Isolation of Microorganisms from Soil Lab Report
In this lab report of isolation of microorganisms from soil ,we will learn how to isolate and count bacteria from soil to make a bacterial pure culture.
Aim
To plate soil sample of various dilution on culture media to count cfu/ml and isolate bacterial pure culture from soil for further studies
Principle
Soil contains enormous numbers and kinds of microorganisms in addition to bacteria there are also protozoa, fungi, molds, algae and microscopic worms in unbelievable numbers. The types of microorganisms will depend on the composition of the soil, moisture, PH and other related environment factors. Therefore no one such technique can be used for the isolation and enumeration of these varieties of microorganisms
There are different methods to isolate soil microorganisms but the viable plate count method is one of the most commonly used procedures for the isolation of most prevalent microorganisms.
This method is based upon the principle that the material containing microorganisms is cultured, each viable microorganisms will develop and grow into a colony. Hence the number of colonies appearing on the plates represents, the number of living organisms present in the sample.
In serial dilution agar plate method in known amount of material is suspended on agitated in a known volume of sterile water to make microbial suspension serial dilution are made by pipetting measured volume.
Finally 0.1 ml of adequate dilutions are added to the sterile petri plates in which 20 ml of cooled nutrient agar media is added. Plates are incubated in an inverted position for 24-48 hours at 37 °C. The number of colonies appearing on dilution plates are counted and the CFU are counted multiplied by the dilution factor to find out number of cells/gram
Requirements
- Soil sample
- Nutrient agar
- Test tubes
- Petri plates
- Sterile 1 ml pipette
- Spreader
- Distilled water
- Cotton
- Bunsen burner
- Microscope
- Colony counter
Procedure
- At first soil sample were collected at randomly minimum fine from a field and mixed thoroughly to make a composite sample for microbiological analysis.
- Leveling of the test tubes and petri plates as 10-1 , 10-2 , 10-3 , 10-4 , 10-5 were done with a marker and each test tubes were filled with 9 ml of distilled water.
- For main stock1gram sample of finely air dried soil was added into 10 ml of distilled water in a conical flasks to make 1: 10 dilution (1g/ml)
- The dilution was shaken vigorously on a magnetic shaken for 20 to 30 minutes for obtaining uniform suspension of microorganisms
- 1ml of suspension was transferred from flask of main stock into 10-1 marked test tube with sterile pipette under aseptic conditions. To make 10-1 dilution of main stock and it was shaken well for about 5 minutes
- Another dilution was prepared by pipetting 1ml of the suspension from 10-1 to 10-2 test tubes by using a fresh sterile pipette to make 10-2 dilution of main stock and it was shaken well
- Further dilutions were made by pipetting 1ml suspension into additional water blanks as prepared before.
- Approximately 100 ml of the cooled medium was prepared and poured into each petri dish under aseptic condition.
- 0.1ml of each stock solutions were transferred from each diluted test tubes into sterile petri plates as marked same as test tubes
- All the plates were incubated in an inverted position at 37 °C for bacteria for 24 to 48 hours.
- Growth was observed in each plates ,cfu was counted and isolated for pure culture for further studies
Observation
Different number and various size of mixed bacterial colonies were observed in each petri plates
Observation table
| Test tubes | Sample(ml) | Distilled Water(ml) | Dilution Factor | Numbers of Colonies |
|---|---|---|---|---|
| 1 | 0.1 ml of main stock | 9 | 10-1 (ten to the power -1) | Too much to count |
| 2 | 0.1 ml of test tube 1 | 9 | 10-2 (ten to the power -2) | 780 |
| 3 | 0.1 ml of test tube 2 | 9 | 10-3 (ten to the power -3) | 710 |
| 4 | 0.1 ml of test tube 3 | 9 | 10-4 (ten to the power -4) | 640 |
| 5 | 0.1 ml of test tube 4 | 9 | 10-5 (ten to the power -5) | 207 |
Calculations
The main formula of CFU is
Colony forming unit = ( Number of colonies in plate X Dilution Factor)/ Sample Volume
So, for test tubes-3, cfu/ml = ( Number of colonies in plate X Dilution Factor)/ Sample Volume
= (710 * 10-3 ) / 0.1
= 7.1 x 10-6 cfu/ml
| Test tubes | 1 | 2 | 3 | 4 | 5 |
|---|---|---|---|---|---|
| Dilution Factor (10 to the power -n ) | 10-1 | 10-2 | 10-3 | 10-4 | 10-4 |
| CFU/ml | Not studied | 7.8 x 10-6 | 7.1 x 10-6 | 6.4 x 10-6 | 2.07 x 10-6 |
Conclusion
From the result we conclude that in higher dilution more growth was observed than the lower dilution .Bacterial colonies in higher dilution was difficult to count. Separated colonies in lower dilution soil sample plated was studied and colony was counted to count cfu/ml, which is in calculation part of this practical.
References
https://in.pinterest.com/pin/131167407882882532/
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