Introduction
To capture a microscopic image of microbial consortium culture. This is required to have preliminary authentication of consortium.
Objective
To detect whether a standard culture is contaminated with any other strain and major colonies are present.
Methodology
- Preparation of agar plate
- Inoculation of culture on agar plate and incubate for 2-3 days at room temperature
- Identification of bacteria and fungi
- Subculture the colony on another plate
- Slant preparation.
Firstly, we thought it was a consortium, but we didn’t have microscopic images, so we visited to The Department of Mycology at SPPU Pune University to learn about the methodology for identifying bacteria and fungi from the consortium. There, Mr.Mahesh Borde sir explained the methodology for identification.
Materials and Methods
REQUIREMENTS:
Available BARC Culture, PDA and NA Media ,Petri plates,Spreader,Microtips and micropipette,Autoclave,Incubator Laminar, Beaker.
PREPARATION FOR PLATING :
1.POTATO DEXTROSE AGAR MEDIA:
12.5 g PDA powder
250 ml water
- Add 12.5 g of PDA powder to a 500 ml flask or container.
- add 250 ml of distilled water to the flask while stirring with a glass rod.
- Continue stirring until the powder is fully dissolved.
- then boil the mixture , the cool it for 5-7 minutes.
- Autoclave the media at 121 degree Celcius for 20 min at 15 lbs pressure.
- Pour the PDA media into sterile containers, such as petri dishes .
- Allow the media to cool and solidify at room temperature.
2.NUTRIENT AGAR MEDIA:
7.5 g Nutrient Agar powder
250 ml distilled water
- Add 7.5 g of Nutrient Agar powder to a 500 ml flask or container.
- add 250 ml of distilled water to the flask while stirring with a glass rod.
- Continue stirring until the powder is fully dissolved.
- Bring the mixture to a boil, then cool it for 5-7 minutes.
- Autoclave the media at 121 degree Celcius for 20 min at 15 lbs pressure.
- Pour the PDA media into sterile containers, such as petri dishes .
- Allow the media to cool and solidify at room temperature.
3.SERIAL DILUTION :
Culture
Sterilized distilled water
Sterilized dilution test tubes (10 ml)
Pipettes
Agar plates (eg. PDA and NA media)
Table Of Serial Dilution of Soil:
Take a 1ml aliquot of the culture using the sterile pipette to a tube containing 9
ml of sterile distilled water. This is a 1:10 dilution.
Mix the contents of the tube thoroughly and repeat the dilution process with additional
tubes.
| Sr.No. | Test Tube | Dilution volume | dilution factor |
| 1. | 1st | 1ml culture + 9ml Distilled water | 1:10 |
| 2. | 2nd | 1ml culture + 9ml Distilled water | 1:100 |
| 3. | 3rd | 1ml culture + 9ml Distilled water | 1:1000 |
| 4. | 4th | 1ml culture + 9ml Distilled water | 1:10000 |
| 5. | 5th | 1ml culture + 9ml Distilled water | 1:100000 |
Mix each tube thoroughly and labelled them with the dilution Factor.
Take a small volume for example 0.1 ml from each dilution tube and spread it onto an
agar plate using a sterile spreader.
Incubate the plates at the appropriate temperature (37°C) for 2-5 days .Observe under the microscope.
Observation
Bacterial colonies growth in 3 days on Nutrient Agar plate.
