. aureus                                                              2.      Coagulase-negative   a.       Acid produced from trehalose   1)       Acid from xylose S. arlettae 2)       No acid from xylose S. saprophyticus b.       No acid from trehalose S. capitis B.       No acid from mannitol                                                                1.      Acid produced from trehalose S. saprophyticus                                                              2.      No acid produced from trehalose s. epidermidis   ***Note: in order to complete the WEEK TWO activities, you must make inoculate and incubate a broth culture with your isolate at least 24 to 48 hours before the lab.*** Bacterial Growth Curve 2.       From Johnson & Case. 2010. Laboratory Experiments in Microbiology, Exercise 20 [Determination of a Bacterial Growth Curve] 1.       Inoculate a broth culture with your isolated bacteria 24 hours before this experiment is to be conducted. 2.       In order to measure the growth of your strain under two different environmental conditions (25oC, 37oC, or 42oC; stationary or aerated; NB, TSB, BHI), inoculate 2 tubes of media with 0.1 ml from a 24 hour culture of your bacteria. 3.       Place these tubes in the spectrophotometer and measure the absorbances at time 0. 4.       Keep these tubes under the appropriate environmental conditions. 5.       Record the absorbance every 10 or 15 minutes for 2 or 3 hours. 6.       Graph the data you obtain. 7.       Swab ½ of a MSA plate. Using a sterile loop, streak back and forth into the swabbed area a few times, then streak   Disinfectants and Antibiotics 3.       From Johnson & Case. 2010. Laboratory Experiments in Microbiology, Exercise 24, 25 [Chemical Methods of Control: Disinfectants & Antibiotics; Antimicrobial Drugs] 4.       Characterize your strain of bacteria by testing it for antibiotic resistance and susceptibility to la b disinfectant. DISINFECTANT 1.       Using sterile water prepare a dilution of disinfectant in a sterile tube, diluted to the strength at which it is normally used. Transfer 5 ml of the diluted test substance to a sterile tube. 2.       Divide one plate of NA into 5 sections and label the sections 0, 2.5, 5, 10 and 20 minutes. 3.       Inoculate the 0 sector with the broth culture of your bacterial isolate. 4.       Aseptically add 0.5 ml of your bacterial isolate broth culture to the tube of disinfectant. Transfer one loopful from this tube to the corresponding sector on the NA plate at 2.5 Minutes, 5 minutes, 10 minutes, and 20 minutes. 5.       Incubate the plates, inverted at 35oC until the next lab period. Observe the plates for growth. Record the growth as (-) = no growth; (+) = minimum growth; (2+) = moderate growth; (3+) = heavy growth; and (4+) = maximum growth. ANTIBIOTICS 6.       Aseptically swab your isolate onto a MH agar plate. Swab in three directions to ensure complete plate coverage. 7.       Place the chemotherapeutic-impregnated disk by pushing the dispenser over the agar. Sterilize your loop and touch each disk with the sterile inoculating loop to ensure better contact with the agar. Record the agents and the disk codes. 8.       Incubate the plates, inverted, at 35oC until the next period. Measure the zones of inhibition using the ruler on the underside of the plate. Record the zone size and, based on the table of antibiotic sensitivity, indicate whether the isolate is sensitive, intermediate or resistant to each antibiotic.   RESULTS Results: Students should record data and observations here (approximately 50 – 100 words and all necessary Tables and Figures). ·         Data should be reported in tabular form. All tables must have a title and be numbered (e. g. “Table 1”). ·         If graphs are necessary, they should be done using EXCEL. All graphs must have a title and labeled axes and be numbered (e. g. “Figure 1”).  ·         Drawings should be neat and well labeled. All drawings must have a title and be numbered (e. g. “Figure 1”). When drawing microscopic observations, the magnification must be noted. ·         Text in the RESULTS section should explain what happened in the experiment, referring to Figures and Tables where necessary.  Do not put your CONCLUSIONS in this section. [30 points]                                 Should probably include but are not limited to: ·         Characterization and Identification of the Bacterial isolate ·         Growth Curves (use EXCEL) ·         Information on Disinfectant and Antibiotic Sensitivity CONCLUSIONS                Conclusion: Students should explain precisely what the data that have been obtained indicate. ·         Students must relate the results to any hypotheses or questions raised in the INTRODUCTION in the form of a summary statement. ·         Students must be careful to draw only those conclusions indicated by the data. Stating conclusions that are not justified or ignoring conclusions that are justified by the data will result in loss of credit. [25 points]   DISCUSSION Discussion: In this section, students should discuss the experiment, noting problems, sources of error, and possible follow up investigations.  The DISCUSSION may include some or all of the following: ·         Students may identify possible sources of experimental error. This does not mean human mistakes but rather error due to the limited sensitivity of laboratory instruments, the necessity to accept some lack of precision in certain measurements, the inability to manage all controlled variables, etc. ·         Students may propose ways to improve the experiment or to gather additional data ·         CONCLUSIONS from this experiment may suggest other hypotheses to test and further investigations that might be carried out by other researchers. These might be included in the DISCUSSION.  [20 points]                                   Should probably include but are not limited to: ·         analysis of techniques used ·         additional methods that might be useful in these exercises ·         clinical significance of isolates REFERENCES       References: Students should reference all appropriate sources that were used for the completion of the laboratory report. ·         Textbooks, lab manuals, and journal articles used should be listed at the end of the report using the CBE format. ·         Footnotes, in the CBE style, may be included in the text. ·         Journal articles must be referenced for the formal lab report. Material from these articles may be referenced in the INTRODUCTION to explain the reason for performing and experiment or to provide support for a hypothesis. These articles may also be referenced in the DISCUSSION to help explain experimental results or to support suggestions for further investigation. [10 points]