Isolation and Mass Multiplication of Trichoderma viride.
Date : 04/08/2021
AIM:
Isolation and mass multiplication of Trichoderma viride.
Indroduction
Trichoderma Viride is mainly used in biofertilizer as biocontrol agent for plant pathogenic diseases and for their ability to increases root growth and development crop production in plant.
It is used for seed and soil treatment for suppression of various diseases caused by fungal growth .
Need of Project
In Vigyan Ashram Trichoderma Viride as biofertilizer is available but now we are trying we do mass multiplication to produce in large scale.
Objective:
To isolation Trichoderma viride
To mass multiply strain of Trichoderma viride
Production of 5 kg Trichoderma viride
Date: 05/08/2021
Requirement:
| Sr No. | Material | Amount |
| 1. | Tamarind Seed | 250gm |
| 2. | Trichoderma spp culture | 5 gm |
| 3. | PDA Media | 500 ml |
| 4. | Tap water | 400ml |
Preparation of Media :
Potato dextrose agar and potato dextrose broth are common microbiological growth media made from potato infusion , and dextrose . Potato dextrose agar is the most widely used medium for growing fungi .
Composition of PDA :- PDA composition for 1 Lit.
| Dextrose | 20 g |
| Potato extract | 4 g |
| Agar | 15 g |
- Weigh around 16.5 gm PDA agar in 500 ml flask and adjust volume 400 ml.
- Autoclave media, petri plates, test tubes, at 121 degree C at 15 lb pressure for 15 mins.
3. After sterilization media,test tube, petri plates are kept in laminar air flow.
4. For Maintaining Aseptic condition: At the same time, clean Laminar Air Flow using Acetone /70% ethanol and ON UV light for 15 min . After 15 min off the UV light . After on blower and day light .
5.Then all remaining procedure carried out under asceptic condition.
6.Pour the media in petri plates. After cooling media 20 Sterile petri plates were made.
Result:
Contamination is seen due to improper handling of instrument and petri plates and gloves were not sterilized.
Date:19/08/2021
Step 1:
Tamarind seeds content mostly starch and polysaccharides. These starch and carbohydrates support good growth of fungus. The tamarind seed coat content polyphenols which has antimicrobial properties. Tamarind seeds are support very good growth of fungus like Trichoderma.
chemical constituents tamarind seed powder include as below:
Carbohydrate – 73.68
Protein – 14.38
Ash – 3.28
Moisture – 8.67
Procedure:
1.Take 50gm tamarind seeds in a conical flask and soak in tap water for overnight.
2.Next day autoclave the flask at 121 degree C temp and 15 lb pressure for15 min.
3.After sterilization 1 gm of Trichoderma powder was add in 5 flask by using Laminar Air flow.
4.After cooling inoculate with available Trichoderma strain and incubated at room temperature on aluminum racks.
5. Observed the growth for 5-6 days.
Date:24/08/2021
Date:28/08/2021
We got Trichoderma viride growth only in one flask . Rest of the three flask contaminated result hence they were dispose.
Growth is observed after 10 days of incubation
Date:29/08/2021
Step 2
1.Again Take 50gm tamarind seeds in a conical flask and break it into small peaces and soak in tap water for overnigh.
2.Next day autoclave the flask at 121 degree C tem and 15 ib pressure for15
min.
Date:30/08/2021
3.Subculturing of Trichoderma viride by using previous growth culture.
Date:08/09/2021
Observed the after 5 -6 dyas. full growth of fungus is observed. After air dry seed in room temperature and crush with the mixture grinder to make a fine powder and 109 gm powder is ready .
We got Trichoderma viride growth only in two flask which account for 109 gm of net weight . Rest of the three flask contaminated result hence they were dispose.
Date:08/09/2021:
I got idea to use plastic tray because each low cost and ease availed to farmers.
- Using 6 conical flask (500 ml capacity). I soak 50 gm. tamarind seed in each flask overnight.
- Then we dispose the water from each flask.
- We sterilize every each flask in autoclave 121 degree C for 15 min by using autoclave.
- Then we sterilize plastic trey by using sodium hypochlorite washing and UV treatment.
- I transfer all tamarind seed into tray and added Trichoderma viride stain availed in soil lab and covered tray with aluminum foil to prevent contamination.
- And incubate for 5-6 days.
- After 5-6 days we observed green color layer of growth of Trichoderma viride.
Date:13/09/2021
- Then removed the foil paper and air dry the tamarind seed.
Date:17/09/2021
After drying grinded the tamarind seed to obtain 249 gm. of Trichoderma powder.
Date:09/09/2021
Preparation of media:
1. Weight 11.7 PDA agar in 300 ml D.W
2. Autoclave media, petri plates, test tubes, at 121 degree C at 15 lb pressure for 15 mins.
3. After sterilization media,test tube, petri plates are kept in laminar air flow.
4. For Maintaining Aseptic condition: At the same time, clean Laminar Air Flow using Acetone /70% ethanol and ON UV light for 15 min . After 15 min off the UV light . After on blower and day light .
5. Then all remaining procedure carried out under asceptic condition.
6 .Pour the media in petri plates. After cooling media 20 Sterile petri plates were made.
. After cooling media 20 Sterile petri plates were made.
Date:10/09/2021
Isolation of Trichoderma from soil.
Soil is collected from polyhouse .
Serial Dilution Plate Techniques :
Trichoderma isolated from soil sample by using serial dilution plate techniques were grow on potato dextrose agar media for proper isolation. Stock culture of sample is prepared by dissolving 1g of soil sample into 9 ml of D.W . Next serial dilution of sample were prepared as 10-1 10-2 …….10-10 .
Date:11/09/2021
Spread Plate Method:
Pipette out 0.1 ml from the appropriate desired dilution series onto the center of the surface of an agar plate.
Dip the L-shaped glass spreader into alcohol.Flame the glass spreader over a Bunsen burner.
Spread the sample evenly over the surface of agar using the sterile glass spreader, carefully rotating the Petridish underneath at the same time.
Test tube no 10-1 take o.1 ml sample transfer to test tube no10-2 this process repeated upto 10-10
Date:16/09/2021
Observation:
Observed the growth for 5-6 days.
Result:
After 5-6 days growth are observed at 25 degree C .
Date:17/09/2021
Microscopic Observation :
septate hyaline hyphae, conidiophores , and conidia are observed.
Date:30/08/2021
We order Trichoderma Viride strain from NCIM (National Collection of Industrial Microorganism) .It took around one month (30 July-30 Aug) arrived in Vigyan Ashram.
Till then arrived strain from NCIM we utilized in house strain available in Vigyan Ashram.
Date:06/09/2021
After receiving strain from NCLM first trail done by Sonal Shinde mam and plate got contaminated due to handling error ( sub culturing was done using slant obtain form strain on PDA with petri plates).
Date:15/09/2021
On our second trail we followed the same procedure and didn’t get any contamination. And 5-6 days we observed 75% growth in petri plates.
Date:20/09/2021
Now I did third attempted obtain by using from second attempt sub culturing (by using 4Th quadrant method).
I incubate the petri plates for 5-6 days then I got 100 % strain growth and my observation are as following days
Second day we observed white growth.
On fourth day color change to light green.
On 6th day it mature with dark green color.
Date: 19/09/2021
1.Using 13 conical flask (500 ml capacity) .
2. I soak 50 gm Tamarind seed in each flask overnight. Next day we dispose the water from each flask in autoclave at 121 degree C for 15 min.
3.After sterilization flask and wire loop and glove were sterilize by using laminar air flow .
4.Then we add Trichoderma viride culture from previous PDA plates and incubates for 5-6 days .
Sub culturing of Trichoderma viride:
Date:28/09/2021
Result:
Then Trichoderma viride growth is observed.
Date:09/10/2021
Observed the after 5 -6 dyas. full growth of fungus is observed. After air dry seed in room temperature and crush with the mixture grinder to make a fine powder and 600 gm powder is ready .
Date:21/09/2021
We can go for this experiment in two ways.
1 Pressure cooker.
2 Plastic Tray.
A (pressure cooker)
1. Let’s utilized cooker itself.
2. Soak 200gm tamarind using tap water overnight. Sterilized the tamarind seed for 15 min by using pressure cooker.
3. Cool the mixture in the cooker to room temp. Now we expose cooker in UV light for 15 min.
4. Using Wire loop scarch the Trichoderma viride strain mix into the tamarind seed with made in pressure cooker.
5.Now we will remove pressure cooker whistle in place of whistle insert rubber tube and cap it of using pvc hollow tube around 2 inch on the rubber tube and plug it using sterile cotton plug.
6.Now will keep the cooker without disturbing it for 5 days for multiplication of fungus.
Date:28/09/2021:
Result:
No growth is observed in cooker.
Using plastic tray (master tray)
1.After strile tamarind seed we can master tray/ plastic tray to grow the trichoderma viride strain.
2.Wash the trey with sodium hypochoride after the wash sterile in UV for 15 min.
3.Then we transfer strile tamarind seed from pressure cooker in tray.
4.Using Wire loop scarch the Trichoderma viride strain mix into the tamarind seed in tray.
5.And trey cover with aluminum foil. Now will keep the tray without disturbing it for 5 days for multiplication of fungus.
Date:28/09/2021
After 5-6 days we observed green color layer of growth of Trichoderma viride.
Next day dry Trichoderma viride.
Date:-09/10/2021
After air dry seed in room temperature and crush with the mixture grinder to make a fine powder and 64 gm powder is ready .
Date:04/10/2021
1.Using 10 conical flask (500 ml capacity) .
2. I soak 50 gm Tamarind seed in each flask overnight. Next day we dispose the water from each flask in autoclave at 121 degree C for 15 min.
3.After sterilization flask and wire loop and glove were sterilize by using laminar air flow .
4..Then we transfer sterile tamarind seed from flask in tray.
5.Then we add Trichoderma viride culture from previous PDA plates and incubates for 5-6 days .
Date:-12//10/2021
Result:-
After 5-6 days we observed green color layer of growth of Trichoderma viride.
Next day dry Trichoderma viride.
Result:
After air dry seed in room temperature and crush with the mixture grinder to make a fine powder and 600 gm powder is ready .
Date:-05/10/2021
1.Using 10 conical flask (500 ml capacity) .
2. I soak 50 gm Tamarind seed in each flask overnight. Next day we dispose the water from each flask in autoclave at 121 degree C for 15 min.
3.After sterilization flask and wire loop and glove were sterilize by using laminar air flow .
4..Then we transfer sterile tamarind seed from flask in tray.
5.Then we add Trichoderma viride culture from previous PDA plates and incubates for 5-6 days .
Date:-12/10/2021
Result:-
After 5-6 days we observed green color layer of growth of Trichoderma viride.
Observed the after 5 -6 dyas. full growth of fungus is observed. After air dry seed in room temperature and crush with the mixture grinder to make a fine powder and 600 gm powder is ready .
Date:-06/10/2021
1.Using 9 conical flask (500 ml capacity) . And 2 conical flask(250 ml capacity)
2. I soak 50 gm Tamarind seed in each flask overnight. Next day we dispose the water from each flask in autoclave at 121 degree C for 15 min.
3.After sterilization flask and wire loop and glove were sterilize by using laminar air flow .
4.Then we add Trichoderma viride culture from previous PDA plates and incubates for 5-6 days .
Date:-13/10/2021
Result:
Next day dry Trichoderma viride.
Result:
After air dry seed in room temperature and crush with the mixture grinder to make a fine powder and 300 gm powder is ready .
Date:-04/10/2021.
MASS MULTIPLICATION OF TRICHODERMA VIIRIDE BY USING SUBMERGERED METHOD
Submerged fermentation is the techniques of cultivation of microorganism in liquid broth which breaks down the nutrient to release the desired bio-active compound into solution. In this method, selected microorganism are grown in closed vessels containing a broth rich in nutrients and high concentration of oxygen. In SmF substrate are utilized quite rapidly hence need to be constantly replaced or supplemented with nutrients
Material:-
Potato Dextrose Broth
Trichoderma viride strain
Procedure:
- Take approximately 4 potatoes and weigh it .
- Wash it and boil and remove peels and oven dry it at 60 degree for 4-5 hours .
- After drying , grind it using grinder and make fine powder .
- Add 50 g of potato infusion powder to 200 ml D/W.
- Add 10 g of dextrose to it . And your broth is ready .
- Then we add Trichoderma viride strain in broth and keep it on rotary shaker for 5/6 days.
After 5-6 days. This liquid broth was used for field trials in Vigyan Ashram.
Date:-15/10/2021
Calculation Of CFU of Trichoderma viride powder by using powder.
What is CFU?
A colony-forming unit (CFU,)is a unit used in microbiology to estimate the number of viable bacteria or fungal cells in a sample. . The visual appearance of a colony in a cell culture requires significant growth, and when counting colonies it is uncertain if the colony arose from one cell or a group of cells.
10 test-tube were prepared using serial dilution technique by using Trichoderma viride powder .
10-8 , 10-9 , 10-10 dilutions were spread on sterile PDA agar plates . these plates were incubated for 72-96 hours for optimum growth
After 4 days optimum growth was observed and CFU count was taken by using colony counter .
The CFU/ml can be calculated using the formula
CFU/ml = (no. of colonies x dilution factor) / volume of culture plate.
CFU count of Trichoderma viride powder is 2.5 x 1012.
Date:-18/10/2021
Mix well talcum powder powder for final product of Trichoderma viride .100 gm Trichoderma viride powder is mixed in 900 gm talcum powder to make it 1 kg .
In house strain Trichoderma :-
So, 350 gm of Trichoderma powder is mixed in 3150 gm talcum powder is mix and make it 3.5 kg of Trichoderma powder by using in house strain in Vigyan Ashram.
Trichoderma viride : –
So, 1650 gm of Trichoderma viride powder is mixed in 14850 gm talcum powder is mixed and make it 16.5 kg Trichoderma viride powder. And 500 gm of Trichoderma viride powder was ready when we will add 4500 gm of talcum powder then we will got 5 kg Trichoderma viride powder.
- Total 20 kg Trichoderma viride powder was ready.
