Activity 3: Lab: Culturing Yeast Cells on Media

Materials Needed

Part 1: Serial Dilutions of Yeast Broth Equipment and materials for students
Equipment and notes	Quantity per group (recommend 2-4)
□ Test Tube Rack	1
□ Test Tubes	4
□ Flask (recommend 1000mL) with water	1
□ Pipette Pump (10 mL)	1
□ Pipette (10 mL)	1
□ Yeast Broth	1 ml
□ water	50 mL
□ Lab Tape	2 inches

Part 1: Serial Dilutions of Yeast Broth Equipment and materials for teacher preparation
Equipment and notes	Quantity
□ Baker’s yeast ~Saccharomyces cerevisiae (Red Star® was used when preparing this activity)	1 gram per student group
□ Warm water	50 mL per student group
□ Beaker (500 mL)	1
□ Microwave or hot plate	1

Part 2 and 3: Inoculation of Media with Yeast Cells on Media Equipment/Observation of Cell Colonies Equipment and materials for students
Equipment and notes	Quantity per group (recommend 2-4)
□ Petri dish with YED, YPD or YEPD medium	1
□ Disposable Loop	1
□ Lab Coat or Apron	1 per person
□ Safety Glasses	1 per person
□ Safety Gloves	1 pair per person
□ Incubator (optional)	1 for entire class
□ Marking pen	1
□ 70% Ethanol solution (in spray bottle)	1-2 for entire classroom use

Part 2: Inoculation of Media with Yeast Cells on Media Equipment and materials for teacher preparation
Equipment and notes	Quantity
□ Petri dishes	1 per student group
□ Magnetic stirrer/hot plate	1
□ 500-1000mL glass beaker	1
□ Magnetic stirring bar	1
□ Distilled/sterile water	500 mL per 15 plates
□ YEPD agar or equivalent recipe	35 grams per 15 plates
□ Scale	1
□ Weigh Boat	1

Preparation Instructions

• Set up the test tube racks with four test tubes per group.
• Place the other equipment and supplies at the student work stations or in a central location where they can be located by group members and taken to their work stations.
• Prepare the yeast broth by dissolving one (1) gram of Baker’s Yeast in fifty (50) milliliters of warm water. The broth can then be placed in small plastic cups to be dispersed to each group.
• Before the class meets, prepare the media and pour in petri dishes. Allow for at least one dish per two students. More can be used if time and materials allow.
• The teacher will need to prepare media for culturing cells. The most effective media for growing yeast cells is YPD (Yeast Peptone Dextrose) or YED (Yeast-Extract Dextrose).
  • Here is a recipe for 1 Liter of Yeast-Extract Dextrose:
  • 10 g Yeast Extract
  • 20 g Anhydrous Dextrose
  • 20 g Agar
  • 1 Liter Water
  • Using a hot plate, stirrer and glass beaker, boil until all powders are completely dissolved into the solution. Allow the media to cool and then pour into dishes. After plates have been poured, allow them to cool and solidify at room temperature. Store the plates in a plastic sleeve, upside down, in the refrigerator.
• YED media is available from Carolina Biological with agar added. Follow the guidelines that come with the media to ensure successful media preparation. On YED media, the yeast cells will culture at room temperature in forty eight hours. Or, they will culture in twenty four hours in the incubator at 30° Celsius.

Class Demonstration

Students should be familiar with the Serial Dilutions technique prior to conducting this activity. If they are not, conduct the Pre Lab Serial Dilutions Exercise first.

Review the concept that serial dilutions reduce the concentration of the sample in small steps or fractions. If the yeast broth was directly placed on the growth medium in the petri dish, the growth would be too great to measure and there would be very little difference from group to group. This step will ensure a more accurate result. Also, diluting the sample will allow groups to see individual colonies of yeast cells that grow on the media.

If this is the students’ first experience with media, perform a demonstration on proper handling of the materials and aseptic technique. Set up an area in the class room that is a “clean zone.” Spray the area with 70% ethanol and wipe with paper towels. Explain to students that the techniques being demonstrated will help to ensure that the only cells that grow on the petri dishes are yeast. Being careless in handling and inoculating the dishes could result in contaminating them with germs from the classroom. Remind students to keep the plates closed except when inoculating them. Also, review the proper streak method and have students practice the method, using their pencils, on their lab sheets. Wearing gloves will help reduce contamination from their hands. Have a labeled “biohazard” bag for disposal of the loops.

Recommended streak pattern:

Pre Lab Definitions

The teacher can use the Power Point to review the pre lab definitions that the students have on their lab sheet.

• Petri Dish: A round, shallow dish used to grow bacteria.
• Culture: To grow living organisms in a prepared mixture of nutrients (media).
• Media: Substance containing nutrients needed for cell growth.
• Agar: Jelly to which food has been added for the growth of microbes.
• Inoculate: To introduce a microbe to an environment where it can grow.
• Sterile/Aseptic Technique: Laboratory procedures used in handling cultures, media and equipment that prevent contamination.
• Colony: Group of bacteria.

Conducting the Lab

Students will follow the directions on the lab sheet to perform the serial dilutions for their sample. Be sure students mix the samples thoroughly in between each step. Mixing can be done with a vortex or by pipetting the contents of the tube up and down several times.

Summary of the Serial Dilutions Student Directions:

The student directions call for groups to use the fourth serial dilution in order to inoculate the dishes. If there is sufficient time and materials, groups could inoculate dishes using samples from the other dilutions. This will allow students to see how the greater the dilution, the easier it is to count the cell colonies. The less the dilution, the greater the growth, but it is much more difficult to count colonies. It will also allow more students to experience inoculating a plate.

The teacher will need to decide if groups will come to a specific “clean area” in the classroom in order to inoculate or if each group will be responsible for sanitizing and preparing their own work area. A “biohazard” bag should be used to dispose of loops.

Be sure that students mix the tubes well prior drawing the samples to inoculate the plates.

Plates can be stored at room temperature (growth will take approximately 48 hours) or placed in an incubator at 30° Celsius (growth will take approximately 24 hours).

When the appropriate amount of time has elapsed for growth to occur, return the plates to the student groups for them to complete the lab analysis portion of their answer sheet and to conduct Activity 4: Making a Wet Mount Slide and Observing Yeast Cells under the Microscope.

For the analysis portion of the student answer sheet, the students will need a black marking pen in order to count their yeast cell colonies. Students should turn their dishes over and use the marking pen to mark and count their colonies.