Microfiltration method of removal of bacterial contaminants and their monitoring by nitric oxide and Limulus assays

• Published in: Nitric oxide 2013-01, Vol.28, p.1-7
• Main Authors: Zídek, Zdeněk, Kmoníčková, Eva, Kostecká, Petra, Jansa, Petr
• Format: Article
• Language: English
• Subjects: Animals; Bacillus subtilis - chemistry; Bacillus subtilis - isolation & purification; Cell Line; Cell Survival; Centrifugation; Decontamination; Escherichia coli - chemistry; Escherichia coli - isolation & purification; Female; Filtration; Limulus Test; Lipopolysaccharide; Lipopolysaccharides - analysis; Lipoteichoic acid; Mice; Mice, Inbred C57BL; Microfiltration; Nitric oxide; Nitric Oxide - biosynthesis; Nitric Oxide - metabolism; Peptidoglycan; Peptidoglycan - analysis; Rats; Rats, Wistar; Teichoic Acids - analysis
• ISSN: 1089-8603; 1089-8611
• DOI: 10.1016/j.niox.2012.08.078

► Limulus and NO assays indicate presence of both G− and G+ bacterial products. ► LPS, lipoteichic acid/LTA and peptidoglycan/PGN activate NO in rat peritoneal cells. ► Polymyxin B can be used for neutralization of LPS merely, not for PGN and LTA. ► 3–100kDa microfiltration removes all traces of LPS, PGN and LTA. ► Microfiltration is universal, feasible and reliable method of sample decontamination. Similar to lipopolysaccharide (LPS), a product of Gram-negative bacteria, the signal macromolecules of Gram-positive bacteria lipoteichoic acid (LTA) and peptidoglycan (PGN) possess multiple biological activities. They may be a source of misinterpretation of experimental findings. We have found that not only LPS but also LTA and PGN can be detected by the Limulus amebocyte lysate (LAL) assay. All of them stimulate the high output in vitro nitric oxide (NO) production of in rat peritoneal cells. The onset of the NO enhancement was observed with 25–100pg/ml of LPS and 25–100ng/ml of PGN and LTA. Polymyxin B (PMX), if applied at concentration 10,000-fold higher than that of LPS, can completely inhibit the NO and LAL binding responses of LPS. The NO-stimulatory and LAL-binding properties of LTA and PGN are not eliminated by PMX. Handling of LPS contamination with PMX may be associated with serious problems because it possesses intrinsic biological activity and becomes cytotoxic at concentration >25μg/ml. The present findings suggest a convenient alternative avoiding these issues. As monitored by the NO and LAL assays, even high amounts of LPS as well as PGN and LTA can be removed by molecular mass cutoff microfiltration. All types of the filters (3kDa to 100kDa) are equally effective. It is suggested that the microfiltration procedure may be considered as a preferable, general and easy method of sample decontamination.