Black swimming dots in cell culture: first report of Bradyrhizobium as a cause

Abstract

Introduction

Black swimming dots (BSDs) are a frequent concern for scientists performing cell culture experiments. Despite this, there is very limited literature on this phenomenon. There is one report of Achromobacter species as the causative organism of black swimming dots in cell culture. In this study, we have revealed a 16S ribosomal RNA (bacterial rrs gene) full gene-based confirmation of black swimming dots as Bradyrhizobium species in our cell culture experiments. Bradyrhizobium is primarily a soil-dwelling bacteria, carrying out nitrogen fixation in legume roots.

Methodology

After inoculating the patient blood in L929 cell lines, DNA (deoxyribonucleic acid) isolation was performed on the seventh day from the trypsinized L929 cells. The culture flask under the tissue culture inverted microscope showed small moving dots in the field. The DNA extracted from the infected cell culture was then subjected to conventional PCR (Polymerase Chain Reaction) with the three sets of pan-bacterial primers targeting the full-length 16S ribosomal RNA gene.

Results

Our study reveals the contamination of mammalian cell culture with the black swimming dots caused by Bradyrhizobium species bacteria.

Conclusion

The 16S rRNA full gene analysis revealed that the black swimming dots in our cell culture flasks were due to Bradyrhizobium species. This study further clarifies that the BSDs in cell culture caused due to Bradyrhizobium species could not be cleared by 10 μg/ml concentration of both the antibiotics, piperacillin and ciprofloxacin.