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The following points highlight the two methods used for measuring the growth in fungi. The methods are: 1. Linear Method (Agar Plate) 2. Mycelial Dry Weight.
1. Linear Method (Agar Plate):
Principle:
Fungal inoculum kept in the centre of the agar plate will grow radially and the rate of growth can be measured each 24 hours.
Requirements:
1. Culture of Fusarium oxysporum on PDA slants.
2. Czapek-Dox agar plates (5).
3. 8 mm cork Borer.
4. Inoculation needle.
5. Alcohol (95%).
6. Bunsen flame.
Procedure:
1. Prepare a seed agar plate either by spot inoculation or spread 1 ml of suspension from slant on agar plate and incubate at 28°-30°C (room temperature) for 96 hours.
2. Sterilize cork borer by making it red hot, dipping it in alcohol and flame heating it.
3. Punch the culture from the periphery (growing region).
4. Make the needle hot and touch the centre of blank agar plates (5).
5. Lift each cylinder and keep it in this central region, press it gently in order that the inoculum comes in contact with the medium.
6. Incubate the plates at 28°-30°C (room temperature) for 144 hour.
7. Measure the diameter of each colony at 2-3 places and take the average.
8. This measurement can be done after 24, 48, 72, 96, 120 and 144 hours.
9. Prepare a graph with time against diameter of colony.
2. Mycelial Dry Weight:
Principle:
On liquid medium (both stationary and aerated agitated cultures) the mycelial growth can be measured as dry weight.
Requirement:
1. Fusarium oxysporum cultures on PDA slants.
2. Czapek-Dox agar plates.
3. Czapek-Dox liquid medium intriplicate.
4. 250 ml conical flasks with 50 ml czapek’s liquid medium in two triplicates sets.
5. Whatman No. 1 Filter Papers (weighed)
6. Funnel.
7. Flasks.
8. Oven 105°C.
9. Shaker.
10. Bunsen flame.
Procedure:
Stationary mat cultures and aerated cultures:
1. Inoculate F. oxysporum on Czapek’s agar plates and incubate at 28°-30°C (RT) for 96 hours.
2. Punch discs of inoculum using sterile cork borer.
3. Lift one disc to each of the three 250 ml flasks with 50 ml liquid medium. Incubate at 28°-30°C (RT) for 144 hours.
4. Make suspension of F. oxysporum and add 1 ml of suspension to the other three 250 ml flasks with medium. Incubate them at 28°-30°C on a shaker (2500 rpm) for 144 hours.
5. Harvest both stationary and agitated cultures filter through previously weighed filter papers, dry them at 105°C for 48 hours.
6. Reweigh the filter papers with dry mycelium, subtract the weight of filter paper and prepare growth curve with time (hours) against weight of mycelium.