Isolation of phylloplane microorganism by serial dilution plate method
The leaf disks are serially diluted and then water samples are plated onto nutrient agar medium.
- Fresh leaves
- Sterile polythene bags
- sterile distilled water
- pipettes (10 ml, 1 ml capacity)
- Czapek Dox Agar plates (for fungi)
- Nutrient agar plates (for bacteria)
- cork borer (5 mm diam)
- Bunsen burner
- Magnetic stirrer or electric shaker
- glass marking penil
- Collect fresh leaf from a healthy plant, transfer in sterile polythene bag and bring to the laboratory.
- take 3 flasks (250ml capacity) and label them 1,2, and 3
- transfer 100 ml distilled water in flask 1, and 90 ml each in flask 2 and 3.
- autoclave the lasks containing water at 121 C for 30 minutes.
- with the help of sterile cork borer cut leaf discs o 5 mm diameter (50 – 60 discs from each leaf).
- transfer 50 leaf discs in a flask containing 100 ml sterile distilled water. (10 g of leaf discs / leaves are transfered when CFUs/g leaves are to be measured).
- stir the flask for about 30 minuted either on a magnetic stirrer or electric shaker.
- transfer 10 ml of water from flask 1 to flask 2 and shake flask 2 for 5 minutes.
- again transfer 10 ml of water from flask 2 to flask 3 with the help of sterile pipette (10 ml), shake it properly.
- pour 1ml suspension, taking from flask 2 and 3, onto surface of Czapek Dox agar plates and nutrient agar plates (in 3 replicates ) for fungi and bacteria, respectively.
- gently shake the plates with slide tilting so as to spread the water sample properly.
- incubate the nutrient agar plated for 24 – 48 hours and Czapek Dox agar plated for 5 – 6 days at 25 C.
first observe the nutrient agar plates and count total number of colony forming units (CFUs). calculate average number of CFUs both of fungi and bacteria. observe fungi after proper staining with cotton blue plus lactophenol and identify to generic or species level. if required purify the fungi in agar slants.
calculate microbial population/cm2 by using the following formula:
Dr. R. C. Dubey – Practical Microbiology.