Your favorite patient has just had surgery. After surgery, Bactroban was applied. After a few days, you notice an infection around incision. Even though more bactroban is applied, the infection persists. You want to identidy the bacteria causing the infection to determine what treatment is best. First, you need to collect a sample.

1) What kind of medium do you place your swab into to grow your bacteria?

2) After incubating for 24 hours, will you have a pure culture? give an explanation for this!

3) what is the next step and what is the purpose of this?

After isolating different species of bacteria, you see two distinct types of colonies.

-One has medium-sized shiny colonies that are mucoid with entire margins, convex elevation and a whitish translucent appearance. This colony type is the dominant type on the plate.

-The other has circular, pinhead colonies which are convex with entire margins

and have a yellowish-white color. There are only 2 colonies that appear this way.

You make a pure culture of each type of colony using half of one colony, labeled with its colony description. You take the other half of the colony you used to make your pure culture, and perform a simple stain to determine the size and shape of the different bacteria.

4. What kind of simple stain would you perform and why? What color will the cells appear when looking through the microscope and why?

5. Do you think the cells’ shape has been affected by the stain? Why or why not?

6. You determine that the cells from the whitish translucent colony are rod shaped. What term do you use to describe this cell type? ____________

You find that the cells from the smaller, yellowish-white colonies are circular and seem to appear in clumps. What cell type is this? ________________

7. After you incubate your pure culture for 24 hours, you perform a gram stain. You find that your circular cells have stained purple. What does this result mean? Don’t just answer gram+ or gram-. Give a brief explanation what this reaction is based on!

8. You find that your rod shaped cells have stained pink. What does this result mean? Don’t just answer gram+ or gram-. Give a brief explanation what this reaction is based on!

Summarize your results:

Colony Appearance

Cell Shape

Gram Stain

9. Based on the gram stain results, which bacteria do you think is likely causing the infection? Give your reason.

10. If you plate the bacteria causing the infection, will it grow on

a. LB agar?

b. GMS agar?

c. PE agar?

11. What tests can you now perform to help identify the problem bacteria?

a. You decide to start with fermentation tests. You incubate your tubes for 24 hours. In both the PRB glucose tube and PRB lactose tube you see yellow broth and an air bubble in the Durham tube. What does this mean?

Summarize your results:

Glucose

Appearance Result

Lactose

Appearance Result

Now that you know the fermenting capabilities of your bacteria, you decided to run the IMViC tests. The first test you try is the Citrate Utilization test.

a. When making the Simmon’s citrate media, you added glucose along with the citrate. Will your test yield accurate results if you use this media? Why or why not?

b. If you don’t add glucose to the Simmon’s citrate media, will your test results be accurate? Why or why not?

c. After running the test properly, you find that the media is a deep blue color and there is growth on the slant. What does this result mean?

d. Next, you perform the Indole test. You find out that your bacteria cannot degrade tryptophan. What does this tube look like?

e. You inoculate your bacteria into 2 MRVP tubes and incubate them for 72 hours. When you perform the methyl red test with one of the 2 MRVP tubes, the media retains a yellow color. What does this result mean? Give 2 possible explanation for this result!

i. Now you perform the Voges-Proskauer test using the second MRVP tube, and the media develops a rose color. What does this result mean and does this result help to interpret the result of the methyl red test? Give a detailed explanation!!!

Summarize your results:

Methyl Red

Appearance Result

Voges-Proskauer

Appearance Result

Indole

Appearance Result

Citrate

Appearance Result

12. Knowing all these different test results can you clearly identify the bacterium that is causing the infection? If not what additional test(s) would you do to identify the bacterium? Give a detailed explanation! (Hint: You can use your results from class to help answer this. You may need to do some extra searches outside of the book.)

13. You decide to perform the litmus test as your final test. You inoculate a tube containing litmus milk medium and incubate the tube for 24hrs. What would you expect to see when you look at the tube the next day? Give a detailed explanation!!!!!

14. Based on what you know so far, what antibiotic might you recommend? Why?

15. Can you transform these cells following the protocol in Chapter 19 of your lab manual? Why or why not?

16. In order to conclude this series of experiments you would like to extract genomic DNA from your identified bacterium. Your supervisor tells you to get 5 X 109 healthy bacterial cells for the extraction. What would you do to get these cells? Give a detailed explanation!!!