Materials:
Step 1:

1. Five covered deep tubes of sterile prepared nutrient agar.
2. One-600 ml beaker.
3. Hot plate.
4. Five sterile Petri dishes.
5. One sterile stirring rod.
6. Five milliliters of automobile oil.
7. One gram of the following bacterial sources:
• Soil.
• Benthic soil.
• Parking lot oil shavings.
• Used automobile oil.
• Optional sources
8. One squeeze bottle of the following:
• Distilled water.
• Soap.
• Lysol.
9. Pipette

1. Hot Plate
2. Five sterile Petri dishes.
3. Five milliliters of prepare Miracle Grow (2.5g/100ml)
4. The five Erlenmeyer flasks from step one.
5. Five covered deep tubes of prepred water agar.
6. One-600 ml. beaker to melt the prepared agar.
7. One milliliter motor oil.
8. Innoculating loop.
9. Twenty sterilized disks. (prepared by punching holes into filter paper, soaked in 70% ETOH, then dried.
10. Ten milliliter pipette.
11. One squeeze bottle of the following:
• Distilled water.
• Soap.
• Lysol.

1. Acquaint yourself with the location and procedure for proper bacterial disposal.
2. Disinfect lab area with lysol.
3. Obtain all necessary materials.
4. Pour 550 ml of tap water into the 600 ml beaker.
5. Place the water filled beaker on the hot plate and place the hot plate temperature on high (or the temperature designated by your instructor*).
6. Bring the water to boiling.
7. Label deep tubes number one through number 5.
8. Place the deep tubes into the boiling water until the agar is liquefied.
9. Measure 1 ml of new automobile oil and place into deep tube number one.
10. Mix until agar and oil solution is uniform.
11. Allow deep tube to cool according to your instructor's guidelines*.
12. Measure approximately one gram of soil.
13. Add to deep tube number one and mix.
14. Pour the contents of deep tube number one into a sterile petri dish according to instructor's guidelines*.
15. With a grease pencil label Petri dish "a. soil".
16. Repeat steps 9 through 14 for each of the following sources:  "b. benthic soil", "c. parking lot oil shavings" and "d. used automobile oil".
17. Label each Petri dish appropriately.
18. Repeat steps 9-10, and pour directly into a petri dish.  Label "control".
19. Place Petri dishes according to your instructor's guidelines*.
20. Clean and sterilize your work area according to your instructor's guidelines*.

1. Set up disposal areas for "contaminated glassware", "contaminated broken glassware", "waste container".
2. Obtain five flasks
3. Label each flask according to the sample that will be put in each.  For example label one "soil", one "parking lot shavings" etc.
4. In each flask put the following
• 10 mL of dilute nutrient solution (dilute miracle grow)
• 80 mL of distilled water
5. Put 0.5 g of the appropriate sample into each flask.
6. Place the flasks according to your instructors guidlines.*
7. Clean and sterlize your work area according to your instructors guidlines.*

Step 2:

1. Disinfect lab area with Lysol.
2. Pour 350 ml. of tap water into the 600 ml. beaker.
3. Place the water filled beaker on the hot plate and place the hot plate temperature on high.
4. Bring the water to boiling.
5. Label deep tubes one through five.
6. Place the deep tubes into the boiling water until the agar is liquefied.
7. Measure one milliliter of Miracle Grow solution into each of the five deep tubes.
8. Mix agar and fertilizer solution in all five tubes.
9. Label each of the Petri plates to match the names on the flasks.
10. Empty the tubes into the plates.
11. Streak the plates with the culture from each tube.
12. Lightly coat the filter disks with motor oil.
13. Place one of the coated disks into each of the four quadrants in each Petri dish.
14. Wait 72 hours for results.

Conclusion Questions:

1. Do your results support your hypothesis?  If yes or no answer why or why not.
2. If there were mistakes or problems with the activity, have the student explain and what could be done to correct the mistake.
3. Explain five techniques that worked well and why.
4. What would be done to continue this activity and what methods would be used.

1. Set up disposal areas for "contaminated glassware", "contaminated broken glassware", "waste container".
2. Prepare a minimum of five deep tubes for each lab group of two or three students in advance.
3. Deep tubes should be placed in the water on the hot plate.  The water should be brought to boiling to allow the agar to liquefy.
4. Once liquefied the oil may be added.  Allow the tube to cool to 550 or until you are able to hold the tube in your hand without discomfort.  At this time the    bacterial sources may be added to the tube and mixed thoroughly.
5. Liquid deep tube agar mixture should be poured into Petri dishes and covered immediately.
6. Allow to solidify.
7. Incubate culture upside down for at least 48 to 72 hours.
8. Sterilize student work areas with ethanol.  Have them wash their hands thoroughly before and after the lab.

1. Set up disposal areas for "contaminated glassware", "contaminated broken glassware", "waste container".
2. Prepare the miracle growth solution for the lab by a ratio of 2.5g/100 mL
3. Sterilize student work areas with ethanol.  Have them wash their hands thoroughly before and after the lab.

 Step 2

1. Set up disposal areas for "contaminated glassware", "contaminated broken glassware", "waste container".
2. Prepare a minimum of five deep tubes for each lab group in advance.
3. Use an innoculate loop to streak agar plates.