Name__________________TA___________________ Date__________________

Microbiology Lab EPOB 3400

Procedure: The first half of the test is not associated with practical stations. The second half of the test is associated with practical stations. The last question on the exam involves a demonstration with your TA.

Non-Practical Questions

_F__1. True or False: An isolated colony is derived from around 5 original cells. (2 pts.)

_b_2. Many of the treatments that were employed for the food-microbiology lab generated large numbers of bacteria after the plates were grown for 24 hours, but we detected very few fungi. Based on what you know to be true about fungi, this could be because: (2 pts.)

a. Fungi in the environment are not readily culturable

b. Fungi typically grow more slowly than bacteria

c. Bacteria outnumber fungi in all environments

d. Fungi require media with lower carbon to nitrogen ratios than bacteria

e. a and c

_e__ 3. Which of the following is/are methods for enumeration on bacterial cell densities? (2 pts.)

I. turbidity Analysis

II streak plates

III. MPN

IV. inhibition zone measurement

a. I and II

b. II and III

c. II, III, and IV

d. III and IV

e. I and III

4. PCR is a technique that allows researchers to amplify a particular segment of DNA. PCRs all contain the same basic ingredients. Circle all of the ingredients that are required for a successful PCR. (2 pts.)

a. Taq polymerase

b. SYBR green

c. Agar

d. Primers

e. Ca²⁺ ions

5. A __presumptive___ test is a general name for a test used to quickly screen for indicator microbes that are potentially present and how many. (2 pts.)

6. You wish to observe colony morphology on a plate of media. In this situation, you would use a __dissecting or stereo___ microscope. (2 pts.)

7. ___magnification_____ is how many times larger a sample appears when viewed with a microscope. ____resolution_____ is a metric of the fineness of detail a microscope is capable of showing without distortion. (2 pts.)

8a. What type of organism does Taq polymerase come from? (2 pts.)

Answer is variable: Thermus aquaticus or bacteria that live in thermal hot springs.

8b. Why is this important to PCR procedure? (2 pts.) Enzymes do not denature at high temperatures so when cycle increases temp to 95 degrees, enzymes are still intact and only need to be added once instead of every cycle.

9. Suppose you are attempting to perform the indole test on the bacteria from a swab of your ear. You have enriched the bacteria in a tube of TSB. You have plates of tryptic soy agar, Indole cards, mannitol salt agar plates, and TSA slants. What is your next step? (2 pts.)

I will quadrant streak to isolate pure colonies, as isolated colonies are required for all metabolic tests.

10. To quantify results for the food microbiology exercise, we used the pour plate method, and using our experimental samples, we constructed a series of dilutions between 10² and 10⁶ in order to get at least one plate containing between 30 and 300 colonies. Briefly indicate why a plate with >300 colonies is problematic, and why a plate with <30 colonies is problematic. (2 pts.)

Statistically counts in this range give the most reliable data. Counts less or greater are less reliable and are more prone to error.

11. In order for bacteria to be capable of taking up DNA, the cells must be competent. List two possible ways to increase transformation efficiency, and then address how these processes specifically affect the cells to enhance their ability to take up DNA from the environment? (4 pts.)

12. Three procedural controls are essential when performing the Kirby-Bauer method so that the results can be compared either with the literature or with other researchers’ results.

a. List two of the three controls. (2 pts.) Agar poured to uniform thickness, 24 hour culture, 0.5 McFarland Standard

b. Indicate how 1 of the two controls listed acts as a control. (2 pts.)

Thickness for diffusion, 24 hours for log phase growth, McFarland Standard for concentration uniformity.

13. When using the Kirby-Bauer method to determine anti-microbic resistance for a species of bacteria, zones of inhibition are measured and the diameter obtained is assigned to one of the following results: “Sensitive,” “Resistant,” or “Intermediate.” How is the Kirby-Bauer method calibrated so that these assignments can be made with confidence? (4 pts.)

MIC used in conjunction with disc diffusion tests with media.

14a. How do fluorescent dyes work and give an example of their use? (2 pts.)

Excited light is absorbed by dye, energy is lost, light sent back out at a lower energy in the form of a longer wavelength.

14b. Give an example of the use of fluorescent dyes. (2 pts.) Many answers possible.

15. Epidemics are the result of the fact that most diseases, left untreated, can spread through populations rather quickly. Give an intrinsic feature of bacteria and an extrinsic environmental factor that could be responsible for rapid spread of bacteria caused diseases.

a. Intrinsic: (2 pts.) many answers possible

b. Extrinsic: (2 pts.) many answers possible

16. Suppose you are designing an experiment to estimate the number of microbes in a solution. Diagram how you would obtain a series of plates with dilution factors of 10^-3, 10^-5, 10^-7, and 10^-8 (to calculate the cell/ml of the stock solution). Note: specify all volumes needed. (4 pts.)

Many answers possible.

17. A researcher at CU is studying microbial life in and around deep-sea volcanic vents. Using traditional culture methods she has isolated 107 different unidentified strains of what she thinks are a mix of Bacteria and Archaea. Discuss how useful a Gram stain would be in narrowing down what types of microorganisms are represented by her sample. (4 pts.)

Many answers are possible as long as logical reasoning is there. Gram stain not useful for Archae and Gram negative bacteria but is useful for separating out gram positive bacteria.

Practical Questions

Station 1: Three microscopes set up for brightfield labeled A, B, and C. All three scopes have good ocular micrometer. Each scope with a stage micrometer and a slide of bacteria labeled A, B, and C. Oil, lens paper,

1. Choose any of the three microscopes labeled A, B, and C. Write the letter corresponding to the microscope in the space provided.

______ Microscope Letter

a. Calibrate the ocular micrometer – show your work. (2 pts.)

___________ b. Estimate the size of a typical cell from the microscope slide. (2 pts.)

Station 2: A microscope with a badly done Gram Stain. Do not decolorize well so stain is iodine colored.

2. The slide under the microscope has been gram stained, but the student who stained it is having trouble obtaining information from it. Can you help him and troubleshoot his technique? (Offer some suggestions and explain why the suggestion should help. ) (3 pts.)

Station 3: A bottle of Mueller-Hinton Agar and a calculator.

3. You are setting up for an experiment that requires exactly 72 plates of Mueller-Hinton Agar. As media is expensive, you must calculate how much media to make. You will be pouring exactly 25ml of media into each plate. How much media powder and how much water will you mix to prepare this media? (2 pts.)

_____________ media powder _________________ water

Station 4: two plates, one containing multiple organisms (including fungi if possible – Plate A, and the other containing only one or two species of bacteria that are also on the first plate (Plate B).

4. Given: A gardener had two eggplants growing in separate 1 gallon pots. One eggplant began to wither and die, while the other appeared quite healthy. Wondering what might be the matter the gardener sampled the soil and used the pour plate method to see what could be cultured. Plate A is from the dead eggplant and Plate B is from the healthy eggplant.

Using the dissecting scope, examine the plates and devise a hypothesis that could explain the observations. (2 pts.)

What else do you need to know before you can tell the gardener what likely happened? (2 pts.)

Station 5: Have 5 pipettors out. A = P1000 set at 1,000, B = P1000 set at 100, C = P200 set at 100, D = P10 set at 10, E = P2 set at 1.

____C possibly B_________ 5. Which pipette or pipettes could you use to dispense 100ml. (2 pts.)

Station 6: Color diagram with a plate and some colonies. ½ of colonies are blue and ½ of the colonies are white.

6. Given: A student accidentally developed a new plasmid with a special gene that could break down oil efficiently. The plasmid also had a b galactosidase gene and an ampicillin resistance gene. The student tried to transform some bacteria with the plasmid. The transformed bacteria were plated on LB broth with ampicillin, then X-gal. A diagram of the resulting plate is in front of you.

What went wrong? (3 pts.) Did not fully spread out X-gal

Station 7: Two EMB plates labeled 1 and 2 with a green sheen. 1 EMB plate labeled 6 with pink colonies. 2 EMB plates labeled 7 and 8, one with pink colonies and one with greenish colonies.

7. Given: A student was testing a sample of water. She inoculated three tubes of double-strength, lauryl-tryptose broth with 10 ml of the sample (Tubes 1, 2, and 3), three tubes of single strength lauryl tryptose broty with 1 ml of the sample (tubes 4, 5, 6), and three tubes of single-strength, lauryl –tryptose broth with 0.1 ml of sample (tubes 7, 8, 9). Then she inoculated EMB plates from the positive tubes. The plates are present.

_________ What is the MPN of coliforms from the presumptive test only? (2 pts.)

________ What is the MPN of E. coli? (2 pts.)

Station 8: Have three identical stations labeled A, B, and C. At each station have a spec, Cuvette A = culture and PGYB. Cuvette B = culture and TSB, Cuvette c = Sterile Water. Cuvette d = Sterile PGYB, Cuvette e = empty, Cuvette E = McFarland Standard, cuvette F = TSB, Standard Curve with number of bacteria (Bacillus subtilis) compared with absorbance. Culture A should be fairly dense.

8. Suppose Dr. Sal Monella created a new medium in which to culture Bacillus subtilis called peptone glucose yeast broth (PGYB). He contended that Bacillus subtilis grew twice as fast in the broth than in trypticase soy broth (TSB). So he inoculated both types of media with Bacillus subtilis for 12 hours represented by culture A and culture B.

Which cuvette should you use to blank the spectrophotometer when determining absorbance for culture “A”? (2 pts.)

What is the absorbance for culture B? (2 pts.)

Are the results of this study consistent with Dr. Sal Monella’s contentions? Explain why with specific numbers and evidence from the study. (4 pts.)

Station 9: A gel with 5 lanes. Lane 1 a ladder, lanes 2 and 3 a PCR product with a band. Lane 4 is megapure water as a negative control with a smear. Lane 5 is just DNA as a positive control. The positive control goes a different distance on the gel than the PCR products.

9. A CU researcher is attempting to use the 16s gene, a gene commonly used to identify prokaryotes, to identify isolated bacterial colonies she has grown on solid media. She has extracted DNA from the colonies using an extraction kit, and now has pure genomic DNA in a buffer solution. Before sequencing of the 16s is possible, the gene must be amplified with PCR, to create the many copies of the isolate’s individual 16s genes required for sequencing. The researcher performed a PCR, then used gel electrophoresis to verify that the reaction was successful and to measure her amplified fragment. Her gel looked like this after staining with SYBRGreen:

Lane 1 was the DNA ladder.

Lanes 2 and 3 used extracts from two colonies as templates, and these were the PCR products she intends to sequence.

Lane 4 was a reaction that used nuclease-free megapure water with no DNA as a template, to serve as a negative experimental control.

Lane 5 was an extract of E. coli DNA, which she used as a positive experimental control.

Describe what each lane of the gel indicates. (2pts) Lane 1 has a ladder. Lane 2 has a nice band from a PCR product. Lane 3 has a smeared band from the PCR product. Lane four has a smeared band from the water control. Lane five has a nice band from the known control.

Can she use this PCR for sequencing and reliable identification of the unknown bacteria she has isolated? Why or why not? (2pts) No, because the negative control of the sterile water has a smeared band indicating it is not pure. So the PCR products in both lanes 2 and 3 could be contaminated and should not be used until the

What could have caused the PCR to turn out the way it did? (2pts) The negative control is contaminated with a band about the same place as the PCR products, indicating that the PCR products could have come from the contaminated water.

Station 10: Illustrator of plate with amp and X-gal with white colonies and ampicillin with green colonies.

10. Plate A has ampicillin and X-gal. Plate B has ampicillin without X-gal. Draw examples of possible plasmids that each type of bacteria could have been transformed with that would result in each phenotype. (3 pts.)

Station 11: A plate containing between 30 and 300 colonies on it (preferably of some colorful species.

11. Given: An inspector obtained 0.25g of a tissue sample from a chicken that apparently died from pustulent flesh wounds at an organic chicken farm in Petaluma, CA. The inspector macerated the tissue in sterile water, and brought the final volume up to 50 mL. Then, 1 mL of the chicken solution was further diluted by adding 1 mL to 19 mL sterile water. The inspector then plated 500 ml of the latter solution onto the plate you see here, and allowed it to grow for 24h at 37 ºC.

Relative to the starting tissue sample, what was the final dilution factor (per gram of sample) that was achieved on the plate? (2 pts.)

Calculate the number of bacteria per gram of the tissue sample. (2 pts.)

What species are most likely present on an infected chicken? (2 pts.)

Station 12. Agar plates, loops, needles, culture, bunson burner (shall we make them light the bunson burner?), striker,

Given: You suspected that your stock culture was contaminated and you wanted to get back a pure stock. So you grew a broth culture from your slant overnight. The broth culture is in front of you. You are now ready for the next step, which you will perform for your TA and your TA will grade you on your technique.

_________ 12. Perform the technique with the culture provided. Assume that you have already disinfected your desk.

0.5 Mix tube

0.5 Flame loop

0.5 Flame lip of tube

0.5 Hold cap properly while dipping loop – don’t set down

0.5 Flame lip of tube again before recapping

0.5 Open plate using “clamshell” technique

1.0 Proper streaking technique

1.0 Plate after incubation