Incubation time and culture medium affect gram staining accuracy: A Cellpose image analysis study.

Area of Science:

• Microbiology
• Clinical Diagnostics
• Computational Biology

Background:

• Gram staining is a fundamental technique in clinical microbiology for rapid bacterial identification.
• The impact of culture media and incubation time on Gram staining accuracy has not been fully quantified using automated image analysis.

Purpose of the Study:

• To quantitatively assess the influence of culture medium and incubation duration on Gram staining fidelity.
• To evaluate the performance of Cellpose 2.0 in analyzing Gram-stained bacterial images.

Main Methods:

• Acquired 270 Gram-stained images of Staphylococcus aureus and Escherichia coli.
• Utilized Cellpose 2.0 to extract 90 quantitative variables related to staining intensity, cell wall, and morphology.
• Analyzed data using ANOVA, Pearson correlation, and principal component analysis (PCA).

Main Results:

• Both culture medium and incubation time significantly impacted staining variables (p < 0.001).
• Extended incubation (>48 hours) led to decreased peptidoglycan retention and reduced Gram-positive classification accuracy.
• PCA effectively separated bacterial species and incubation time points, with Blue/Red ratio correlating strongly with Gram-positive percentage.

Conclusions:

• Extended incubation (>48 hours) and selective media introduce systematic bias in Gram staining, especially for S. aureus.
• The Cellpose framework supports 24-48 hour cultures from nutrient-rich media for reliable Gram stain-based identification.
• This study provides a framework for laboratory quality assurance in Gram staining.