On this page is a set of general review questions for
which there is presently no posted key.
1. Relevant to just plain, old Common Sense: Technicians A and B work in a water quality lab. They received a sample of water from the Vilas Park Beach and they found that they were out of sterile saline which they usually use for a diluent, so they used the next best thing on hand: sterile Brain Heart Infusion Broth.
Technician A made decimal (1/10) dilutions of the water sample and then inoculated plates from the dilutions. Technician B did the same, but he got distracted and let his first 1/10 dilution sit around for a few hours before making subsequent dilutions and platings.
After incubation of the plates, Technician A got a count of 4.4 X 102 CFU/ml and Technician B's count was 4.9 X 106 CFU/ml. Who had the more reliable count of the water sample? Why?
(Note: Both technicians performed specific techniques regarding dilutions, inoculations and incubations correctly. The answer has nothing to do with those techniques.)
2. Relevant to our enrichment/isolation experiments (9.3, 10.2, 11.1, 11.2, 11.3): Fill in the blanks on the table given here. When we get to other specific groups of bacteria (enterics, coliforms, etc.), the same considerations apply – that is, we can extend the table to accomodate these organisms and fill in the blanks with the relevant items.
3. Relevant to the energy-generating processes discussed in Experiments 5.1, 7.1 and 11.1: You should be able to match the energy-generating processes in column b with the items in column a. (One letter per blank. Some items in column a have more than one blank.)
Two processes which are responsible for the patterns of growth seen in Thioglycollate Medium that help in defining "oxygen relationships."
Two processes which are possessed by the purple non-sulfur photosynthetic bacteria. We set up a test for these (in Experiment 11.1, Period 3) which was similar to the test for "oxygen relationships."
The process associated with nitrate reduction.
Three processes which are associated with anaerobic growth.
The process responsible for creating anaerobic conditions in our photosynthetic enrichments (and in media overlayed with mineral oil in the enteric experiment).
A. aerobic respiration
B. anaerobic respiration
D. oxygenic phototrophy
E. anoxygenic phototrophy
4. More about the nature and definitions of "cells" and "colony-forming units": First consider the organism Aerococcus viridans and the characteristic arrangement of its cells. Now, suppose you had a broth culture in a tube which you vortexed vigorously for a minute or two. Just before and immediately after this vortexing, you made dilution plates to determine the CFU/ml of the culture. After incubation of the plates, you find that the pre-vortexing count was approximately 2 X 108 CFU/ml, and the post-vortexing count was approx. 8 X 108 CFU/ml. As you realize the number of cells in the culture would not have changed between the inoculations of the two sets of plates, how do you account for the increase in the count?
5. Relevant to Experiment 11.2 – relating the age of a Bacillus culture to the staining and morphological characteristics of the cells: You believe you have isolated a species of Bacillus, and an isolated colony is on a plate which was incubated for two days. What two staining procedures could you perform on the colony which would help to confirm your diagnosis? Also, what portion of the colony (center or edge) would you use for each stain, and why?
6. Comparing growth curves (which are drawn according to periodic counts made for broth cultures) and colony development – similar in part to the above question: You have a colony of Bacillus cereus which has been growing for two days on a plate, constantly expanding outward ("colonizing new territory") as the vegetative cells continue to grow and divide. Might various points from the center to the edge of the colony correspond to particular points or stages on a growth curve which could be made for a broth culture of B. cereus? How might this relate to differences one may see in the gram reaction and presence of endospores between the center and the edge? (Note: Can one truly say "old" cells and "young" cells? Recall the introduction to Experiment 1.)
7. Relevant to Experiment 14 and the negative consequences of bad streaking and isolation: Here is a "faulty diagnosis" question, showing how following correct procedures (such as the minimum aseptic and safety procedures in Appendix B of the lab manual) can be very critical.
A patient was admitted with signs of a severe bacteremia (bacterial blood infection). Technician A took a blood culture and determined that the cause of the infection was a typical Salmonella. The patient was then treated for Salmonella blood infection but did not respond to treatment. Classic signs of another disease began to appear.
Technician B came in and took another blood culture and correctly determined that (1) Salmonella was not present and (2) there were actually two different organisms in the blood, one of which was Yersinia pestis, the causative agent of plague.
Consider the following table as you answer the questions below. (Note: Maltose, mannose and mannitol are sugars or sugar-like compounds which can be fermented by some organisms to produce acid.)
8. Relevant to the most-probable number method and Experiment 9: You decide to do a most probable number analysis to enumerate bacteriophages in soil, specifically those which can lyse E. coli strain B. A 1/10 dilution of soil was made, and this suspension was passed through a filter in order to remove microbial cells and soil particles. From the filtered material, two more 1/10 dilutions were made (up through 10–3).
From each of these three dilutions, flasks of Nutrient Broth were inoculated; each flask was inoculated with either 10, 1 or 0.1 ml as shown in the table below. Immediately following this procedure, each flask was inoculated with E. coli strain B.
After incubation, the flasks were checked for turbidity (growth of the host culture), and the results were tabulated as shown in the table below. From these results, what would be the most probable number of plaque-forming units (PFUs) per gram of the soil?
(A hint: What actually constitutes a "positive reaction" in these flasks? Utilize the bottom row of the following table. Figure that out and use the MPN table accordingly!)
|dilution of soil||10–1||10–2||10–3|
|amount of the dilution inoculated into each of three flasks||10 ml||1.0 ml||1.0 ml||1.0 ml||0.1 ml|
|number of flasks showing turbidity||0||1||2||3||3|
|number of flasks showing a "positive reaction" can be indicated here, for convenience|
9. Relevant to our phage-infectivity and conjugation experiments: In Experiment 9.2, we found that the Hfr strain we used in Experiment 8.2 is resistant to phage JL-1, and the F– strain is sensitive to the same phage strain. If we were to find the opposite to hold true regarding another phage strain (we can call it JL-2), how could we set up an experiment to demonstrate the result of conjugation and recombination involving the transfer of phage resistance genes from the Hfr strain to the F– strain? Recall what was done in Experiment 8.2. Included in your experiment would be a consideration of the selective medium we would use to detect the desired recombinants.
10. Relevant to dilution plating and Experiment 15: Is this set-up OK?
11. Relevant to dilution plating and Experiment 15: Consider the following set-up, and again recall our old definitions (from Appendix C) of "dilution factor" and "plated dilution."
12. Relevant to differential media and Experiment 17: For your Experiment 17 unknown, you find you have two colony types (=2 different gram-negative rods) on your MacConkey plates, and you need to find your third unknown. Looking at the colonies on your HIA plates, how would you go about finding your third unknown?