Aim: To perform pour plate technique
Introduction- Pour plate method is most commonly used to quantitate the number of microorganisms present in the sample.
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.
- 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
Examine all the plates and count the number of colonies by using colony counter. The number of microorganisms are calculated by the above formula
Isolated colonies are examined on the surface of plate and calculate the number of colonies with dilution factor.
- 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.
- 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.