MICRB 202 (F04) Practicum II study guide
MICRB 202 (F07)
Study Guide for Lab Practicum II (15% final grade)
1. Know what a bacteriophage plaque looks like for T4 phage. Does the density of plaque forming units (PFUs) reflect the density of phage particles (virions) in the lysate or diluted lysate used to infect the host bacterium?
2. How are phage isolated, and specifically how is the lysate made? Is it highly probable that phage in a lysate made with one host bacterium can infect a different species of bacterium?
3. Know the differences between lytic and lysogenic cycles, and virulent and temperate phage. What condition can initiate the lytic cycle of a temperate phage?
4. How does transduction happen? What is a transducing particle versus a phage particle? How did we detect transductant colonies in our experiment?
5. What happens in conjugation? Know possible conjugation results for the following matings: F+ x F-, Hfr x F-. Given locations (F-plasmid versus chromosome) and types of antibiotic resistance genes (AmpR, StrR, NalR) for different E. coli strains, be able to predict which will grow on different antibiotic containing media and media without antibiotics.
6. Know how to interpret hand scrubbing results, and what they may mean with respect to transient versus normal microbiota of the skin.
7. How did we assess if bodily fluids contained lysozyme? Was lysozyme more effective on Gram negative or Gram positive bacteria and why (know lysozymes function, i.e. which specific compound does it target and what is the consequence)?
8. How is a Kirby Bauer assay performed? How is the level of resistance to a specific antimicrobial agent determined for a specific bacterium in the Kirby Bauer assay? Why can there be differences in the level of resistance between bacterial populations of same species?
9. Know the colony characteristics of a coliform bacterium grown on ENDO agar and XLD agar. Know descriptions of colony characteristics of most Salmonella spp. and Shigella grown on XLD agar.
10. Be able to identify the test and interpret results (positive and negative) for the following tests: 1) motility and H2S production on SIM stabs; 2) glucose and lactose fermentation on Russell’s double sugar (RDS) slant; 3) urease; 4) tryptophanase (i.e. indole production); and 5) citrate utilization. Know the principle and functionbehind each test (microbial process and/or enzyme involved, indicator dyes for pH, etc…).
11. Be able to use a separation outline (dichotomous key) for identification of enteric bacteria to genus based on test data (see #10 above) collected.
12. Know principle and function of the BioLog system. What is an advantage of using a multi-test system like BioLog? Know that the BioLog and other multitest kit approaches are only as good at identification of unknown bacteria as the database created and tested for them. Know how to determine the growth conditions for a BioLog assay and read the results (assuming the software ever works again!).
13. Know the general steps for the DNA extraction protocol we used, and be able to explain the purpose, i.e. what is happening, in each major step.
14. What are the three main steps of PCR, and what happens in each step? What are the ingredients for a PCR reaction? Why can we run repeated cycles of PCR without destroying the DNA polymerase?
15. What are restriction endonucleases? How are they used in generating a Restriction Fragment Length Polymorphism (RFLP), i.e. DNA “fingerprint”?
16. How does agarose gel electrophoresis work in separating DNA fragments by their size. What are the units of DNA size? After electrophoresis, what general steps must be done to visualize and document your results? Which reagent is most hazardous in this procedure (HINT: it’s involved in staining the DNA)?
17. Given photo-documentation of gel results, how do you collect quantitative data on DNA fragment (band) size for unknowns? Know how to generate a standard curve of DNA size versus migration based on marker DNA fragments of known size, and how to infer unknown DNA size from migration distance.
18. Why did we amplify the 16SrRNA? Did we amplify the whole gene? Which restriction enzymes did we use?