Direct microscopic count yields a count of all cells both dead and alive. Plate count methods facilitate the counting of viable (and able to reproduce) cells only.
In most viable count methods, a dilute sample of bacteria or other organisms is dispersed on the surface of a solid medium.
Each organism or cluster of organisms grows on the surface of the medium to develop into distinct colonies.
The original number of viable organisms in the sample can be calculated from the number of colonies formed and the dilution used for the spreading.
Plating techniques are simple, sensitive, and widely used to count microorganisms in various samples in food, soil, and water.
It is not absolutely certain to state that each colony develops from a single cell; hence the result is expressed as colony forming units (CFU) rather than a number of organisms.
Enumerating the organisms by plate count method can be done by spread plate or pour plate methods.
What is Spread Plate Technique?
This technique usually involves the spreading of 0.1 ml dilution on the solid nutrient medium.
If organisms are present and able to form visible colonies after appropriate incubation colony-forming units are counted.
The number of colonies developed on the plate multiplied by the respective dilution factor gives the bacterial load of the sample.
In this type of technique, we are using the serial dilution technique to form a diluted sample.
This technique is an isolation method used for the quantitative estimation of cells.
Requirements of Spread Plate Technique
- Sterilized 9 dilution tubes with 9 ml 0.85% NaCl as a diluent
- Sterile 1 ml and 10 ml pipettes
- Sterile nutrient agar plates
- Glass spreader
Procedure of Spread Plate Technique
- Serial dilutions of the sample are prepared via the serial dilution technique.
- The diluted sample (0.1 ml) of the respective dilution is plated on the nutrient agar plates in duplicates or triplicates.
- The glass spreader was sterilized by dipping in alcohol and held in flame for sterilization.
- The drop of the spread on the surface of the nutrient agar.
- The plates were incubated at 37°C for 24 hours.
- The colonies were counted for the estimation of CFU/ml in the sample.
- Calculate the CFU value of the sample with the formula.
Uses of Spread Plate Technique
- This technique is used to count the total number of CFUs (colony forming units) on a single plate.
- The spread plate technique is also used for the calculation of the concentration of cells in the tube.
Calculations of Spread Plate Technique
Bacterial density of original sample = No. of CFU/ml of selected dilution x dilution factor