Xenobiotics Research Unit, Biological and Health Sciences Centre, The Pontifical Catholic University of Paraná, Curitiba, Brazil.
J Microbiol Methods. 2011 Feb;84(2):299-306. doi: 10.1016/j.mimet.2010.12.018. Epub 2010 Dec 20.
Due to technical problems, biofilm biomasses are difficult to be precisely determined. One reliable strategy is based on the colorimetry of formazan compounds derived from tetrazolium salt reduction. XTT presents some desirable properties that make the biofilm measurements easier. However, cells entrapped within the extracellular matrixes normally do not metabolize the tetrazolium equally, leading to underestimation of cell contents. This study evaluated the effectiveness of D-glutamine, a plerotic substrate of tricarboxilic acid cycle (TAC), as inducer of XTT reduction. The metabolic activities of aerobic and anaerobic 48 h-old monospecific biofilms of Pseudomonas aeruginosa ATCC®27853™, Klebsiella pneumoniae ATCC®13883™, Staphylococcus epidermidis ATCC®12228™, Streptococcus mutans ATCC®25175™, and Candida albicans SC5314 were evaluated. Results showed that D-glutamine 50 mM (for P. aeruginosa, K. pneumoniae, and S. epidermidis) and 25 mM (for S. mutans and C. albicans) may enhance the detection of soluble formazan in a significant manner, what becomes the XTT reduction assay more robust.
由于技术问题，生物膜生物量难以精确确定。一种可靠的策略是基于四唑盐还原生成的甲臜化合物的比色法。XTT 具有一些理想的特性，使生物膜测量更容易。然而，通常被困在细胞外基质中的细胞不能平等地代谢四唑盐，导致细胞含量的低估。本研究评估了 D-谷氨酰胺（三羧酸循环（TAC）的填充底物）作为 XTT 还原诱导物的有效性。评估了需氧和厌氧 48 小时龄的铜绿假单胞菌 ATCC®27853™、肺炎克雷伯菌 ATCC®13883™、表皮葡萄球菌 ATCC®12228™、变形链球菌 ATCC®25175™和白色念珠菌 SC5314 的单种生物膜的代谢活性。结果表明，50mM 的 D-谷氨酰胺（用于铜绿假单胞菌、肺炎克雷伯菌和表皮葡萄球菌）和 25mM 的 D-谷氨酰胺（用于变形链球菌和白色念珠菌）可以显著增强可溶性甲臜的检测，使 XTT 还原测定更稳健。
