To stain or not to stain: unraveling potential bacterial contamination in germ-free husbandry using bacterial viability staining

AIMS: Hygienic monitoring (HM) of germ-free (GF) mouse colonies is exceptionally challenging. The test accuracy of the applied diagnostic methodology has to be outstanding to provide proof of absence of all living microorganisms confirming the GF status. In this context, microscopy of native intestinal content serves as a highly sensitive diagnostic tool for the detection of bacterial contaminants. However, with this method residual microorganisms may be detected. To overcome this risk of false-positive results, we complemented our analyses with a bacterial viability staining of the intestinal content of GF mice. METHODS AND RESULTS: Intestinal contents of GF mice (n = 13) from five isolators were analyzed by bacterial culture and phase-contrast microscopy. Additionally, 16S rRNA gene PCR analysis and metagenomic sequencing were performed. To distinguish between live and dead bacteria, intestinal content was stained by a Bacterial Viability Kit and analyzed by fluorescence microscopy. While culture medium proved sterility of the sample material, increased amounts of scattered bacterial structures were detected during microscopic analysis, indicating potential contamination. Molecular techniques pointed to a presence of environmental bacteria. However, viability staining revealed the presence of only dead (double-stained) bacteria in all samples. Likewise, non-viable bacteria have been identified in samples obtained from irradiated feed, probably being the source of bacterial structures found in GF mice. CONCLUSIONS: Altogether, detected bacterial structures were proven to be non-viable and therefore should not be interpreted as isolator contaminants. Thus, in our hands, with the herein described report of suspected contamination, we prove that bacterial viability staining served as a highly valuable screening tool, enhancing diagnostic quality of the HM of GF colonies.