Accurate estimation of microbial concentration is essential for identification, isolation, cultivation, and characterization. Quantitative measurements underpin all microbiological disciplines, from clinical diagnostics and industrial microbiology to environmental sampling and academic research.
The foundation of serial dilution and plating techniques dates back to 1883 when German physician Robert Koch published his pioneering work on infectious agents. Koch’s methodologies remain the gold standard for microbial enumeration today, applicable to both single species and complex microbial communities.
What is Serial Dilution?
Serial dilution is a stepwise reduction in concentration of a known or unknown substance, such as a microorganism, achieved by successive resuspension into fixed volumes of a liquid diluent. The original sample is referred to as solution₀, with subsequent dilutions creating a series of predictable concentrations.
Diluents typically consist of 0.45% saline, which maintains osmotic balance. Volumes are often chosen as multiples of 10 to allow logarithmic reduction for easy calculations.
Example of a 10-Fold Serial Dilution
For example, a stock solution (solution₀) containing 100 E. coli cells in 10 mL of nutrient broth can be diluted as follows:
- 1 mL of solution₀ into 9 mL of saline → solution₁ (10 cells)
- 1 mL of solution₁ into 9 mL of saline → solution₂ (1 cell)
Each step represents a 10-fold dilution, simplifying calculations of colony-forming units (CFU).
Figure 2: Streak plating technique. The plate is divided into sections, and streaking is reduced with each section to isolate colonies.
Figure 3: Spread plating of serially diluted samples to enumerate colony-forming units.
Plating Techniques for Microbial Enumeration
After serial dilution, plating techniques allow for microbial enumeration and isolation. Two common methods are streak plating and spread plating.
Streak Plating for Isolation
Streak plating is used to isolate individual colonies from mixed populations. A diluted sample is introduced onto agar, and a sterile loop is used to streak in a controlled zig-zag pattern. Quadrant streaking reduces cell density across the plate, producing distinct colonies.
Spread Plating for Enumeration
Spread plating distributes a measured aliquot of diluted sample evenly across an agar plate. Colonies arise from single cells, allowing calculation of CFU/mL based on dilution factor and volume plated.
Calculating Colony Forming Units (CFU)
CFUs are calculated using plates with 30–300 colonies for statistical reliability. Plates with fewer than 30 colonies are too few to count (TFTC), and those with more than 300 are too numerous to count (TNTC).
The formula for CFU/mL is:
CFU/mL = (Average colony count × Dilution factor) / Volume plated (mL)
Plotting log₁₀ CFU/mL versus time allows visualization of bacterial growth phases and calculation of generation times.
Application to Winogradsky Column Microbial Communities
Serial dilution and plating are particularly useful for studying microbial communities in Winogradsky columns. These columns contain distinct aerobic, microaerophilic, and anaerobic zones. Samples harvested from each zone can be serially diluted and plated to evaluate population diversity and abundance.
Streaked plates often reveal mixed populations with diverse colony morphologies, while plates inoculated with a known species, such as E. coli, show uniform colonies. Isolated colonies can then be used for enrichment assays, identification, or further physiological studies.
Step-by-Step Laboratory Procedure
1. Laboratory Setup and Safety
- Wear PPE: lab coat, gloves, goggles.
- Sterilize workspace with 70% ethanol.
- Keep a flow chart of materials and stepwise protocol in your lab notebook.
2. Media Preparation
- Prepare LB agar and LB broth according to manufacturer recommendations.
- Autoclave at 121°C for 15 minutes at 15 psi.
- Pour agar plates (≤15 mL per plate) and allow to solidify.
3. Diluent and Serial Dilution
- Prepare ten 20 mL test tubes labeled T1–T10, each with 9 mL of 0.45% saline.
- Perform serial dilutions of the target organism or Winogradsky column sample.
- Vortex thoroughly after each transfer to ensure uniform suspension.
4. Plating
- Spread plating: pipette 100 µL of diluted sample and spread evenly with a sterile rod.
- Streak plating: streak samples on designated quadrants using zig-zag patterns to isolate colonies.
- Incubate aerobic organisms at 37°C and anaerobic organisms in an anaerobic chamber at 37°C.
5. Data Collection and Analysis
- Count colonies from 30–300 colonies per plate.
- Calculate CFU/mL using the average colony count, dilution factor, and plated volume.
- Plot log₁₀ CFU/mL against time to analyze bacterial growth phases.
Summary
Serial dilution and plating remain cornerstones of microbiological methodology. These techniques enable precise enumeration, isolation, and characterization of microorganisms, from single-species cultures to complex environmental communities. When combined with growth curve analysis and careful experimental design, they provide profound insights into microbial physiology, ecology, and population dynamics across clinical, industrial, and research contexts.
Explore more about bacterial growth curves and microbial isolation techniques on The Science Notes.
