- Soil contains diversity of microbes include bacteria, fungi, algae and protozoa.
- Bacteria is the most abundant and important microorganism.
- They are unicellular, non-chlorophyll containing, very small and primitive microorganism.
- There are various methods for isolation and enumeration of microorganism from any material but serial dilution is the most simple and commonly used method.
- Principle of this method is that when any sample containing microbes spread on agar plate, each single microorganism will develop into colony.
- Since the no of microorganism in soil is high, so spreading the sample in an undiluted manner leads to the dense growth of microorganism with overlapped colonies makes harder to isolate and study.
- So it is necessary to dilute the sample before spreading and this is done by the serial dilution method.
- Serial dilution made the concentration low to 10, 100, 1000 and more folds. The extent of dilution is depends on the type of sample or material.
- Sterile culture tubes
- Nutrient agar petriplates
- Micropipettes and tips
- Autoclaved water
- Cotton plugs
- Glass spreader
- First prepare stock solution by adding 1 g of soil to 10 ml autoclaved water in a test tube.
- Take 5 test tubes and mark 10-2, 10-3, 10-4, 10-5 … with a glass maker and add 9 ml autoclaved water to each test tube.
- From the stock solution pipette 1 ml solution with the help of micropipette and transfer it to the second test tube containing 9 ml of autoclaved water marking 10-2.
- From second test tube transfer 1 ml solution to the next tube, repeat this to desirable dilution.
- Now from each test tube pipette out 80-100μl solution and spread on the solidified agar media plates with a spreader.
- Incubate the plates for 18-24 hours at 37OC.
- After incubation observe the number of colonies and also observe the different growing patterns of the colony as well as morphology.
- By using the following formula calculate the number of microorganism per gram of the soil.
- Viable cells/ gram of soil = X dilution factor