Herrera Guadalupe, Martinez Alicia, Blanco Manuel, O'Connor José-Enrique
Centro de Citometría, Departamento de Bioquímica, Facultad de Medicina, Universidad de Valencia, Valencia, Spain.
Cytometry. 2002 Oct 1;49(2):62-9. doi: 10.1002/cyto.10148.
Flow cytometry has become a choice methodology for microbiological research. However, functional cytometric assays in live bacteria are still limited. This is due, in part, to the cell wall impairing penetration of vital dyes in bacteria, thus imposing permeabilization procedures. These manipulations may affect cell physiology, provoke cell aggregation or lysis, and they are time-consuming. Escherichia coli B strains have been used for mutagenic assays because of an altered lipopolysaccharide that provokes increased membrane permeability. We assessed the use of these strains as possible alternatives for flow cytometric assays to avoid the permeabilization steps.
Suspensions of E. coli K-12 (strain AB1157) and E. coli B (strain WP2 uvrA/pKM101, denoted as strain IC188) were stained with several fluorochromes, including fluorescein isothiocyanate, propidium iodide, Nile Red, bis-(1,3-dibutylbarbituric acid) trimethine oxonol, hydroethidine, and dihydro-dichlorofluorescein diacetate, under basal conditions and following permeabilization, impairment of membrane potential, inhibition of dye efflux pump, and oxidative stress. Fluorescent staining of both strains was compared by epifluorescence microscopy and flow cytometry.
The E. coli B strain IC188 exhibited more efficient staining with vital fluorochromes than the E. coli K-12 strain AB1157 and maintained a similar membrane potential. In addition, IC188 showed higher sensitivity than AB1157 to reveal oxidative stress when challenged with prooxidants.
E. coli B strains may be useful for biochemical and toxicological studies based on flow cytometry and fluorescence microscopy.
流式细胞术已成为微生物学研究的一种选择方法。然而，对活细菌进行功能性细胞计数分析仍然有限。部分原因是细胞壁会阻碍活性染料进入细菌，因此需要进行通透化处理。这些操作可能会影响细胞生理，引发细胞聚集或裂解，而且耗时较长。大肠杆菌B菌株因其脂多糖改变导致膜通透性增加，已被用于诱变分析。我们评估了使用这些菌株作为流式细胞术分析的可能替代方法，以避免通透化步骤。
在基础条件下以及通透化、膜电位受损、染料外排泵抑制和氧化应激后，用几种荧光染料对大肠杆菌K-12（菌株AB1157）和大肠杆菌B（菌株WP2 uvrA/pKM101，记为菌株IC188）的悬浮液进行染色，这些荧光染料包括异硫氰酸荧光素、碘化丙啶、尼罗红、双（1,3-二丁基巴比妥酸）三甲川氧杂羰花青、氢乙锭和二氢二氯荧光素二乙酸酯。通过落射荧光显微镜和流式细胞术比较两种菌株的荧光染色情况。
与大肠杆菌K-12菌株AB1157相比，大肠杆菌B菌株IC188在用活性荧光染料染色时表现出更高的效率，并且保持了相似的膜电位。此外，在用氧化剂挑战时，IC188比AB1157表现出更高的氧化应激敏感性。
大肠杆菌B菌株可能有助于基于流式细胞术和荧光显微镜的生化和毒理学研究。
