Serial Dilution and Standard Plate Count

Serial Dilution and Standard Plate Count

• Serial Dilution: A technique used to obtain a viable cell or standard plate count.
• Standard Plate Count: A method for determining microbial density in a culture by counting the number of colonies formed when a portion of the broth is transferred to an agar plate.
• Confluent Growth: When bacterial growth completely covers the plate, making it impossible to differentiate individual colonies. Undiluted broth cultures often produce confluent growth.
• Purpose of Serial Dilution:
  1. Reduces cell density to achieve countable plates.
  2. Provides a mathematical framework to link the unknown original cell density with the known number of colonies on a plate.

Countable Plate

A countable plate contains between 30 and 300 colonies.

• Each colony originates from a single cell that underwent multiple rounds of division.
• Each colony is counted as a CFU (colony forming unit).

Serial Dilution Process

1. Original inoculum (containing an unknown number of bacteria) is diluted into a series of tubes.
2. Each tube contains 9 mL of sterile diluent (a sterile solution used for making dilutions).
3. 1 mL of the inoculum is transferred from the original sample to the first tube.
4. The tube is mixed thoroughly, and 1 mL is transferred to the next tube containing 9 mL of sterile broth.
5. Sequential dilutions are continued until each tube contains 1 mL from the tube before it, along with 9 mL of diluent.

Dilution Factor

• Definition: A means of tracking the amount of the original broth in each tube.
• Example: Adding 1 mL of original inoculum to 9 mL of diluent results in a 1:10 dilution factor.
  • The original inoculum represents 1/10th of the new solution.
• Taking 1 mL of the 1:10 diluted broth and adding it to 9 mL of diluent results in a 1:100 dilution.
  • The original inoculum is now 1/100th of the solution.
• Serial dilution involves diluting the original sample multiple times over a series of tubes.
• A "1 in 10" serial dilution involves diluting 1 mL of sample in 9 mL of diluent (totaling 10 mL).
• A "1 in 2" dilution would involve adding 5 mL of original inoculum to 5 mL of diluent.
• The dilution factor is calculated as: \frac{\text{mL transferred}}{\text{total mL in solution}} = \frac{\text{amount transferred}}{\text{amount transferred + diluent}}
• The dilution factor can be expressed as a fraction (e.g., 1/10), a ratio (e.g., 1:10), or an exponent (e.g., 10^{-1}).

Plating and Colony Counting

• A small portion of liquid is removed from each tube and plated to allow bacterial growth.
• With proper dilutions, the number of colonies on the plates should decrease with each greater dilution.
• In a “1 in 10” serial dilution, each plate should ideally have one-tenth the number of colonies compared to the previous one.
• The goal is to find a countable plate with 30-300 distinguishable colonies.
• Plates with lower dilutions (e.g., 1:10, 1:100) may have too numerous to count (TNTC) colonies or confluent growth.
• Higher dilutions may have too few colonies.
• Confluent growth appears as large areas or patches of growth without discernible colonies.

Calculating Original Cell Density

• Count the number of colonies on the countable plate.
• Use the dilution factor of the tube from which the bacteria were plated to calculate the original cell density.
• Formula:
  \text{original cell density} = \frac{\text{colonies counted}}{\text{(volume plated)} \times \text{(dilution factor)}}
• Example:
  • Countable plate: 1:10,000 dilution with 32 colonies.
  • Volume plated: 1 mL.
  • Dilution factor: 1/10,000 or 10^{-4}.
  • Calculation:
    \text{original cell density} = \frac{32 \text{ colonies}}{(1 \text{ mL}) \times (10^{-4})} = 320,000 \text{ bacteria/mL}
• Reference:
  • Serial dilution and standard plate count are discussed in OpenStax Microbiology, Chapter 9.1, pages 331-333.

Spread Plate Technique

• Purpose: To spread the liquid containing bacteria evenly on the agar plate for individual colonies to grow.
• Materials:
  • Alcohol lamp.
  • Alcohol.
  • Glass spreader (bent glass rod).
• Steps:
  1. Sterilize the glass spreader by dipping it in alcohol and igniting it with the alcohol lamp to burn off the alcohol.
  2. Use the sterilized spreader to spread the bacterial culture over the entire surface of the agar.

Additional Notes

• 1 milliliter (mL) = 1,000 microliters (μL)
• 1 liter (L) = 1000 milliliters (mL)

Common Issues Affecting Colony Count

• Contamination leading to more colonies.
• Inaccurate dilution due to improper mixing.
• Errors in volume transfer.
• Non-sterile equipment.
• Miscalculation of the final result.

Practice Problems

1. Plating 500 μL of a sample diluted by a factor of 10^{-3} produced 86 colonies. What was the original concentration of the sample?

   • Solution:
     \text{original cell density} = \frac{86 \text{ colonies}}{(0.5 \text{ mL}) \times (10^{-3})} = 172,000 \text{ bacteria/mL}

2. Plating 0.2 mL of a sample diluted by a factor of 10^{-3} produced 117 colonies. What was the original concentration of the sample?

   • Solution:
     \text{original cell density} = \frac{117 \text{ colonies}}{(0.2 \text{ mL}) \times (10^{-3})} = 585,000 \text{ bacteria/mL}

3. You were instructed to add 1.0 mL of an undiluted sample to 9 mL of sterile water. Instead, you add the entire sample (5.0 mL) to the 9 mL of diluent. What was the intended dilution factor, and what was the actual dilution factor?

   • Intended dilution factor: 1 mL / (1 mL + 9 mL) = 1/10
   • Actual dilution factor: 5 mL / (5 mL + 9 mL) = 5/14