George Teena, Sivam Visnuvinayagam, Vaiyapuri Murugadas, Anandan R, Sivaraman Gopalan Krishnan, Joseph Toms C
Microbiology, Fermentation and Biotechnology Division, ICAR-Central Institute of Fisheries Technology, Willingdon Island, Kochi, 682 029, Kerala, India.
Cochin University of Science and Technology, Kochi, 682022, Kerala, India.
Arch Microbiol. 2025 Feb 14;207(3):59. doi: 10.1007/s00203-025-04251-0.
Precise quantification of biofilm is critical as the formation and persistence of biofilm have significant implications in the environmental, therapeutic and industrial contexts. The microtiter plate assay using crystal violet with 33% glacial acetic acid or 94-100% ethanol as the resolubilising agent is widely used for the categorisation of biofilms into weak, moderate and strong categories. But, the use of varying wavelengths for the measurement of biofilm resulted in discrepancies in categorisation across the studies due to the difference in the extinction coefficient of CV. This study emphasises the importance of measuring the biofilm at the absorbance maximum (λ) of resolubilized CV, identified as 585 nm for 33% acetic acid and 580 nm for 94-100% ethanol. To address the challenge of harmonizing the results across studies, a method was developed to adjust the biofilm categorisation threshold based on the extinction coefficient ratios of CV at different wavelengths enabling consistent classification regardless of the wavelength used. Validation with E. coli and S. aureus demonstrated that the adjusted thresholds produced results similar to that obtained with the λ. This standardised approach not only enables the researchers to obtain accurate and consistent results in the future studies, but also facilitates the comparison of previously published data on biofilm research, which is essential for the exploration of newer therapeutic strategies against biofilm related infections.
生物膜的精确量化至关重要，因为生物膜的形成和持续存在在环境、治疗和工业背景中具有重大意义。使用结晶紫与33%冰醋酸或94 - 100%乙醇作为再溶解剂的微量滴定板测定法被广泛用于将生物膜分类为弱、中、强三类。但是，由于结晶紫（CV）消光系数的差异，在不同研究中使用不同波长测量生物膜导致分类存在差异。本研究强调在再溶解的CV的最大吸光度（λ）处测量生物膜的重要性，对于33%乙酸确定为585 nm，对于94 - 100%乙醇确定为580 nm。为应对跨研究统一结果的挑战，开发了一种方法，根据CV在不同波长下的消光系数比率调整生物膜分类阈值，从而无论使用何种波长都能实现一致的分类。用大肠杆菌和金黄色葡萄球菌进行验证表明，调整后的阈值产生的结果与在λ处获得的结果相似。这种标准化方法不仅使研究人员能够在未来研究中获得准确和一致的结果，还便于比较以前发表的关于生物膜研究的数据，这对于探索针对生物膜相关感染的新治疗策略至关重要。
