Materials Included In Kit

Additional Materials Required

Prelab Preparation

Making and pouring the nutrient agar should be done at least one day before the lab. Add only enough nutrient agar to cover the bottom of each Petri dish (about 20 mL).

Preparation of nutrient agar: Mix 15 g of nutrient agar with 600 mL of water in an Erlenmeyer flask. Place a foam plug in the mouth of the Erlenmeyer flask to prevent evaporation of the water. Bring the solution to a boil, stirring occasionally to completely dissolve the agar. Sterilize the nutrient agar using an autoclave or pressure cooker. Sterilize according to the instruction manual or at 121 °C at 15 psi for 15 minutes.

To help prevent contamination before and after pouring the plates, follow these steps:

• Disinfect work areas using a spray bottle containing a bleach solution or liquid Lysol^®.
• Open the sealed sleeve of plastic, sterile, Petri dishes once the nutrient agar is read to be poured. (You may wish to keep this sleeve for storing the plates after they have been poured and cooled.)
• Without removing lids, put the dishes in a single line on the outer edge of the work area.
• Using insulated gloves, pour approximately 15 mL of nutrient agar into each Petri dish by picking the lid straight up, keeping the lid between you and the liquid agar. Hold your breath while pouring each dish to help prevent airborne contamination. Add only enough nutrient agar to cover the bottom of the dish. Replace the lid. Avoid excessive movement to prevent creating air drafts.
• Allow the nutrient agar to cool without disturbing the dishes in any way. (Depending on room temperature, this could take up to 30 minutes.) When the dishes are completely cooled, tape them shut with two pieces of tape. Turn the dishes upside down and put them back in the plastic sleeve. Tape the sleeve shut and put the entire sleeve in a refrigerator or other cool location. Do not freeze.
• Prior to handing out the dishes to students, check for contamination on the nutrient agar surface. If contamination is present, tape the dish(es) shut and dispose of accordingly. Set the incubator to 37 °C a couple of hours before beginning the activity.

Safety Precautions

Although Serratia marcescens is a non-pathogenic bacterium, there is always the potential for contamination by pathogens when dealing with microorganisms. Instruct students to wear goggles and gloves and to follow aseptic techniques when handling microbes. Do not allow students to open the Petri dish lids after inoculation. Remind students to disinfect the work surfaces and to wash their hands thoroughly with antibacterial soap and water after working with microorganisms. Sodium hypochlorite provided in the kit for disinfection is corrosive and toxic. Avoid contact with acids, which can release toxic chlorine gas.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Upon finishing work with bacterial cultures, label the stock tubes and instruct students to disinfect their work areas (including the incubator handle). Use a spray bottle containing the bleach solution included with the kit (if the surfaces are compatible with bleach), or a 70% isopropyl alcohol solution or Lysol^® solution to sterilize all work surfaces. Microbiological cultures may be disposed of according to Flinn Suggested Biological Waste Disposal Method Type I.

Teacher Tips

• Enough materials are provided in this kit for 30 students working in pairs, or for 15 groups of students. Bacterial colonies should be visible after 48–72 hours. The Prelab Questions should be completed before beginning the activity, and students should be allowed an extra day after making observation to answer the Post-Lab Questions.
• Tyndallization can be used if no autoclave is available. Boil the nutrient agar for 20 minutes in the covered Erlenmeyer flask. Cool the agar and incubate at 37 °C for a day. Repeat the boiling, cooling, incubation procedure for three consecutive days followed by boiling again. The three incubations force many of the heat-resistant spores to grow due to heat shock. These newly growing bacteria and fungi are killed in the next boiling step.
• The “25 °C” dishes may be stored at room temperature, which is generally in the range of 20–25 °C. If preferred, the exact temperature of the room may be measured and written on the dish rather than 25 °C. Optimal color is observed in the range of 24–28 °C.
• Cultures grown at room temperature should have smooth, dark red bacterial colonies present. Cultures grown at 37 °C should display white or pale pink colonies. To achieve all white colonies, subculture the 37 °C Petri dish onto a fresh Petri dish and incubate at 37 °C for 48 hours.
• Stock cultures of S. marcescens contain red-colored colonies. However, if the bacteria are shipped in hot weather they may be pink or white upon arrival. If you do not want students to see the original color of the bacteria, place black electrical tape around the tube and dip the sampling swabs into the tube for the students.
• As an alternative or follow-up procedure, students may experiment with growing S. marcescens cultures at different temperatures between room temperature and body temperature to determine the temperature cutoff for the expression of the red pigment. Cultures grown at temperatures in between those tested in this activity are often pink in color. This is because some cells are still expressing the prodigiosin but many are not.

Answers to Prelab Questions

1. Why is it considered proper practice to label the bottom of the Petri dish rather than the lid?

   Lids may be accidentally interchanged between Petri dishes. The bottom of the dish cannot “move” and therefore will identify the bacteria sample and “owner” in the event that the lid is misplaced, cracked, etc.

2. Explain the term gene expression in your own words.

   Gene expression refers to the transcription of gene DNA to produce mRNA, which is ultimately translated into the proteins required for specific cellular processes. The genotype encoded in the gene is “expressed” in the phenotype.

3. Write a hypothesis for what you predict will be observed at the two different temperatures based on information in the Background section.

   The culture grown at 25 °C may contain the prodigiosin pigment and appear red in color. Cultures grown at 37 °C may lack the enzyme precursors that form prodigiosin and therefore appear white or colorless. This hypothesis is based on the sentence in the Background section that states, “Studies indicate that the prodigiosin protein may have anti-fungal properties, which may help the bacteria ward off fungal “competitors” that utilize common resources in the soil.” Soil is likely to be a much cooler environment than the human body.

Answers to Questions

1. Draw what was observed on each of the two plates. Use colored pencils, or describe in words, the colors observed.
   {10824_Answers_Figure_2}
2. Based on the observed results of this activity, predict what the result would be if a culture of Serratia marcescens were grown at a temperature between the two temperatures tested in this activity. Explain your reasoning.

   In the Background section, it states that the bacteria may appear red, pink, white or colorless. In a temperature range midway between 25 °C and 37 °C, the bacteria are likely to appear pink as the enzymes responsible for the synthesis of prodigiosin are not being transcribed at a lesser rate.

3. If someone had a population of Serratia marcescens present on their skin and they came into contact with rhizopus mold, would the presence of S. marcescens offer resistance to the mold? Explain.

   If the bacteria were present on the hand, then they would be thriving at 32–33 °C (average surface temperature of the body) and prodigiosin would likely not be produced by the bacteria, or be produced at low rates. Since it is specifically the prodigiosin protein that may have anti-fungal properties, not the S. marcescens bacteria in general, it is not likely that any anti-fungal properties will be in effect.

4. Suggest a possible advantage for the S. marcescens to have a temperature-sensitive protein like prodigiosin? In your opinion, why would a temperature dependant pigment be and evolutionary advantage?

   For energy and resource conservation—so that energy for the production of the prodigiosin protein is conserved for times when the bacteria may have greater competition for resources.

5. Based on the incubation temperature in which prodigiosin was produced, what do you believe the optimal growth temperature is for competitors of S. marcescens?

   Around 25 °C

Introduction

Explore the effect of temperature on the expression of specific genes in Serratia marcescens bacteria.

Concepts

• Microbiology
• Gene expression
• Enzymes

Background

All living organisms contain many genes encoded in their DNA sequences. Each gene contains the information needed for the cell to produce a specific protein or enzyme required to carry out a particular biochemical function or to fulfill a particular cellular process. Factors such as pH, light, and temperature may cause certain genes to be “turned on” or to be “turned off” under certain conditions. In other words, although the genes are always present in the organism’s DNA, a variety of stimulants or inhibitors may cause gene expression—the transcription of the gene DNA to produce mRNA, which is ultimately translated into the proteins required for specific cellular processes—to start or stop. If gene expression is turned off, the appropriate mRNA is not produced and translation will not occur. The corresponding proteins are not produced and the cellular function controlled by the protein stops. Therefore the gene is no longer expressed.

In this activity, the study organism is Serratia marcescens, a Gram-negative common soil bacterium, which may be red, pink, white or colorless. The presence of several specific enzymes within S. marcescens are required for the dark, red-colored pigment prodigiosin to be produced. Studies indicate that the prodigiosin pigment may have anti-fungal, antibacterial and antiprotozoan properties, which may help the bacteria ward off “competitors.”

Although the bacteria will grow and thrive over a fairly wide temperature range (17–43 °C), the gene for prodigiosin is only expressed in part of that temperature range. If the bacteria are grown at a temperature in which the genes are expressed, then the enzymes responsible for the synthesis of prodigiosin are produced, the pigment will be present and the bacteria will appear red in color. Alternatively, when the bacteria are grown at a temperature in which the enzymes responsible for the synthesis of prodigiosin are not transcribed, no pigment is made and the bacterial colonies will appear white or colorless.

Experiment Overview

In this activity, S. marcescens will be grown on nutrient agar at room temperature (approx. 25 °C) and at human body temperature (37 °C). The effect of temperature on differences in gene expression will be defined based on the presence or absence of the prodigiosin pigment.

Materials

Prelab Questions

1. Why is it considered proper practice to label the bottom of the Petri dish rather than the lid?
2. Explain the term gene expression in your own words.
3. Write a hypothesis for what you predict will be observed at the two different temperatures based on information in the Background section.

Safety Precautions

Although Serratia marcescens is a non-pathogenic bacterium, there is always the potential for contamination by pathogens when dealing with microorganisms. Wear goggles and gloves when handling microbes, and always use sterile or aseptic techniques. Tape all Petri dishes shut and do not open the Petri dish lids after they have been inoculated with bacteria. Sterilize all work areas and surfaces after handling the bacteria, as instructed by your teacher, and wash your hands thoroughly with antibacterial soap before leaving the laboratory.

Procedure

1. Obtain two nutrient agar Petri dishes and a wax pencil. On the bottom of one of the dishes write 25 °C, along with the names of the group members and the date. On the bottom of the second Petri dish write 37 °C, along with the names of the group members and the date.
2. Carefully open the wrapper of a sterile cotton swab. Do not allow the cotton tip to contact any surface, including your fingers. Lightly touch the cotton tip to a container of sterile water so that the tip is moist but not saturated.
3. Obtain a sample of Serratia marcescens by gently touching the wet cotton tip to the surface of the slant culture. Lift the lid of the Petri dish labeled 25 °C a few centimeters and gently streak the surface of the agar in a zigzag pattern using the tip of the cotton swab (see Figure 1). Immediately close the Perti dish lid.
   {10824_Procedure_Figure_1}
4. Place the used cotton swab back into the original wrapper and throw it away in the trash according to your teacher’s instructions.
5. Seal the Petri dish closed by wrapping masking tape around the sides.
6. Repeat steps 2 and 3 using the 37 °C dish and a fresh cotton swab.
7. Store the 25 °C dish on a counter or table at room temperature and place the 37 °C plate in an incubator as directed by your teacher.
8. After 48 hours, check the Petri dishes and observe any differences in the appearance of the bacterial growth on the plates.
9. Disinfect all work areas per your teacher’s instructions.

Student Worksheet PDF

10824_Student1.pdf