Differential Staining

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Differential Staining

Purpose:The purpose of this experiment was to become familiar with subtypes of culture media and the uses for each, learn and employ the streak and pour dish techniques, and generate a pure culture of a specific organism.

Set Up:For this experiment I needed: 1 Distilled water, 1 Paper towels, 1 10%-bleach or 70% alcohol solution, 1 Zip bag, 1 Pan to heat agar, 1 Isopropyl alcohol (rubbing alcohol), 1 Cultures: S. epidermidis and L. acidophilus, 1 Gloves, Disposable, 1 Pencil, marking, 11 Petri dish, 60 mm, 2 Candles (flame source), 1 Thermometer-in-cardboard-tube,6 Test Tube(6), 16 x 125 mm in Bubble Bag, 1 Test tube holder, 1 Test-tube-rack-6x21-mm, 1 Pipet Graduated Small (5 mL), 1 Baker’s Yeast Packet – Saccharomyces cerevisiae, 1 Agar, MRS - 18 mL in Glass Tube, 4 Agar, Nutrient - 18 mL in Glass Tube, 1 Broth, Nutrient - 5 mL in Glass Tube, 2 Inoculation Loop, Plastic, 1 Mask with Earloops (11) in Bag 5" x 8"

of Individual Colonies

Methodology: Exercise 1: Isolation Using the Pour Plate Method
Part I: Preparation of Solid Media
1. Disinfect the work area.
2. Melt the agar tubes. Refer to the Preparation of Solid Media section in the Introduction for further instruction. 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 (9 mL) 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 solidifyfor 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: Isolation Using the Pour Plate Method
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. Isolation of In8. Pour the contents of L. acidophilus #1 into the corresponding Petri dish and cover the dish immediately. Repeat for L. acidophilus #2 and #3. 9. Allow the agar to solidify at room temperature.

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.

Exercise2: Isolation and Enumeration by Dilution to Extinction

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...
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