This study evaluated methods for accurately counting bacterial spores using microscope counting slides. Cook and Lund compared two slide types: shallow chambers (0.02 mm depth) and deeper chambers (0.10 mm depth). They analyzed various sources of error affecting spore counts, including chamber depth variation, cover-glass fit, sampling distribution, and counting technique. Testing Bacillus subtilis spore suspensions, they found that 0.10 mm slides produced more reproducible and accurate counts than 0.02 mm slides. The 0.02 mm slides yielded counts 15-36% higher than 0.10 mm slides, likely due to inconsistent cover-glass contact reducing actual chamber depth. Statistical analysis showed spore distribution followed expected patterns. Validation through percentage viability comparisons—combining total counts with colony formation and slide culture techniques—confirmed 0.10 mm slides provided more reliable results. The authors recommend 0.10 mm slides for bacterial spore enumeration, as they minimize systematic errors from cover-glass fitting while maintaining acceptable precision with reasonable replication.

Key findings

• 0.10 mm depth counting slides produced more reproducible spore counts than 0.02 mm depth slides due to better cover-glass contact and proportionally smaller fitting errors
• Inconsistent cover-glass fit on 0.02 mm slides caused systematic undercounting, with actual chamber depths varying by up to 12.4% from nominal specifications
• 0.10 mm slides yielded total counts 15-36% lower than 0.02 mm slides, which was validated by comparing percentage viability using both plate counts and slide culture methods
• Spore distribution in counting chambers followed Poisson distribution, indicating random distribution and supporting the validity of microscope counting methodology
• Variation between replicate counts on 0.10 mm slides approached theoretical sampling error limits, indicating minimal additional systematic error sources