Aim: To perform pour plate technique
Introduction- Pour plate method is most commonly used to quantitate the number of microorganisms present in the sample.
Requirements:
Apparatus: Sterile plates, inoculating loop, sterile pipettes, test tubes, test tube rack, water bath, Glass marking pencil, Busen burner.
Bacterial culture: Staphylococcus aureus, Chromobacterium indica
Media: Melted agar
Instruments: Colony counter with a magnifying glass, Autoclave, Hot air oven, Incubator.
Procedure:
- Dilute the bacterial sample in such a way that the final concentration of bacteria will be in the range of 30-300 cfu/0.1-1.0ml
- Each diluted sample is poured in Petri dish. Mark the number of dilution factors on Petri dishes like 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, 10-7, etc.
- Add melted agar medium in Petri dishes and gently swirling the plate on the tabletop for proper mixing of the sample with media.
- Inoculated Petri plates are allowed to cool at room temperature (till temp. reaches to 45°C).
- Incubate the plates at 37°C for 24-48 hours for cultivation.
- At the end find the number of colony-forming unit (cfu) per millimetre of the sample. To find this, the number of colonies is multiplied by the dilution factor.
The number of CFUs per ml of sample = number of colonies x the dilution factor of the plate counted.
For Example: If a plate containing a 1 x 10-3 dilution of the original sample and shows 120 colonies then the number of CFUs per ml in the original sample can be found by multiplying 120x 1000
Observation:
Examine all the plates and count the number of colonies by using colony counter. The number of microorganisms are calculated by the above formula
Result:
Isolated colonies are examined on the surface of plate and calculate the number of colonies with dilution factor.
Advantage:
- This method is used to count viable cells.
- This method is used to enumerate the bacteria in milk, water, food, soil etc.
- Distinct separate colonies are obtained on a solid medium.
- No requirement of spreaders.
- Aerobic, anaerobic and facultative aerobic organisms can grow on the media.
Disadvantage:
- For each dilution, you need separate tubes.
- Need to control the specific temperature, otherwise, too hot media kills the bacteria present in the sample.
- If colonies get overlap it creates problems in isolation.
- It is a time-consuming method especially for the preparation of dilution.
- Aerobic bacteria which get trapped inside the agar fail to survive due to lack of oxygen.
