Nakamura R M, Kinomoto M
Department of Cellular Immunology, National Institute of Health, Tokyo, Japan.
Kekkaku. 1990 May;65(5):365-8.
Acid-fast staining is broadly used for the detection of mycobacteria in smears or pathological preparations, but it does not distinguish viable and non-viable bacteria. Enzymatic hydrolysis of fluorescein diacetate (FDA) was reported as a tool to detect viable mammalian cells or protozoa. We have applied this method to mycobacteria samples to detect viable or non-viable bacteria in the smear. Mycobacterium bovis BCG (Tokyo), 20 mg/ml suspension of standard vaccine, was used as the sample of viable bacteria. A part of the same suspension was heated at 100 degrees C for 30 min and used as the sample of non-viable bacteria. Three samples, viable, non-viable, and 1:1 mixture of the two, were stained with acid-fast staining and FDA-EB (ethidium bromide) mixture, respectively. For FDA/EB staining, stock solution of FDA (5 mg/ml acetone) was diluted 1:50 in PBS and 25 microliters of FDA and 25 microliters of EB (20 micrograms/ml PBS) were mixed with 50 microliters of bacterial suspension. Preparations were observed with either light or fluorescein microscope. Living bacteria were stained in yellow green in FDA/EB staining while non-viable bacteria were red. Mixed sample of live and dead bacilli showed differential staining with green or red in FDA/EB staining, but no difference was shown in acid-fast staining between viable and non-viable bacteria.
抗酸染色广泛用于涂片或病理制剂中分枝杆菌的检测，但它无法区分活菌和死菌。据报道，荧光素二乙酸酯（FDA）的酶促水解可作为检测活的哺乳动物细胞或原生动物的一种工具。我们已将此方法应用于分枝杆菌样本，以检测涂片中的活菌或死菌。牛分枝杆菌卡介苗（东京株），标准疫苗的20mg/ml悬浮液，用作活菌样本。将同一悬浮液的一部分在100℃加热30分钟，用作死菌样本。分别用抗酸染色和FDA-溴化乙锭（EB）混合物对三个样本（活菌、死菌以及两者1:1的混合物）进行染色。对于FDA/EB染色，将FDA储备液（5mg/ml丙酮溶液）在PBS中按1:50稀释，取25微升FDA和25微升EB（20微克/ml PBS）与50微升细菌悬浮液混合。用光学显微镜或荧光显微镜观察制片。在FDA/EB染色中，活菌被染成黄绿色，而死菌被染成红色。活菌和死菌的混合样本在FDA/EB染色中呈现出绿色或红色的差异染色，但在抗酸染色中活菌和死菌之间没有差异。
