10 Eneumeration of Microbes

Procedure:

Make a 10 fold serial dilution with your saliva

1. Initial and date the Petrifilm and label with the following dilutions (Make sure to label in the corner of the film so that it doesn’t interfere with counting): 10^-4, 10^-5, 10^-6, 10^-7, and 10-8 and control indicating the dilutions you will put onto each Petrifilm
2. Label 10 Eppendorf tubes with the following label/dilutions: 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7, 10^-8  and “control”
3. Fill all your Eppendorf tubes with 1080 μl diluent using an Eppendorf pipette (you can pipette 2 x 540 μl as you CANNOT pipette 1080 μl with your P1000)
4. Take a new Eppendorf tube and label it “saliva”. Collect some of your spit (saliva) in a that Eppendorf tube ~ 200μl – this is your undiluted sample stock. You want to find out the CFU/ml in that sample
5. Make a 1:10 dilution series: Mix saliva by shaking the tube and aseptically transfer 120 μl of the saliva to your tube labeled 10^-1. This is a 1/10 or 10^-1 dilution.
6. Close the tube and vortex the tube containing the 10^-1 dilution
7. Aseptically transfer 120 μl of the 10-1 dilution to the next tube, labeled 10^-2. This is a 1/100 or 10^-2 dilution. Mix.
8. Using the same pipette tip, repeat this process until you complete the dilution series. (1 to 2, 2 to 3, 3 to 4, 4 to 5, etc.)
9. Do not add any saliva to the tube labeled “control”. Fill 1080 μl of your sterile diluent (PBS) into the tube called “control”.
10. Add 1 ml of sterile PBS from your “control” Eppendorf tube to your “control” Petrifilm

Plating on Petrifilm

1. After the last tube has been diluted, start with the 10^-8 dilution tube, mix, open
2. Place the 3M Petrifilm AC Plate on a flat, level surface
3. Lift the top film and with the pipette perpendicular dispense 1 mL of sample suspension onto the center of bottom film
4. Drop the top film down onto the sample to prevent trapping air bubbles, tapping gently with your fingers to spread the sample out
5. Continue the same process with the Petrifilm labeled 10^-7 . Gently spread out the liquid after you close the Petrifilm.
6. Then inoculate the remaining Petrifilms, moving from highest dilution to lower dilution, transferring 1 mL the 10^-6, 10-5 10-4 dilution tubes. You can use the same tip for the procedure.
7. Make sure you inoculate one Petrifilm “control” with your diluent only.
8. Stack the Petrifilms, and leave them for 48 hours at room temperature. The colonies will appear as red dots.

Results:

Petrifilm Growth:

CFU Calculations: