Prescott's Microbiology
10th Edition
ISBN: 9781259281594
Author: Joanne Willey, Linda Sherwood Adjunt Professor Lecturer, Christopher J. Woolverton Professor
Publisher: McGraw-Hill Education
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Textbook Question
Chapter 36, Problem 4CHI
Legionella pneumophila is a bacterium that is often found in water systems (e.g., shower heads, air-cooling towers). The dispersal of these bacteria can lead to the development of Legionnaires’ disease, a particularly virulent pneumonia in the elderly and immunocompromised. Water systems are routinely monitored for L. pneumophila contamination using GVPC medium, which contains glycine and the antibiotics vancomycin, polymyxin B, and cyclohexamide. However, this method is known to underestimate the number of L. pneumophila cells in the environment. Flow cytometry (FC) is one approach to count cells. How would you go about determining if FC is a good way to monitor L. pneumophila in the environment? Specifically, how would you collect your samples and compare your results to those obtained by the currently accepted approach (i.e., plating on GVPC medium)? What controls would you need to perform? Based on this information, how do you think the results from FC would compare to those of GVPC cultures obtained from the same samples?
Read the original paper: Allegra, S. 2008. Use of flow cytometry to monitor Legionella viability. Appl. Environ. Microbiol. 74:7813.
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1) Look at the ideal results. Were your predictions accurate, and how did they compare with your results?
2) You used aseptic technique during this lab. Why is it important to work in a sterile manner when working with bacteria in the lab?
3) Why are the cells incubated at 42°C?
Overview of Transformation Protocol
-Prepare competent bacteria for transformation:
Treat starter E. coli bacteria with CaCl2and Competent Cell Solution (CCS). Store on ice until transformation procedure.
Competent cells are cells that are likely to take up foreign DNA and be transformed. This step increases the likelihood that the E. coli cells will take up the introduced vector and be transformed.
-Transformation procedure:
Obtain two microcentrifuge tubes containing your competent cells. Label one tube +DNA and one -DNA.
Add CaCl2 to both tubes.
Add the transformation mix containing the plasmid DNA to the tube labeled +DNA. Do not add any plasmid DNA to the -DNA tube.
Incubate both tubes on ice for 10 minutes. Then, place both tubes in a 42\deg C water bath for 45 seconds. Replace the tubes in an ice bucket for 2 minutes.
Add recovery broth to both tubes.
Incubate both tubes in a 37 C water bath for 5 minutes.
Questions:
1) What differences would you expect to see between the…
Overview of Transformation Protocol
-Prepare competent bacteria for transformation:
Treat starter E. coli bacteria with CaCl2and Competent Cell Solution (CCS). Store on ice until transformation procedure.
Competent cells are cells that are likely to take up foreign DNA and be transformed. This step increases the likelihood that the E. coli cells will take up the introduced vector and be transformed.
-Transformation procedure:
Obtain two microcentrifuge tubes containing your competent cells. Label one tube +DNA and one -DNA.
Add CaCl2 to both tubes.
Add the transformation mix containing the plasmid DNA to the tube labeled +DNA. Do not add any plasmid DNA to the -DNA tube.
Incubate both tubes on ice for 10 minutes. Then, place both tubes in a 42\deg C water bath for 45 seconds. Replace the tubes in an ice bucket for 2 minutes.
Add recovery broth to both tubes.
Incubate both tubes in a 37 C water bath for 5 minutes.
Questions:
1)What is the selectable marker in this experiment? How…
Chapter 36 Solutions
Prescott's Microbiology
Ch. 36.3 - You have detected aerobic, Gram-positive cocci...Ch. 36.3 - Prob. 2MICh. 36.3 - Prob. 1RIACh. 36.3 - Describe in general how biochemical tests are used...Ch. 36.3 - Prob. 3RIACh. 36.3 - Prob. 4RIACh. 36.3 - Describe a dichotomous key that could be used to...Ch. 36.3 - How can nucleic acidbased detection methods be...Ch. 36.3 - How can a suspect bacterium be fingerprinted?Ch. 36.4 - Prob. 1MI
Ch. 36.4 - Figure 36.10 Agglutination Tests. (a) Tube...Ch. 36.4 - Prob. 3MICh. 36.4 - Prob. 4MICh. 36.4 - Prob. 1RIACh. 36.4 - Prob. 2RIACh. 36.4 - Prob. 3RIACh. 36.4 - Prob. 4RIACh. 36.4 - Prob. 5RIACh. 36.4 - Name two types of immunodiffusion tests and...Ch. 36.4 - Prob. 7RIACh. 36.4 - Prob. 8RIACh. 36.4 - Prob. 9RIACh. 36 - As more ways of identifying the characteristics of...Ch. 36 - Prob. 2CHICh. 36 - ELISA tests usually use a primary and secondary...Ch. 36 - Legionella pneumophila is a bacterium that is...Ch. 36 - Prob. 5CHI
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