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NDT Advance Access originally published online on November 22, 2006
Nephrology Dialysis Transplantation 2007 22(2):612-616; doi:10.1093/ndt/gfl675
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

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

Nobuyasu Yamaguchi1, Takashi Baba1, Sachiko Nakagawa1,2, Akira Saito3 and Masao Nasu1

1Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, 2Toray Medical Co., Ltd., 1-2-1, Kinshi, Sumida-ku, Tokyo 130-0013 and 3Department of Medicine, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan

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{at}phs.osaka-u.ac.jp

Keywords: carboxyfluorescein diacetate; dialysis fluid; endotoxin; fluorescent staining; microbial monitoring; microcolony method



   Introduction
Top
 Introduction
Subjects and methods
 Results
 Discussion
 Acknowledgement
 References
 
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 >2000 CFU/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 colony-forming cells are sometimes detected in dialysis fluids with significantly low endotoxin level [4]. Simple, rapid and accurate methods of enumeration without long-time (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 4',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 detected macroscopically on agar medium, however, they can form microcolonies with 10–50 µm diameter under conventional conditions [6]. The number of culturable cells can be rapidly and easily determined by counting these microcolonies.

The present study applies fluorescent vital staining and microcolony methods to rapidly enumerate total, physiologically active and culturable bacteria in dialysis fluids, and results are compared against a standard culture-based method.



   Subjects and methods
Top
 Introduction
 Subjects and methods
Results
 Discussion
 Acknowledgement
 References
 
Dialysis fluid samples and determination of endotoxin concentration
Dialysis fluid samples were collected from 16 haemodialysis clinics in Japan. Dialysis fluid delivery systems of all haemodialysis centres were multiple patient (central) systems. Dialysis fluid samples (1500 ml) were obtained from the tube before an endotoxin cut filter (a hollow fibre filter to absorb low-molecular weight impurities) of the haemodialysis monitoring machine, by using sterilized equipment. Samples for bacteriological determination were stored at 4°C, and samples for endotoxin determination were stored with stabilized reagents [0.64% (w/v) triethanolamine, 0.16% (w/v) polyethylenglycol 6000 and 0.1 mol/l trisodium citrate] at 4°C. These were analysed within 24 h.

Endotoxin contents of dialysis fluids were determined by means of endotoxin-specific limulus reagent (Endospecy [8]; Seikagaku Corp., Tokyo, Japan). The detection limit of endotoxin was 1 EU/l.

Bacteriological determination
CFUs were determined by conventional plate-counting technique on R2A agar (Difco). Each dialysis fluid was spread on R2A agar and incubated at 25°C for 7 days. Bacterial colonies were counted macroscopically.

The numbers of total and physiologically active bacteria were determined by DAPI–CFDA double staining [6]. Bacterial cells in 500 ml of dialysis fluid samples were vacuum-filtered onto polycarbonate black filters (ADVANTEC, Tokyo, Japan; pore size 0.2 µm) and then incubated in staining buffer [0.1 mol/l phosphate buffer (pH8.5), 5% (w/v) NaCl, 0.5 mmol/l EDTA (ethylenediaminetetraacetic acid)] [9] containing DAPI (Sigma; final concentration, 1 µg/ml) and CFDA (Sigma; final concentration, 150 µg/ml) in a filtration funnel for 5 min at room temperature (~25°C). The staining buffer in the funnel was then removed by vacuum filtration. The filters were mounted on glass slides with immersion oil and fluorescing cells were counted at 1000 x magnification using an E-400 epifluorescence microscope (Nikon, Tokyo, Japan), equipped with a 100 W Hg lamp. A total of 20–30 microscopic fields were observed per sample under both ultraviolet and blue excitation, and bacterial cells in each microscopic field were enumerated. Only bright and clear fluorescent cells were counted in order not to obtain false positive results. Bacterial numbers were calculated as described by Kirchman et al. [10]. The filter-blocks of E-400 for viewing cells stained with DAPI and CFDA were UV-2A (Nikon) and B-2A (Nikon), respectively.

The numbers of microcolony-forming units (mCFU) were determined as described by Kawai et al. [6]. Bacterial cells in 500 ml of dialysis fluid samples were vacuum-filtered onto a polycarbonate filter (ADVANTEC; pore size 0.2 µm), which was immediately removed from the funnel, and placed on R2A agar. After 48 h incubation at 25°C, the filter was placed on filter paper (No.2; Whatman) soaked with 4% (w/v) formaldehyde, for 10 min at room temperature. The formaldehyde-treated filter was placed on filter paper soaked with sterilized water to wash the underside of the filter gently, then placed uppermost on the filter paper soaked with double- and single-stranded nucleic acid-staining dye, SYBR Gold (Invitrogen; final concentration: 1/1000 dilution of supplied product), containing polysorbate 80 [Nacalai Tesque, Kyoto, Japan; final concentration: 2% (w/v)] for 5 min (polysorbate 80 was added to enhance staining efficiency). The stained microcolonies were enumerated by epifluorescence microscopy as well as CFDA-stained cells.

In this study, 500 ml of each dialysis fluid was filtered for direct counting by fluorescent microscopy, and the lower detection limit of DAPI–CFDA double staining was 84 cells/ml [10]. In the microcolony method, the whole area of each filter was scanned by epifluorescent microscopy with low magnification objective lens (20x or 40x), and the lower detection limit was 4 cells/ml.



   Results
Top
 Introduction
 Subjects and methods
 Results
Discussion
 Acknowledgement
 References
 
Bacterial cells in dialysis fluid samples were effectively stained and easily observed by fluorescent microscopy (Figure 1). Both active and inactive cells emitted blue fluorescence under ultraviolet excitation (Figure 1A), and only enzymatically active bacteria emitted green fluorescence under blue excitation (Figure 1B) in each microscopic field, by double staining with DAPI and CFDA. No particles autofluoresced under epifluorescent microscopy when bacterial cells trapped onto a filter were observed without the double staining. Microcolonies in dialysis fluids were also clearly observed by SYBR Gold staining (Figure 1C).


Figure 1
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  		Fig. 1. Bacterial cells in dialysis fluid. (A) Total bacterial cells stained with 4',6-diamidino-2-phenylindole (DAPI) and observed under ultraviolet excitation. (B) Esterase-active cells stained with carboxyfluorescein diacetate (CFDA) and observed under blue excitation. (C) Microcolonies stained with SYBR Gold and observed under blue excitation.

 
Table 1 shows the numbers of total bacterial cells, esterase-active ones, mCFUs and CFUs determined for 16 dialysis fluid samples.


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  		Table 1. Bacterial number and endotoxin level in dialysis fluids

 
The total bacterial numbers determined by DAPI staining were 9.9 x 101 – 4.2 x 103 cells/ml, and the number of CFDA-stained bacteria with esterase activity was 47% of the total bacterial number in the highest sample (average: 15%). DAPI-staining enabled detection of low level of bacterial contamination in dialysis fluids which could not be determined by endotoxin measurement (sample 1). The numbers of esterase-active cells in 16 dialysis fluid samples were always higher than those of CFU.

Bacterial cells in 10 dialysis fluids formed microcolonies within 48 h. Certain bacterial cells in dialysis fluids formed relatively large microcolonies (overgrowth), and each microcolony was too close together; it was thus difficult to discriminate each microcolony.

The number of macroscopic colonies was <30/ml in most of the dialysis fluids. Compared with the fluorescent microscopic counts, numbers of culturable bacteria in dialysis fluid samples were underestimated.

The endotoxin levels of samples 6, 12 and 13 were higher than 15 EU/l. However, numbers of CFUs in these samples were similar to those of dialysis fluids with low endotoxin levels. Endotoxin levels of these dialysis fluid samples seemed to correlate with the number of DAPI-stained cells (Figure 2; R = 0.54).


Figure 2
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  		Fig. 2. Correlation between number of DAPI-stained cells and endotoxin level in dialysis fluids (n = 16).

 
Ultrapure dialysis fluid samples were also collected and bacterial numbers in these samples were below the detection limit (DAPI-stained cells: <8.4 x 101 cells/ml; CFDA-stained cells: <8.4 x 101 cells/ml; microcolonies: <4 mCFU/ml; CFU: <30 CFU/ml) as well as endotoxin level (<1 EU/l).



   Discussion
Top
 Introduction
 Subjects and methods
 Results
 Discussion
Acknowledgement
 References
 
Repeated haemodialysis with bacteria-contaminated dialysis fluids induces chronic inflammation through cytokine production, stimulated by endotoxin in haemodialysis patients [11]. It is considered to be one of the main causes of the progress of atherosclerosis, ß2-microglobulin amyloidosis, and malnutrition in long-term dialysis patients [12,13]. Accurate and rapid detection of bacteria and endotoxin concentration in dialysis fluids is therefore important to prevent the progression of those complications by improving dialysis fluid purity as rapidly as possible.

Tryptone glucose extract agar (TGEA) media or trypticase soy agar (TSA) media were often used to detect bacteria in dialysis fluids. Pass et al. [14] reported that many bacterial cells in dialysis fluids cannot be cultivated on nutrient-rich media such as TSA, and it may result in a significant underestimation of the bacterial population of water and dialysis fluid [14]. Ledebo and Nystrand [4] also reported that a nutrient-poor medium is suitable to detect bacteria in dialysis fluids. In this study, R2A medium, which is nutrient-poor and more suitable for detection of bacteria in aquatic environments, was used to detect a higher number of culturable bacteria in dialysis fluid samples than TGEA or TSA media. However, plate- counting was time-consuming and it underestimated the numbers of active cells. The number of culturable cells can be determined faster by microcolony method with R2A media (within 48 h), compared with the conventional plate-counting method with the same media (7 days). Moreover, viable bacteria were detected as microcolonies in samples 3 and 13, while no macroscopic colony was detected in these samples. It is well known that many bacteria in an aquatic environment form microcolonies, but not macroscopic colonies [6] despite the fact that the same media are used.

In this study, certain culturable cells formed relatively large microcolonies within 48 h. Incubation time for microcolony method may be reduced to 24 h [6] for rapidly growing cells in dialysis fluids. The presence of growing cells in dialysis fluids can be ascertained within 24 h by counting rather small microcolonies, and 48 h incubation allows accurate detection of microcolony-forming cells.

Staining with CFDA enumerates physiologically active bacterial cells in dialysis fluids, as it can enter the cells by passive diffusion and is cleaved by non-specific esterases releasing fluorescent carboxyfluorescein within viable cells. CFDA has been commonly used as a vital stain [5], and it has also been reported that numbers of CFDA-stained cells in fresh water were similar to numbers of bacteria with growth potential in those samples determined by quantitative direct viable count method [15].

Our results showed that an average of 15% of bacteria in dialysis fluids retains physiological activity. Conventional colony counting could not detect the most physiologically active bacteria in the samples, whereas fluorescent vital staining allowed enumeration of them. Moreover, endotoxin levels of the dialysis fluid samples seemed to correlate with total bacterial number determined by DAPI-staining. Endotoxin-specific limulus reagent used in this study reacts with lipopolysaccharide, and not ß-glucan. The source of lipopolysaccharide is the cell wall of gram-negative bacteria, and bacterial populations in fresh water environments are usually dominated by gram-negative bacteria [16]. Most of the bacterial cells detected in dialysis fluids by DAPI staining were assumed to be gram-negative, and we supposed that DAPI counts, which indicate the bacterial abundance in the samples, would correlate with endotoxin level, which is the indicator of abundance of gram-negative bacteria. Thus, double staining with DAPI and CFDA might be suitable for routine bacteriological quality control of dialysis fluids, as it is simple and requires <1 h, including staining and enumeration. The data of ultrapure samples showed that no false-positive results were obtained by DAPI–CFDA double-staining method and microcolony method.

Microscopic observation, even by a skilled person, is labour-intensive and time consuming. Automation of this process by digital image analysis systems [17] or specific equipments such as laser scanning cytometer might facilitate the microbial monitoring of dialysis fluids using fluorescent staining.

Further investigation should focus on whether microscopically detected bacteria in dialysis fluids are harmful or not. Kawai et al. [16] reported that dominant bacteria in purified water used in the pharmaceutical manufacturing process could not be detected by plate counting with R2A media, and they identified the dominant bacteria by denaturing gradient gel electrophoresis of polymerase chain reaction (PCR)-amplified ribosomal RNA gene fragments. They also found that the number of non-culturable mycobacteria was increased in a pharmaceutical manufacturing water supply system (a holding tank). Bacterial cells in dialysis fluids can be identified by the same approach, without the need for culture. The source of contaminants such as contact points of the connective devices [18], water for dialysis fluid preparation [19] or bacteria released from biofilms formed in the system (filters, tanks, tubes or taps) [20] should also be determined.



   Acknowledgement
Top
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgement
References
 
We would like to thank the staffs of Medical Corporation Showa-kai and Shinshikai Medical Corporation for sampling of dialysis fluid samples.

Conflict of interest statement. None declared.



   References
Top
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgement
 References
 

  1. Arvanitidou M, Spaia S, Katsinas C, et al. (1998) Microbiological quality of water and dialysate in all haemodialysis centres of Greece. Nephrol Dial Transplant 13:949–954.[Abstract/Free Full Text]
  2. Oie S, Kamiya A, Yoneda I, et al. (2003) Microbial contamination of dialysate and its prevention in haemodialysis units. J Hosp Infect 54:115–119.[CrossRef][ISI][Medline]
  3. Ferrara E, Lemire J, Grimm PC, et al. (2004) Mycobacterial peritonitis in pediatric peritoneal dialysis patients. Pediatr Nephrol 19:114–117.[CrossRef][ISI][Medline]
  4. Ledebo I and Nystrand R. (1999) Defining the microbiological quality of dialysis fluid. Artif Organs 23:37–43.[CrossRef][ISI][Medline]
  5. Davey HM and Kell DB. (1996) Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single-cell analysis. Microbiol Rev 60:641–696.[Abstract/Free Full Text]
  6. Kawai M, Yamaguchi N, Nasu M. (1999) Rapid enumeration of physiologically active bacteria in purified water used in the pharmaceutical manufacturing process. J Appl Microbiol 86:496–504.[CrossRef][Medline]
  7. Nakajima K, Nonaka K, Yamamoto K, et al. (2005) Rapid monitoring of microbial contamination on herbal medicines by fluorescent staining method. Lett Appl Microbiol 40:128–132.[CrossRef][ISI][Medline]
  8. Izuhara Y, Miyata T, Saito K, et al. (2004) Ultrapure dialysate decreases plasma pentosidine, a marker of ‘carbonyl stress’. Am J Kid Dis 43:1024–1029.[CrossRef][ISI][Medline]
  9. Yamaguchi N and Nasu M. (1997) Flow cytometric analysis of bacterial respiratory and enzymatic activity in the natural aquatic environment. J Appl Microbiol 83:43–52.
  10. Kirchman D, Sigda J, Kapuscinski R, Mitchell R. (1982) Statistical analysis of the direct count method for enumerating bacteria. Appl Environ Microbiol 44:376–382.[Abstract/Free Full Text]
  11. Owen WF and Lowrie EG. (1998) C-reactive protein as an outcome predictor for maintenance hemodialysis patients. Kidney Int 54:627–636.[CrossRef][ISI][Medline]
  12. Schwalbe S, Holzhaeffer M, Schaeffer J, et al. (1997) ß2-microglobulin associated amyloidosis: a vanishing complication of long-term hemodialysis? Kidney Int 52:1077–1083.[ISI][Medline]
  13. Stenvinkel P, Heimbuger O, Paultre F, et al. (1999) Strong associations between malnutrition, inflammation and atherosclerosis in chronic renal failure. Kidney Int 55:1899–1911.[CrossRef][ISI][Medline]
  14. Pass T, Wright R, Sharp B, Harding GB. (1996) Culture of dialysis fluids on nutrient-rich media for short periods at elevated temperatures underestimate microbial contamination. Blood Purif 14:136–145.[CrossRef][ISI][Medline]
  15. Yokomaku D, Yamaguchi N, Nasu M. (2000) Improved direct viable count procedure for quantitative estimation of bacterial viability in freshwater environments. Appl Environ Microbiol 66:5544–5548.[Abstract/Free Full Text]
  16. Kawai M, Matsudera E, Kanda H, et al. (2002) 16S ribosomal DNA-based analysis of bacterial diversity in purified water used in pharmaceutical manufacturing processes by PCR and denaturing gradient gel electrophoresis. Appl Environ Microbiol 68:699–704.[Abstract/Free Full Text]
  17. Ogawa M, Tani K, Yamaguchi N, Nasu M. (2003) Development of multicolour digital image analysis system to enumerate actively respiring bacteria in natural river water. J Appl Microbiol 95:120–128.[CrossRef][Medline]
  18. Miller TE and Findon G. (1997) Touch contamination of connection devices in peritoneal dialysis-a quantitative microbiologic analysis. Perit Dial Int 17:560–567.[Abstract/Free Full Text]
  19. Vorbeck-Meister I, Sommer R, Vorbeck F, Horl WH. (1999) Quality of water used for haemodialysis: bacteriological and chemical parameters. Nephrol Dial Transplant 14:666–675.[Abstract/Free Full Text]
  20. Hanlon GW, Denyer SP, Hodges NA, et al. (2004) Biofilm formation and changes in bacterial cell surface hydrophobicity during growth in a CAPD model system. J Pharm Pharmacol 56:847–854.[CrossRef][ISI][Medline]
Received for publication: 16. 4.06
Accepted in revised form: 19.10.06


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