Abstract
The purpose of this exercise to become familiar with subtypes of culture media, to learn how to use streak and pour techniques and to generate a pure culture of a specific organism Hypothesis
This exercise will allow me to gain an understanding of culture media, to use specific techniques such as streak and pour and generate cultures of specific organisms.
Procedures
Exercise 1
Part I
1. Disinfect the work area.
2. Melt the agar tubes.
3. Leave the 18 mL tube of MRS agar in hot water (50°C) for use in Part II.
4. Use the marking pencil to label the bottom of one Petri dish S. epidermidis. Pour one half (9mL) of the contents of a tube of nutrient agar into the S. epidermidis Petri dish and the other half into the bottom of an unmarked Petri dish. Cover the dishes and allow them to solidify for use in Part IV.
5. Pour the remaining melted nutrient agar into the unmarked Petri dishes (half a tube per dish). Cover the dishes and allow them to solidify for use in Part III.

Part II
1. Disinfect the work area.
2. Label the bottom surface of three sterile Petri dishes L. acidophilus #1, #2, and #3, respectively. 3. Disinfect three test tubes by submerging them in boiling water for 5 minutes. The tubes will be hot, so use tongs or tweezers to lift them out of the water. Be careful not to contaminate the tubes by touching their lips or interiors. When the tubes are cool, label them to match the L. acidophilus Petri dishes.
4. Divide the liquid MRS agar into the three test tubes marked L. acidophilus. If the agar has begun to solidify, reheat it until it is fully melted. Set the test tubes of agar in the hot water to prevent them from solidifying.
5. After ensuring the tubes of agar are cool enough not to kill the bacterial culture but are still fully liquid, use aseptic techniques to inoculate the tube labeled L. acidophilus #1 with one loop full of the saved L. acidophilus culture. Gently mix and return the tube to the hot water.
6. Inoculate L. acidophilus #2 with one loop full of the bacteria media mix from tube #1.
Gently mix and return the tubes to the hot water.
7. Inoculate L. acidophilus #3 with one loop full of the bacteria media mix from tube #2.
Gently mix and return the tubes to the hot water.
8. Pour the contents of L. acidophilus #1 into the corresponding Petri dish and cover the dish 10. Incubate the dishes in an inverted position for 24–72 hours at 35oC–37oC.
11. Examine the dishes for isolated colonies. Record the appearance of each dish.
12. Store the culture in the refrigerator for use in future experiments.
13. Soak the Petri dishes in a 10%-bleach solution for 1 hour and then discard them.
14. Soak the test tubes in a 10%-bleach solution for 1 hour and then discard the contents. Clean and rinse the test tubes for future use. immediately. Repeat for
L. acidophilus #2 and #3.
9. Allow the agar to solidify at room temperature.

Exercise 2
1. Disinfect the work area.
2. Prepare an S. cerevisiae culture.
3. Label six test tubes 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6.
4. Label six unmarked agar dishes from Part I 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6.
5. Use a plastic graduated pipet to add 2.25 mL of distilled water to each test tube. times to ensure that the pipet is dry before drawing up water.
6. Mix the yeast-water solution well to disperse the organisms evenly.
7. Transfer 0.25 mL of the yeast solution into the test tube labeled 10-1. Pipet the solution up and down several times to mix it thoroughly and ensure all organisms are rinsed from the pipet into the solution.
8. Transfer 0.25 mL of the 10-1 yeast solution into the test tube labeled 10-2. Pipet the solution up and down several times to mix it thoroughly and ensure all organisms are rinsed from the pipet into the solution.
9. Repeat the transfer process to transfer the yeast solution from the 10-2 tube to the
10-3 tube; from the 10-3 tube to the 10-4 tube; from the 10-4 tube to the 10-5 tube; and from the 10-5 tube to the 10-6 tube as shown in Figure 13. Thoroughly rinse the pipet in water to remove all organisms, inside and out.
10. Beginning with the 10-1 tube, mix the sample by pipetting the solution up and down several times. Then pipet 0.125 mL (four drops) onto the corresponding 10-1 agar dish.
11. Place the cover on the dish and swirl the dish gently to spread the solution evenly over the surface.
12-14. Repeat the steps for the remaining dilutions as shown in Figure 14.
15. For each dilution, count the number of colony-forming units on the dishes. Mark the position of each colony on the bottom of the dish with a marking pen as you count it.
16. Mix 1 tablespoon of bleach into the yeast culture and let it stand for at least 30 minutes to ensure all organisms have been destroyed. Then discard the contents.
17. Soak the Petri dishes in a 10%-bleach solution for 1 hour and then discard them.
18. Soak the test tubes in a 10%-bleach solution for 1 hour and then discard the contents. Exercise 3
1. Disinfect the work area.
2. Use the nutrient agar dish labeled S. epidermidis from Exercise 1.
3. Use aseptic techniques to obtain a loop full of the saved liquid S. epidermidis culture.
4. Streak the inoculum into the first quadrant as shown in Figure 16.
5. Disinfect the inoculation loop. Do not obtain a new inoculum. Instead, use the disinfected inoculation loop to streak several times through Quadrant 1 to pick up some organisms on the loop. Then streak from Quadrant 1 to Quadrant 2 as shown in Figure 16.
6. Repeat the Procedure for Quadrants 3 and 4, respectively. Be sure to disinfect the inoculation loop between each quadrant.
7. Disinfect the inoculation loop.
8. Cover the dish, invert it, and incubate it for 48–72 hours at 35oC–37oC.
9. Identify an S. epidermidis colony.

Exercise 4
1. Label a tube of nutrient broth S. epidermidis Stock Culture.
2. Aseptically transfer an S. epidermidis colony from your Petri dish into the nutrient broth. The culture that grows in the broth will be a pure culture because it originated from only a single colony, which originated from a single organism.
3. Incubate the stock culture for 24–48 hours to establish the culture. Then store the culture in a zip bag in the refrigerator for use in future experiments. You may also store dish cultures in a similar manner.
4. Mix 1 tablespoon of bleach into the original S. epidermidis culture and let it stand for at least 30 minutes to ensure all organisms have been destroyed. Then discard the contents.
5. Soak the Petri dishes in a 10%-bleach solution for 1 hour and then discard them.
6. Disinfect the work area.

Observations