Overview
This lecture covers the process of performing serial dilutions and plate counts to estimate viable bacteria in food samples, using alfalfa sprouts as an example.
Plate Count Basics
- Plate counts estimate viable bacteria by counting colonies formed from individual cells in a sample.
- Only bacteria capable of growing on nutrient agar are counted as viable.
Dilution and Sample Preparation
- Dilutions are necessary when the sample has too many bacteria to count directly.
- Serial dilutions use the same dilution factor for each tube or bottle.
- Common dilution blanks contain 9 mL or 99 mL of sterile water or 0.085% sodium chloride.
- 1 mL into 9 mL creates a 1:10 (10⁻¹) dilution; 1 mL into 99 mL makes a 1:100 (10⁻²) dilution.
- Mixing different dilutions multiplies the dilution factors (add exponents for serial steps).
- Solid samples (1 g) are treated as equivalent to 1 mL for calculations.
Plating Procedure
- Transfer known volumes (0.1 or 1.0 mL) of diluted sample to labeled sterile petri dishes.
- Use the same pipette only when moving from lower to higher dilutions.
- Pour 12-15 mL of melted tryptic soy nutrient agar (45 –50°C) into each plate, swirl gently to mix.
- Allow agar to solidify and stack plates to minimize condensation.
Incubation and Colony Counting
- Incubate plates inverted at 35–37°C for at least 24 hours.
- Colonies may form on or below the agar surface; all must be counted.
- Report results as colony forming units (CFU) due to possible clumping of cells.
- Select plates with 25–250 colonies for counting.
Calculations
- CFU per gram or mL = (number of colonies × reciprocal of dilution factor).
- Dilution factor = all dilution steps × plated volume.
- Formula: CFU/g = colonies counted × (1/dilution factor).
Key Terms & Definitions
- Plate Count — Method to estimate viable bacterial numbers by counting colonies.
- Viable Cells — Bacteria capable of growth and forming colonies.
- Serial Dilution — Stepwise dilution using the same factor at each step.
- Colony Forming Unit (CFU) — Unit representing one viable cell or group capable of forming a colony.
- Dilution Factor — Total dilution of a sample, product of all dilution steps and plated volume.
Action Items / Next Steps
- Record all data accurately.
- Discard used plates for autoclaving as required.
- Review dilution and calculation examples for practice.