- 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
Reference and Sources
- Acid fast staining of bacteria
- Aseptic Transfer Technique
- Bacterial Flagella, Fimbriae and Pili
- Bacterial Growth and Nutrition
- Fimbriae vs Flagella
- Fimbriae vs Pili
- Growth Curve of Bacteria
- Instruments used in Microbiology Laboratory
- MacConkey agar
- McFarland Standards
- Monochrome Staining
- Negative Staining
- Nutritional Requirements of Micro-Organisms
- Preparation and Sterilization of culture media
- Serial Dilution in Microbiology
- Streak Plate Technique