Microbiology

Experiment Procedure

Background: Traditionally many microbes have been identified using what we call differential tests. A differential test is any test that may allow you to differentiate to different types of microbes. Some of the more common differential tests include: Gram stain, Mannitol Salts Agar, and Eosin Methylene Blue Agar. In this experiment we will be testing our two yeast species using a new type of broth I am calling Yeast Carbon Utilization Broth. The purpose of this broth is to test if the microbe will be able to use the carbohydrate included in the broth. The only carbon source available in the broth is the carbohydrate added. This means that we should be able to detect if each yeast can use the three different carbohydrates, I have made broths with. If this broth works as planned you should see the broth get cloudy and change color as the yeast uses the carbohydrate. The color change is based on the response of a pH indicator to changes in pH. As the yeasts use the carbohydrate, they should produce acidic byproducts (carbonic acid is formed from CO2 mixing with water). This should cause the pH to drop which will change the color of the pH indicator. We are seeking the answers to two related questions. First is just if the broth works. It is hypothesized that the broth will work. It is predicted we will see color changes and cloudiness indicating that the broth did work. The second related question is if the pattern of sugar utilization will match the published results for these species. It is hypothesized that the pattern will match what is published and we will see Saccharomyces cerevisiae use only glucose and Xanthophyllomyces dendrorhous to use all three sugars. It is predicted that the S. cerevisiae glucose tube will show both a color change towards the green and cloudiness. X. dendrorhous is predicted to show a color change in glucose, xylose and cellobiose broths towards the green and cloudiness.

Procedure: 1) Disinfect your work area with alcohol or bleach

2) Put on a pair of gloves and gather supplies. You will need at least two loops (more is better if you still have them) and your Yeast carbon utilization broths (they may be packaged in two separate places). You should have a total of six: two with black lines on the lid (cellobiose), two with red on the lid (glucose) and two with green on the lid (xylose). You will also need a slant of each of the yeast species.

3) Label the broth with a date, your initials and what sugar it contains. Then label one set with S. cerevisiae (one glucose, one xylose and one cellobiose). Then label the other set with X. dendrorhous. It’s also fine to use S.c. and X.d. to save on space but write clearly!

4) Make sure to practice aseptic technique as you do the following steps.

5) Open a sterile loop and transfer a small amount of S. cerevisiae to the tube labeled S. cerevisiae glucose. After transferring, close the cap and mix by gently inverting the tube a few times.

6) If you have loops left, dispose of the loop and use a NEW loop. If you are running low on loops use the same loop as step 5 to transfer a small amount of S. cerevisiae to the tube labeled S. cerevisiae xylose. After transferring, close the cap and mix by gently inverting the tube a few times.

7) If you have loops left, dispose of the loop and use a NEW loop. If you are running low on loops use the same loop as step 6 transfer a small amount of S. cerevisiae to the tube labeled S. cerevisiae cellobiose. After transferring, close the cap and mix by gently inverting the tube a few times. Dispose of loop. 8) Open a sterile loop and transfer a small amount of X. dendrorhous to the tube labeled X. dendrorhous glucose. After transferring, close the cap and mix by gently inverting the tube a few times.

9) If you have loops left, dispose of the loop and use a NEW loop. If you are running low on loops use the same loop as step 8 transfer a small amount of X. dendrorhous to the tube labeled X. dendrorhous xylose. After transferring, close the cap and mix by gently inverting the tube a few times.

10) If you have loops left, dispose of the loop and use a NEW loop. If you are running low on loops use the same loop as step 9 transfer a small amount of X. dendrorhous to the tube labeled X. dendrorhous cellobiose. After transferring, close the cap and mix by gently inverting the tube a few times. Dispose of loop.

11) Take a picture of all six of your tubes with the labels visible. Find a relatively warm place to set your tubes to incubate (slightly above room temp 75º F).

12) Clean up your work area and disinfect it after you remove and dispose of your gloves.

13) Mix the tubes every other day if possible. Record any color changes and visible growth as you mix them. I am expecting the color to start to change after about two days and growth to be visible starting around the third day but this may vary depending on how warm they are.

14) After about one week take a picture of all six tubes with the labels visible. This will be the maximum time we will incubate them.

Results from Experiment

Class Average Data of 22 entries

S. cerevisiae   

Cloudiness pH (use indicator color)

D-Glucose Yes 3.8

D-Xylose Yes/No  5.2  

Cellobiose Yes 4.9

X. dendrorhous  

 Cloudiness pH (use indicator color)

D-Glucose Yes 3.8

D-Xylose Yes 5.0

Cellobiose Yes 4.0

Sugar	Saccharomyces cerevisiae	Xanthophyllomyces dendrorhous
D-Glucose	Positive	Positive
D-Xylose	Negative	Positive
Cellobiose	Negative	Positive

Hypotheses - A hypothesis is a statement backed by evidence. Persike, D. S., Bonfim, T. M., Santos, M. H., Lyng, S. M., Chiarello, M. D., & Fontana, J. D. (2002). Invertase and urease activities in the carotenogenic yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma). Bioresource technology, 82(1), 79-85.

Barnett J, Payne R, Yarrow D. Yeasts: Characteristics and Identification. 3rd ed. Cambridge, UK: Cambridge University Press; 2000. (no e-version available; take my word for it)

Vázquez, M. (2001). Effect of the light on carotenoid profiles of Xanthophyllomyces dendrorhous strains (formerly Phaffia rhodozyma). Food Technology and Biotechnology, 39(2), 123-128.

Will need to provide additional four sources

For experimental question Read Experimental Procedure

For Methods: Broth Recipe ● 1 L Deionized water ● 6.7 g Difco Yeast Nitrogen Base (#239210) ● 5 g Sugar (Either Cellobiose, D-Glucose, D-Xylose) ● Adjust pH to 5.7 with HCl or NaOH ● 5 mg Bromocresol Green Combine first 3 ingredients using only sugar per batch. Adjust pH. Filter sterilize and aseptically dispense into 2.5 mL tubes.