Overview
This lecture explains the serial dilution technique used in microbiology labs to estimate the number of microbes in a sample, focusing on achieving a countable number of colonies for accurate measurement.
Purpose of Serial Dilution
- Serial dilution is necessary to reduce microbial concentration for easier colony counting.
- Accurate counts require plates with 30–300 colonies; too few or too many yield unreliable data.
- Dilution allows estimation of colony-forming units (CFU) per milliliter in an original sample.
Serial Dilution Technique
- Begin with 1 mL of the original sample added to 9 mL of sterile medium for a 1:10 dilution.
- Mix thoroughly, then transfer 1 mL from this tube into the next tube with 9 mL sterile medium.
- Repeat this sequential dilution process through multiple tubes (e.g., 5 times).
- After diluting, plate samples (often 0.1 mL) onto agar plates using pour or spread plate methods.
Counting Colonies and Calculations
- Incubate plates and select the one with 30–300 colonies for counting.
- If 50 colonies are found on a 1:10,000 dilution plate using 0.1 mL, calculate total CFU as follows:
- Multiply by 10 to scale up from 0.1 mL to 1 mL.
- Multiply by the dilution factor (10,000).
- Example: 50 colonies × 10 × 10,000 = 5,000,000 CFU/mL in the original sample.
- Plates with >300 colonies are unreliable; use plates within the target range.
Key Terms & Definitions
- Serial dilution — Stepwise dilution of a substance in solution to reduce concentration for easier analysis.
- Colony-forming unit (CFU) — A unit estimating the number of viable microbes capable of forming colonies.
- Pour plate — A method where diluted samples are mixed with agar and poured into plates to grow colonies.
- Spread plate — Technique where diluted samples are spread over the surface of solid agar using a sterile tool.
Action Items / Next Steps
- Watch the next video on pour plates vs. spread plates for further instructions.
- Practice performing serial dilutions and colony counts in the lab.