Experiment 7: Enzymes and Adaptation Lab Notebook Assignment

MCB 301 Spring 2015

Experiment 7: Enzymes and Adaptation Lab Notebook Assignment

24 points total

Purpose/Relevance (2 points)

Data/Results (4 points)

· Completed tables 1-4

Discussion (15 points)

· All results are clearly interpreted, hypotheses are made and evaluated for the obtained results and their deviation from the “expected” results. There is a good synthesis of the experiment and the results, particularly in the scope of a larger context. (10 points)

· What is tryptone? Why is it important and where is it specifically used with regards to the experiment you performed? (3 points)

· Some bacteria require certain growth factor(s) (e.g., purines, pyrimidines, amino acids, vitamins) to be added to the medium in order to grow. Other bacteria can synthesize the factor(s) from another carbon source, so they do not need the preformed factor(s) added to the medium. What two ingredients in some of the media used in the experiment supply vitamins for bacterial growth? (2 points)

Grammar and Format (3 points)

Experiment 8: Lactic Acid Bacteria Lab Notebook Assignment

65 points total

Purpose/Relevance (2 points)

Data/Results (20 points)

· Observation of any bubbles present in enrichment culture

· Measured pH of enrichment

· Shapes and arrangements of cells from enrichment culture

· Observation of colony appearance on TJA plate

· Cell characteristics from TJA plate: Gram reaction; shape and cell arrangement from wet mount; catalase reaction

· Observation of colony appearance on HIA-glucose plate

· Cell characteristics from HIA-glucose plate: Gram reaction; shape and cell arrangement from wet mount; catalase reaction

· Observation of colony appearance on HIA-sucrose/NaN3 plate

· Cell characteristics from HIA-sucrose/NaN3 plate: cell shape and arrangement

Discussion (40 points)

· Explain the purpose of each of the following in the lactic acid bacteria enrichment (4 points):

· Cabbage

· Glucose

· Salt

· Not screwing the cap on the bottle tightly

· Is the HIA-sucrose-NaN3 medium a selective medium, a differential medium, both selective and differential, or neither? Explain your answer (include what selective and differential media are, what organisms are selected/differentiated in our experiment and how?). (6 points)

Grammar and Format (3 points)

Experiment 9: Environmental Effects Lab Notebook Assignment

60 points total

Purpose/Relevance (2 points)

Data/Results (14 points)

· Completed Tables 1-5

Discussion (41 points)

· The discussion should also include whether or not the data you collected as a section was sufficient for you to make sound conclusions about each organisms tolerance for temperature, growth temperature, pH and oxygen use. (8 points)

· Summarize the major classifications of organisms for temperature class, pH and oxygen utilization. (6 points)

· Discuss how the results of the “Effects of Growth Temperature” experiment differs from the results of the “Effects of Temperature Tolerance” experiment. (4 points)

Grammar and Format (3 points)

Experiment 10: Sporeforming Bacteria Lab Notebook Assignment

63 points total

Purpose/Relevance (2 points)

Data/Results (15 points)

Anaerobic plates:

Description of growth

Catalase test results, if performed

Aerobic plates (Period 1):

Description of different colonies

Presence or absence of spores in wet mount

Catalase test results

NA plates (Period 2):

Presence or absence of spores in wet mount

Gram reaction

Catalase test results

Post enrichment:

Calculation of amount of glucose needed for cultures

Location of growth in each of the 3 anaerobic tubes (no shaking)

OD of 5 samples

pH of 5 samples

VP test on 5 samples

Discussion (43 points)

· What ingredients are in Nutrient Agar medium (besides agar) and what is the function of those ingredients? Is the NA medium a selective medium, a differential medium, both selective and differential, or neither? Explain your answer. (5 points)

· Describe how the catalase test works. Explain how the results of the catalase test is used in this Experiment. (6 points)

· Describe two ways that the microscope plays an important role in obtaining a pure culture of Bacillus or Clostridium from an enrichment. (2 points)

Grammar and Format (3 points)

Experiment 12: Transposon Mutagenesis Lab Notebook Assignment

62 points total

Purpose/Relevance (2 points)

Data/Results (17 points)

· State the total number of colonies obtained on each of the 8 transposant plates (LB-Kan)

· State the total number of potential mutants that you saw (whites, pinks or combinations).

· State the total number of colonies on the LB plate you counted.

· Calculate the number of cells/ml of transposants. Show your work.

· Calculate the number of cells/ml of recipients. Show your work.

· Calculate the transposition frequency. Show your work.

· State the number of potential mutant colonies that remained white, pink or a combination thereof after you re-streaked them. If you didn’t have any of your own potential mutants and re-streaked using colonies from someone else's plate, be sure to state this.

· Give the Gram stain results of all colonies that were tested.

· State all biochemical tests performed and the results of those tests, including all of the controls.

Discussion (40 points)

· How does kanamycin work? (2 points)

· Describe how pRL27 confers resistance to kanamycin to its host? (2 points)

· Where is diaminopimelic acid normally found? Be specific. (2 points)

· If a bacterial cell cannot make diaminopimelic acid and it is not available in its environment, then the cell may die. How? (2 points)

· A bacterial cell contains pRL27, but the cell does not contain the pir gene. Describe what happens if you produce a culture of the bacteria by growing it LB broth. (2 points)

Grammar and Format (3 points)

Experiment 13: Biochemical Tests Lab Notebook

43 points total

Purpose/Relevance (2 points)

Data/Results (8 points)

· Known Organism

· Table of each test performed, with result and appearance

· Table of all 12 organisms with a written/illustrated description of each

Discussion (30 points)

· Why is a color change indicating acid production in a fermentation culture more meaningful than no color change in the same medium? (2 points)

· Explain why a pH indicator that has a lower pH range is not as effective as a pH indicator with a higher pH range, when using in a test medium that is buffered well. (2 points)

Grammar and Format (3 points)

Exercise 14: Identification Lab Notebook

47 points total

Purpose/Relevance (2 points)

Data/Results (13 points)

· Isolation of bacteria, both Gram-positive and Gram-negative. TA must initial your lab notebook in order to receive credit.

· Record your observations of any growth on MacConkey agar

· Gram reaction and shape of both isolates

· Dichotomous key, Be sure to include both organisms (Gram-positive and Gram-negative) in your key. Note that the key must be initialed by the TA before collecting any biochemical test media.

· Table of biochemical test results for each isolate

· Identification of each of your unknown organisms

Discussion (29 points)

· What is the key step in any successful identification of a bacterium from a natural source, such as a human with an infection, and why? (2 points)

· Why are morphological characteristics alone not sufficient to identify an unknown bacterium? (2 points)

· How does knowing the source of a bacterial specimen make its identification easier? (2 point)

Grammar and Format (3 points)

Experiment 15: Southern Blot Lab Notebook Assignment

65 points total

Purpose/Relevance (2 points)

Data/Results (20 points)

· Labeled photograph of gel

· Final Southern blot membrane

· Make a table of the bands on your photograph showing:

· The size in kilobases

· The DNA content of each band. Is it vector DNA, insert DNA or a combination of both vector and insert DNA?

· Is the band present on your blot, yes or no?

· Using your data draw a restriction map showing where the sites are located with respect to one another on the pGEX/rpoD plasmid. Use the map of pGEX as a reference. Be sure to include the relative location of each restriction site by specifying the position number or approximate distances in kilobases between all the sites that you have indicated. If any sites cannot be accurately placed on your map, explain the problem.

Discussion (40 points)

· Based on the pattern of bands detected on your Southern blot, what is the identity of your probe? Does your probe contain the rpoD gene sequence? (2 points)

· How do the results obtained using the other probe compare with your results? Are different bands detected? How does this illustrate the principle of probe specificity? (2 points)

· Describe the plasmid used in this Experiment (e.g., size, genes, etc). (3 points)

· Based on your agarose gel results, were there multiple bands in the lane containing undigested plasmid DNA? What would multiple bands represent and why would they migrate differently? (3 points)

Grammar and Format (3 points)

Experiment 16: ELISA Lab Notebook

26 points total

Purpose/Relevance (2 points)

Data/Results (4 points)

· Copy of Table 1

Discussion (17 points)

· Which tubes of the mock HIV were the “infected serum” samples? Explain your reasoning. (2 points)

· Think about the intensity of the reactions with respect to the dilutions on the plate. Is this information helpful in tracking the infection? Why or why not? (3 points)

· An HIV ELISA should be performed after 6 weeks of initial exposure to HIV. Why? (2 points)

Grammar and Format (3 points)

Experiment 17: MIC Lab Notebook

24 points total

Purpose/Relevance (2 points)

Data/Results (3 points)

· Completed Tables 1 and 2

Discussion (16 points)

· Using your data, state concisely the effect in E. coli of cat gene dosage (0 copies, one copy, or multiple copies) on the amount of cellular resistance to chloramphenicol. Your answer should include a description of the effect on the amount of CAT enzyme produced and the consequent effect on ribosomal activity. (6 points)

· Does the procedure we used in this exercise determine if an antibiotic is bacteriostatic or bacteriocidal? Explain your answer. (2 points)

Grammar and Format (3 points)