New And Precise Quantification Cultures Mircoorganism

New And Precise Quantification Cultures Mircoorganism

Experiment 1: Direct Counts Following Serial Dilution

Materials:

60 mL Sterile phosphate buffered saline (PBS) 6 Sterile snap-cap tubes 250 mL Beaker Hot pad 10 Disposable sterile transfer pipette 8 Disposable sterile plate spreader (6) 5 cm. Petri dishes Nutrient agar Parafilm™

1 Pair of gloves 10 mL Graduated Cylinder Permanent marker *Microwave or other boiling water bath *1 tsp. Soil sample *10% Bleach solution

*You must provide

   

 

Procedure

Prepare Agar Plates:

1. If necessary, prepare six Nutrient agar plates by following Steps 1-5 of Lab 2 Experiment 1.

Prepare Stock Soil Solution:

2. Use your 10 mL graduated cylinder to dispense 10 mL of PBS in a 15 mL conical tube; label this tube “Stock Soil Solution”.

3. Add 9 mL of PBS into each of the other 5 tubes. Label the tubes as follows: 10-1, 10-2, 10-3, 10-4, 10-5.

4. Label the 6 petri plates as 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6.

5. Add the soil sample to the Stock Soil Solution tube. Invert the tube 10 times to mix well.

Prepare a Serial Dilution of the Stock Soil Solution as follows:

6. Use a sterile disposable transfer pipette to transfer 1 mL of the soil/saline mixture to the conical tube labeled 10-1 . Invert the tube 10 times to mix well.

7. Using the same transfer pipette, transfer 1 mL of the solution from the 10-1 tube to the 10-2 tube; invert 10 times to mix well.

8. Transfer 1 mL of the solution from the 10-2 tube to the 10-3 tube; invert 10 times to mix well.

9. Transfer 1 mL of the solution from the 10-3 tube to the 10-4 tube; invert 10 times to mix well.

10. Transfer 1 mL of the solution from the 10-4 tube to the 10-5 tube; invert 10 times to mix well.

11. Using a new, sterile transfer pipette for each tube, transfer approximately 0.1 mL ( or 4 drops) from the Stock Soil Solution tube to the plate labeled 10-1. Notice that you are performing a 10-fold dilution of the original sample by only adding 0.1 mL, rather than 1 mL. Spread the diluted soil solution evenly over the plate with a separate sterile spreader. Cover the plate with the lid.

12. Repeat Step 15 for the 10-1 tube, putting 0.1 mL of that solution on the 10-2 plate. Spread the diluted soil solution evenly over the plate with a separate sterile spreader. Cover the plate with the lid. Repeat this process for all the tubes and plates, remembering to use clean pipette for each tube and to plate the solution on the next lower dilution plate (i.e., 10-2 tube solution on 10-3 plate, etc.).

13. Allow plates to air dry, then seal with Parafilm™. Incubate in a warm location (not to exceed 37.7 °C or 100 °F) for 3 days (until well defined colonies appear).

14. Observe the plates and assess which plates should be classified as TNTC or TFTC, and which one(s) are countable plate(s). Count the colonies on the countable plate(s) and determine the population density using the following equation:

Pop. Density = CFU/(dilution x volume plated).

Record your results in Table 1.

15. Take a picture of your cultures (with your name clearly visible in the background of the photo) for your lab worksheet.

16. Pour approximately 5 mL of the 10% bleach solution onto each surface of your culture plates, allowing it to cover the entire surface. Incubate them for 20 minutes, and then pour the bleach down the sink with running water.

17. Seal the petri dishes with Parafilm™ and dispose of them in the trash.

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