Comparison between direct methods for determination of microbial cell volume: electron microscopy and electronic...

Area of Science:

• Microbiology
• Microscopy
• Cell Biology

Background:

• Accurate size determination of microorganisms is crucial for ecological and physiological studies.
• Various microscopy techniques are employed, but sample preparation can induce artifacts like cell shrinkage.

Purpose of the Study:

• To compare size frequency distributions of phototrophic and heterotrophic microorganisms using different sizing techniques.
• To quantify cell shrinkage induced by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and electronic particle sizing (EPS).

Main Methods:

• Microorganisms were analyzed using SEM, TEM, and EPS.
• Cellular volumes were measured and compared across the three techniques.
• Shrinkage factors were calculated, and relationships with cell type and pigment content were investigated.

Main Results:

• Statistically significant differences in size measurements were observed among SEM, TEM, and EPS.
• Electron microscopy techniques resulted in lower mean cellular volumes compared to EPS, with shrinkage factors ranging from 1.1 to 6.2 (mean 2.3).
• SEM induced greater shrinkage than TEM. Phototrophic bacteria showed more pronounced shrinkage than heterotrophic microorganisms, correlated with pigment content, suggesting solvent-induced pigment extraction.

Conclusions:

• Sample preparation methods, particularly for SEM, significantly impact the measured size of microorganisms.
• Shrinkage is not dependent on microorganism size or cell wall type but is influenced by pigment content in phototrophic bacteria.
• Researchers must consider these methodological artifacts when interpreting microbial size data obtained from different techniques.