Question

You collected a sample of soil from your backyard to use for as a source of inoculum for your isolate project, and want to use plating technique to determine the number of microbes present. Based on what you know about microbial cell counts in soil environments, you estimate that there might be up to 1010 cells per g of soil, but that in many soil samples only a small fraction (0.1-1%) of cells are typically able to be cultured on solid medium. Outline a dilution and plating procedure that you could use to obtain an appropriate number of colonies on a plate to quantify microbes in this soil sample. Include the type of plating technique you would use, the type of media that you would use, the specific dilutions and volumes that you would plate, the targeted number of colonies you would want on a plate for accurate quantification, and the units you would use to report your results. Assume that you have sterile buffered saline solution for use as a diluent.

Answer 1

The following protocol can be used to estimate the numbre of organisms present in the soil.

1. weigh 1grams of soil and dissolve in 10mL of buffered saline. allow the sand to mix properly and then allow it to settle down . you can also give a short spin to fasten the process of settling.

2. take 900uL of saline and mix it with 100uL of supernatent of the soul mixture and label the dilution.

3. keep doing this procedure from one tube to the next until the dilution of 10^-8 is obtained.

4. take an agar plate of nutrient rich media and add 100uL of these samples from the last 4-5 tubes and use the spreading technique to properly spread all the bacteria until the plate is dried.

5. incubate the plate at 37 degrees as most of the bacteria colonies are able to grow at this temperature.

6. next day, count the number of colonies in each plate and select the plate with 60-100 colonies approximately.

calculate the number of colonies by multiplying the obtained number by dilution factor to obtain the number of cells in the starting solution.