| A. | Don appropriate personal protective equipment (PPE). |
| B. | Aseptically transfer 1 mL from dilution tube 2 to dilution tube 3 and mix well. This is a 10-5 cumulative dilution. |
| C. | Aseptically transfer 1.0 mL from dilution tube 4 to dilution tube 5 and mix well. This is a 10-7 cumulative dilution. |
| D. | Aseptically transfer 0.1 mL from dilution tube 1 to dilution tube 2 and mix well. The cumulative dilution of the culture is 10-4. |
| E. | Allow the inocula to soak into the plates before proceeding. |
| F. | Aseptically transfer 0.1 mL from dilution tube 2 to plate A1. Using the spread plate technique, disperse the sample evenly over the entire surface of the agar. |
| G. | Aseptically transfer 1 mL from dilution tube 3 to dilution tube 4 and mix well. This is a 10-6 cumulative dilution. |
| H. | Obtain eight plates, organize them int four pairs and label them A1, A2, B1, B2, etc. |
| I. | Keep accurate records in your lab notebook. Clean up the lab benches, doff PPE, and wash hands. |
| J. | Repeate the procedure with plate A2 and label both plates "10-5mL of original sample," or simply "10-5" Notice that transferring 0.1 mL counts as a dilution!! The tube was a 10-4 dilution, but the plate is a 10-5 dilution!! |
| K. | Invert the plates so they are "agar up" to prevent condensation from ruining the plates. |
| L. | Repeat the aseptic transfer, spread plate technique, and label schematic for dilutions tubes 3, 4, and 5, and plates B, C, and D. |
| M. | Incubate the plates at 35â for 24 to 48 hours. |
| N. | Obtain five dilution tubes and label them 1â5, respectively. Make sure they remain covered until needed. |
| O. | Aseptically add 9.9 mL sterile water to dilution tubes 1 & 2. Cover when finished. Aseptically add 9.0 mL sterile water to dilution tubes 3, 4, and 5. Cover when finished. |
| P. | Mix the broth culture an aseptically transfer 0.1 mL to dilution tube 1. Mix dilution tube 1 well. This is a 10-2 dilution |
