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Methodology（Principles）of Enumeration of Viable Bacteria
in Dialysis Fluid Production Process
◆Introduction
It has generally been recognized that the contamination of biologically active agents
in dialysis fluids deteriorates the biocompatibility of hemodialysis therapy.
Bacterial pyrogens,capable of penetrating dialyzer,
are responsible for a systemic inflammatory reaction in hemodialysis patients.
An accurate microbiological examination in hemodialysis centers is important for hemodialysis patients can suffer from pyrogenic reactions
due to bacterial contamination in dialysis fluids.
The one of the source of bacteria is the biofilm.
The biofilm is the flock of bacteria.
The growth and lysis of bacteria at the sites of biofilm formation
leads to the release of a variety of substances, including not only endotoxins,
but also muramyldipeptides and so on.
Therefore, the microbiological contamination in dialysis fluids production
process should be rapidly and precisely determined.
However, recently there are some reports concerning about the existence
of the viable but non-culturable (VBNC) bacteria in the nature.
The microbial cell of VBNC states isn't able to grow up with some stimulation but not dead.
There are some possibilities to grow up again under the suitable conditions.
On the other hand, there are non-culture methods for the bacterial detection.
Non-culture method, the bacteria could be counted directly with some detection tools.
And one of them a fluorescent microscope is used well
for the bacterial count in environmental samples.
D irect E pi f luorescent T echnique (DEFT) is the detection method,
based on various indicators of cells.
The fluorescent dyes are used well as an indicator and they are commercially available.
And the bacterial number in the dialysis fluids could be counted within a few hours
by using the non-culture method.
◆Direct Epifluorescent and microcolony methods（Technical cases）
Direct Epifluorescent method
On of the technical cases of non-culture method is introduced below.
The DNA-binding fluorochrome, 4', 6- d i a midino-2- p henyl i ndole (DAPI)
and 6-carboxyfluorescein diacetate (6CFDA) are used.
DAPI can stain the total bacteria (both living and dead cells)
and the 6CFDA (the intracellular esterase indicator)
could detect the physiologicallyactive cells.
The total and dead cells could be detected by using these reagents.
Microcolony method
There are many bacteria in aquatic environments,
which can not form large colonies and the growth stops
in a small colony state (about 10-60 μm diameter).
The number of culturable cells can be rapidly and easily determined
by counting these microcolonies with microscope.
Fig.1 shows the examples of the cultures and the non-culture methods.
The black cells show VBNC state bacteria and red cells show culturable bacteria.
VBNC state bacteria can't make the colony under the conventional conditions.
Also there are the bacteria which can from only the microcolony.
It is difficult to count them directly with our eyes.
On the other hand, in the case of non-culture method,
the fluorescent staining m ethod is commonly used for the detection of bacteria.
There are many kinds of fluorescent reagents as each viable indicator,
for example, the membrane robustness, intracellular enzymatic activity, respiratory activity.
By using these fluorescent reagents, target bacterial activities could be detected.
Fig.2 shows conventional plate-counting technique. Fig.3 shows DEFT.
The microbial cell is observed rod-shaped under the fluorescence microscopic observation.
From 20 to 30 microscopic fields are observed per sample under the excitation,
and bacterial cells with fluorescent are enumerated. |
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| 【Fig.1】Culture and Non-culture Methods |
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| 【Fig.2】Culture Method |
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【Fig.3】Non-culture Method - Direct Epifluorescent
【Fig.3】Filtration Technique (Escherichia coli ) |
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In order to popularize there techniques, automatization is important.
The development of these devices is also advanced.
Fig.4 shows rapid and automated bacterial detection system. |
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| 【Fig.4】Rapid and Automated Bacterial Detection Systems |
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By using these techniques, microbial cells can be detected and enumerated rapidly
and their physiological status can be estimated at the same time.
There techniques should be useful in microbiological quality
assurance of dialysis fluids production process.
Correspondence to : Tomotaka Naramura, Ide Clinic
7-14-2, Zenkai-Minamimachi, Nishi-ku, Kobe,Hyogo, 651-2109, Japan
Tel. +81-78-976-0528 Fax. +81-78-976-0538 E-mail : naramura@hi-net.zaq.ne.jp |
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