目的:为了评估两种市售活力染料的功效,5-氰基-2,3-二-(对甲苯基)四唑鎓氯化物(CTC)和5(6)-羧基荧光素二乙酸酯(CFDA),使用全纤维荧光计报告活细胞浓度和物种。
结果:四种细菌(两种革兰氏阳性和两种革兰氏阴性)通常与食物中毒或食物腐败有关(大肠杆菌,肠沙门氏菌,金黄色葡萄球菌,和蜡状芽孢杆菌)用CTC或CFDA染色,并使用纤维荧光计来收集完整的荧光发射光谱。对于107至108个菌落形成单位(CFU)ml-1的革兰氏阴性细菌,浓度与荧光强度之间存在良好的相关性。与革兰氏阳性菌的浓度没有相关性;然而,CTC和CFDA光谱中的信息显示了区分革兰氏阴性细胞和革兰氏阳性细胞的潜力,尽管它可能仅反映了本研究染色条件下的总体细菌代谢活性。
结论:浸渍探针分析方法的检测限(LoD)过高,然而,一种测量单细胞荧光的方法的发展可能会改善这种局限性。新的细菌特异性荧光染料的开发也可以解决这种限制。使用这些染料区分细菌的能力可以增加使用CTC和CFDA计数细菌的测量值。
OBJECTIVE: To assess the efficacy of two commercially available viability dyes, 5-cyano-2,3-di-(p-tolyl)tetrazolium chloride (CTC) and 5(6)-carboxyfluorescein diacetate (CFDA), in reporting on viable cell concentration and species using an all-fibre fluorometer.
RESULTS: Four bacterial species (two Gram-positive and two Gram-negative) commonly associated with food poisoning or food spoilage (Escherichia coli, Salmonella enterica, Staphylococcus aureus, and Bacillus cereus) were stained with CTC or CFDA and the fibre fluorometer was used to collect full fluorescence emission spectra. A good correlation between concentration and fluorescence intensity was found for Gram-negative bacteria between 107 and 108 colony-forming units (CFU) ml-1. There was no correlation with concentration for Gram-positive bacteria; however, the information in the CTC and CFDA spectra shows the potential to distinguish Gram-negative cells from Gram-positive cells, although it may simply reflect the overall bacterial metabolic activity under staining conditions from this study.
CONCLUSIONS: The limit of detection (LoD) is too high in the dip-probe approach for analysis; however, the development of an approach measuring the fluorescence of single cells may improve this limitation. The development of new bacteria-specific fluorogenic dyes may also address this limitation. The ability to differentiate bacteria using these dyes may add value to measurements made to enumerate bacteria using CTC and CFDA.