Nielsen S L, Black F T, Storgaard M, Obel N
Department of Medicine and Infectious Diseases, Marselisborg Hospital, Aarhus University Hospital, Denmark.
APMIS. 1995 Jun;103(6):460-8.
A modified method for measurement of intracellular killing of Staphylococcus aureus in human neutrophil granulocytes is described. After phagocytosis of S. aureus the extracellular bacteria were sufficiently removed by repeated centrifugations and washings of the granulocytes. The optimal conditions for incubation of granulocytes for measurement of intracellular killing were found to be 37 degrees C in the presence of 5% CO2. Under these conditions, stable pH, the viability and the capacity of the granulocytes for superoxide anion generation were preserved. The number of intracellular viable bacteria was determined after lysis of the granulocytes, which should be done in H2O at pH 11 to ensure sufficient cell lysis. The kinetics and individual variation of the intracellular killing are described. The intra- or extracellular location of surviving bacteria was studied. After approximately 8 h incubation we observed intracellular growth of S. aureus followed by lysis of granulocytes and extracellular growth of bacteria. Consequently, the incubation period should not be extended beyond 5 to 8 h when the assay is used to study the effects of antibiotics on intracellular killing.
本文描述了一种改良的方法，用于测定人类中性粒细胞对金黄色葡萄球菌的细胞内杀伤作用。金黄色葡萄球菌被吞噬后，通过对粒细胞进行反复离心和洗涤，充分去除细胞外细菌。发现粒细胞孵育以测定细胞内杀伤作用的最佳条件是在5%二氧化碳存在下于37摄氏度。在这些条件下，粒细胞的pH值稳定、活力以及产生超氧阴离子的能力得以保留。在粒细胞裂解后测定细胞内活菌数量，应在pH值为11的水中进行裂解以确保细胞充分裂解。描述了细胞内杀伤的动力学和个体差异。研究了存活细菌的细胞内或细胞外定位。孵育约8小时后，我们观察到金黄色葡萄球菌在细胞内生长，随后粒细胞裂解，细菌在细胞外生长。因此，当该测定法用于研究抗生素对细胞内杀伤作用的影响时，孵育时间不应延长超过5至8小时。
