Rapid monitoring of bacteria in dialysis fluids by fluorescent vital staining and microcolony methods

Abstract

Bacterial contamination of dialysis fluids can cause inflammation and pyrogenic reactions in patients. Bacteria can grow more rapidly in dialysis fluids than in the high quality water used for their preparation, because dialysis fluids contain nutrients and electrolytes. Rapid determination of the microbiological quality of dialysis fluids is important, as it may aid in the reduction of instances of endotoxaemia. Microbiological contamination in dialysis systems should be detected as quickly as possible in order to provide dialysis fluid to patients with safety. Microbial contamination in dialysis fluids is usually assessed by culture-dependent methods such as plate counting. Arvanitidou et al. [1] examined dialysis fluids collected from 85 haemodialysis centres in Greece and found a total of 3500 5000 colony-forming units (CFUs)/ml of heterotrophic bacteria in 40% of measured samples [1]. Oie et al. [2] reported that dialysis fluids are more frequently contaminated than the water, acid or bicarbonate concentrates used for dialysis fluid preparation, and that the bacterial count in 42.5% of 40 dialysis fluid samples was >2000CFU/ml. These data indicate that the microbial contamination levels in a considerable proportion of dialysis fluid samples are relatively high. However, culture-dependent methods often underestimate bacterial numbers, because many bacterial species such as mycobacteria cannot be cultivated by conventional means [3], thus potentially placing the patients at a higher risk of inflammation and pyrogenic reactions. In addition, plate counting can require up to several days to detect bacteria. Therefore, microbiological qualities of dialysis fluids are often evaluated by measuring the endotoxin level. The endotoxin assays are simple and require a few hours to obtain results. However, endotoxin levels are often not correlated with numbers of CFU, and high numbers of colonyforming cells are sometimes detected in dialysis fluids with significantly low endotoxin level [4]. Simple, rapid and accurate methods of enumeration without longtime (e.g. 7 days) culture are essential, in order to maintain microbiological quality of dialysis fluids. Fluorescent staining is widely applied to detect bacteria rapidly [5]. Fluorescent dyes stain the nucleic acid and/or protein within the bacterial cells, and these cells can be counted by fluorescent microscopy, laser scanning cytometry and flow cytometry. Fluorescent vital staining such as 40,6-diamidino-2-phenylindole (DAPI) – carboxyfluorescein diacetate (CFDA) double staining can simultaneously determine the total bacterial count and the number of physiologically active bacteria (bacteria with enzymatic activity), not only in environmental samples (river water, pond water, ground water) but also in medical and pharmaceutical samples (pharmaceutical water [6] and herbal medicines [7]). By this technique, numbers of total bacteria (both active and inactive cells) are determined by DAPI staining, and physiologically active cells are selectively enumerated with CFDA staining, without the need for culture. DAPI is used for total direct counting of microbial cells in aquatic environments, because of its high stainability [5]. CFDA is a popular, ultra-sensitive, fluorescent substrate for estimating esterase activity in live micro-organisms (bacteria, yeast and fungi) [5]. Activity of cytoplasmic esterase of micro-organisms is an indicator of their physiological activity. Not only cells with growth potential but also dormant or injured cells can be stained with CFDA. Many bacteria in water with low nutrient concentrations cannot form large colonies which are Correspondence and offprint requests to: Masao Nasu, Environmental Science and Microbiology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan. Email: nasu@phs.osaka-u.ac.jp Nephrol Dial Transplant (2007) 22: 612–616