Pre-Laboratory Experiment

(5 student groups/30 students )

See "Materials List," for a complete listing of materials for this experiment.

Kit Materials:

Note: Kit contains enough reagents for 10 gels

5 Bacterial Samples:

Escherichia coli

Micrococcus luteus

Serratia marcescens D1

Unknown A (M. luteus)

Unknown B (S. Marcescens WCF)

2	10-20 % SDS Polyacrylamide gels
5	Tryptic soy broth tubes with Density Indicator Strips
5	Tryptic soy agar
5	Sterile transfer pipets
10	Filter papers
10	Sterile cotton swabs
	5 mg Lysozyme powder 10 mL Lysozyme Reconstitution Buffer (10X) 5 mL SDS Solubilization buffer (4X)
1	Low molecular weight protein marker
2	SDS running buffer packs 4X concentrate
2	Bottles, Ward's protein stain (500 mL each)
1	Gel Staining tray
1	Spatula (gel handler)
5	(non-sterile) Plastic transfer pipets (6 mL)
7	Sterile serological pipets (1 mL)
60	Microcentrifuge tubes
5	Sheets of graph paper
1	"Student Study Sheet" Copymaster
1	"Student Analysis Sheet" Copymaster
1	Teacher's Manual

Materials Needed but Not Supplied:

	WARD'S PAGE tank Ward's power supply 20 uL micropipets 1 L Beaker
1	Bubble rack
5	Small funnels

Culture Preparation of a Specific Cell Density

1. Label the outsides of the sterile tryptic soy agar tubes with a corresponding bacterial name.

2. Using sterile technique, untwist the caps of the cryovials. With a sterile pipet, add 0.5 ml of tryptic soy broth supplied with this product to the pellet in each cryovial.

3. Allow 10 seconds for pellets to soften, then mix by drawing the suspensions up and down through a sterile transfer pipet 8-10 times.

4. Dip a sterile cotton swab into each suspension and use it to streak the surface of each correspondingly labeled agar slant.

5. Incubate the tubes (with caps loosened one half-turn) at room temperature (only Escherichia coli at 37 deg.C) for 24-48 hours or until a visible bacterial lawn is present on the surface of each agar slant.

6. With a permanent marker, label the outsides of two sterile tryptic soy broth tubes. (for each of the five bacterial cultures inoculated in step 4.)

7. Using sterile cotton swabs, inoculate the appropriate broth tubes. Use half of the bacterial lawn growth for each of the two tubes for each organism. Do not transfer agar with the bacteria. The bacteria can be released from the swabs into the broth tubes by repeatedly pressing the swabs against the tube walls and then stirring the suspensions.

8. Incubate each broth tube (with caps loosened one half-turn) at room temperature (only Escherichia coli at 37deg. C) until only the darkest (topmost) bar can be seen on each Insta-Ratereg. Density Indicator Strip (approximately 24-48 hours). To read the Density Indicator Strips resuspend the settled bacteria in each broth tube and hold each tube (with the Density Indicator Strip away from the observer) in front of a white card. When the cultures have reached the proper densities, they may be used immediately for sample preparation or they may be refrigerated for several days before use.

9. All contaminated materials must be autoclaved at 121 deg.C and 15 psi for 20 minutes prior to disposal.

Bacterial Sample Preparation:

( 5 student groups/30 students )

Note: You may elect to have each student team prepare one of the following bacterial samples.

1. Set up a hot water bath using a 1 L beaker and a bubble rack. (The bubble rack will float)

2. Add all 5 mg of lysozyme powder to the 10 mL bottle of 10X Lysozyme Reconstitution Buffer. Mix by gently inverting the bottle several times. DO NOT SHAKE THE BOTTLE. Allow the powder to completely dissolve, then mix again by inverting the bottle.

3. Label 10 microcentrifuge tubes in sets of two as follows:

4. 1) E. coli
   2) M. luteus
   3) S. marcescens D1
   4) Unknown A
   5) Unknown B

5. Thoroughly mix each broth culture tube by inverting it. Using a fresh serological plastic pipet for each culture, transfer 0.9 mL (900 uL) of each bacterial culture to its appropriately labelled microcentrifuge tube. Refrigerate the remaining culture for future use. Discard all contaminated pipets in a biohazard bag as directed by your teacher.

6. Using a serological pipet add 0.1 mL (100 uL) of lysozyme solution to each microfuge tube. Swirl gently and incubate the tubes at room temperature for 10 minutes. Lysozyme is an enzyme that breaks down bacterial cell walls by attacking the peptidoglycan fabric. Bacterial cells whose cell walls have been removed by enzymatic action are called protoplasts. A bacterial protoplast consists of an intact plasma membrane, cytosol, and a nucleoid.

7. Following incubation in the enzyme solution, add 0.33 mL (333 ul) of 4X SDS Solubilization buffer with 2-mercaptoethanol to each microcentrifuge tube using a fresh serological micropipet. Mix by gently swirling the microcentrifuge tube. The SDS solubilization buffer solubilizes the lipids and the plasma membrane and emulsifies the cell.

8. Place the ten (five sets of two) labeled microcentrifuge tubes in the bubble rack and place the bubble rack in the 1-L beaker containing the hot water for 10 minutes.

9. Following incubation, remove the bubble rack from the beaker and allow the tubes to cool to room temperature for 5-10 minutes.

10. Each bacterial cell lysate should be filtered to obtain a clear filtrate, free from large cellular debris that can interfere with electrophoresis separation. Use the filter paper provided to form five cones by folding each paper twice in half. (If only one funnel is available, rinse funnel between samples and use with a new cone.) Place each cone into a separate funnel and insert each funnel into an appropriately labelled microcentrifuge tube. Pour each set of the bacterial cell lysate through the filter and let it drain into the microcentrifuge tube. Sample yields may be small, about 1.5 mL, but should be sufficient for loading up to 40 gel lanes. Any unused samples must be kept frozen for future use.

11. In this experiment 5 bacterial samples will be electrophoresed along with a molecular weight protein marker. Provide each student team with a set of samples prepared in advance by the instructor.

    Pre-label student microcentrifuge "reaction" tubes (set of 6 per student group) as follows:

    Tube #1: E. coli
    Tube #2: M. luteus
    Tube #3: S. marcescens D1
    Tube #4: Unknown A
    Tube #5: Unknown B
    Tube #6: Molecular weight marker

12. Using a 20 mL micropipet (see measurement for micropipeting tips), transfer 40 uL of each of the above sample types into corresponding student reaction tubes. (Use the microcentrifuge tubes provided.) Cap the tubes. Note: Any unused bacterial samples should be stored frozen. Bacterial samples have loading dye included. (Loading dye is a solution consisting of bromophenol blue, to enable you to track the migration of the sample down the gel, and sucrose, which helps to keep the sample in the loading well.)

13. Use the enclosed Ward's copymasters ("Student Study" and "Student Analysis") to make worksheet copies. Permission is granted for unlimited copies made for use within a single school building.

Preparation of Running Buffer:

1. Prepare SDS running buffer (4 X) according to the following directions: To make concentrated stock solution, pour the contents of this packet into a 2-L beaker containing about 1-L of deionized or distilled water. Stir until the powder has dissolved completely. Fill the beaker to the 1500-mL mark with additional distilled or deionized water. This is a 4X stock concentrate. To make 1-L of working buffer solution, just prior to use, mix 750 mL of deionized or distilled water with 250 mL of buffer concentrate. One liter volume is enough to run one gel using a single tank; 2-L will run two gels using a dual tank. Note: Although it is not usually necessary, filtration of running buffer helps assure crisp band imaging results. Store hydrated buffer concentrate and/or working buffer in a refrigerator.

Additional Preparation Notes:

• You may wish to designate one area of the laboratory as an "electrophoresis area" where cells and power supplies are connected. Assure that any wall outlet is properly wired, i.e., that correct polarity exists (use a circuit tester). Assure that electrical power cords and patch cords are clear from moisture or wetness.

• Assure that all students are thoroughly drilled on the correct procedure for making electrical connections and that they are directly supervised by you.

• Assure that all students wear the following personal protective equipment: Safety goggles, smock or apron (while loading gels and during electrophoresis) and protective gloves (during staining).

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