Serial dilution: Isolation of Trichoderma spp., from soil

Serial dilution is a common technique used in microbiology to isolate microorganisms from a sample. This technique involves diluting a sample several times to reduce the number of microorganisms present in the sample. Here is a detailed procedure for serial dilution for the isolation of Trichoderma from soil:

Materials Required:

- Soil sample
- Sterile distilled water
- Sterile test tubes
- Sterile pipettes
- Sterile Petri dishes
- Potato dextrose agar (PDA) plates
- Incubator

Procedure:

1. Collect a soil sample from the desired location using a sterile spoon or spatula.

2. Weigh the soil sample and record the weight.

3. Add the soil sample to a sterile test tube containing 9 ml of sterile distilled water.

4. Mix the soil and water thoroughly by shaking or vortexing for 1-2 minutes.

5. Label another set of sterile test tubes as 10-¹, 10-², 10-³, 10-⁴, and 10-⁵.

6. transfer 1 ml of the soil-water mixture to the labelled 10-¹ test tube using a sterile pipette.

7. Mix the contents of the test tube thoroughly by shaking or vortexing for 1-2 minutes.

8. Using a new sterile pipette, transfer 1 ml of the 10-¹ dilution to the labelled 10-² test tube.

9. Repeat steps 7 and 8 for each subsequent dilution until you reach the desired dilution factor.

10. Label sterile petri dishes with the dilution factor and replicate the number.

11. Pour approximately 20 ml of melted PDA into each labelled petri dish.

12. Using a sterile pipette, transfer 0.1 ml of each dilution onto the surface of the PDA in each petri dish.

13. Spread the inoculum evenly on the surface of the PDA using a sterile glass rod or spreader.

14. Incubate the Petri dishes at 25-30°C for 3-5 days.

15. Observe the Petri dishes for the growth of Trichoderma colonies.

16. Count the number of Trichoderma colonies on each plate and record the results.

17. Calculate the number of Trichoderma colonies per gram of soil by multiplying the number of colonies by the dilution factor and dividing by the weight of the soil sample.

18. Repeat the procedure with different soil samples and at various dilution factors to obtain a representative sample of Trichoderma in the soil.

Note: Always use sterile techniques to avoid contamination of the samples and media.