Quantifying Bacteria Concentration using Serial Dilution and Plate Count Method

Overview

• Method involves serial dilution of bacteria culture followed by plate counting.
• Purpose: Determine the concentration of bacteria in a culture.

Materials Needed

• 7 sterile microcentrifuge tubes.
• 7 LB agar plates.
• Micropipettes (20-200 µL and 100-1000 µL).
• LB broth.
• Bacterial culture.
• Inoculation loop or bacterial spreader.
• Incubator set to 37°C.

Procedure

Preparation

1. Labeling:

   • Microcentrifuge tubes: Label with dilution factors: 10^1 to 10^7.
   • LB agar plates: Similarly label with dilution factors: 10^1 to 10^7.

2. Micropipetting:

   • Use a 100-1000 µL micropipette. Set to 900 µL.
   • Pipette 900 µL of LB broth into each microcentrifuge tube using aseptic technique.

Serial Dilution Process

1. Use a 20-200 µL micropipette set to 100 µL.
2. Pipette 100 µL of bacterial culture into the tube labeled 10^1.
3. Mix thoroughly by vortexing or pipetting up and down at least 5 times.
4. Transfer 100 µL of the mixture from the tube labeled 10^1 to the tube labeled 10^2.
5. Mix thoroughly and repeat the dilution process for each successive tube up to 10^7.

Plating

1. First Dilution:
   • Mix the 10^1 dilution tube.
   • Pipette 100 µL onto the corresponding LB agar plate labeled 10^1.
   • Sterilize inoculation loop or spreader by flaming; allow to cool.
   • Spread the culture evenly over the plate surface (do not use streak plate technique).
2. Repeat the plating for each dilution on the matching plate.

Incubation

• Stack plates upside down.
• Incubate at 37°C for 16 to 24 hours.

Analysis

• After incubation, plates will be ready for analysis to determine bacterial concentration.