Contract n°EVK1-CT-2002-00108
Deliverable 02 version 2
Handbook for analytical methods and
operational criteria for biofilm reactors
Updated November, 2005
Non confidential
http://www.safer-eu.com
Contact persons : sylvain.fass@nancie.asso.fr
Jean-Claude.Block@pharma.uhp-nancy.fr
EVK1-CT-2002-00108 Surveillance and control of microbiological stability in drinking water distribution networks
Handbook for analytical methods and operational criteria for biofilm reactors
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SAFER
EVK1-CT-2002-00108
Revised
Prepared by:
Ilkka Miettinen
National Public Health Institute Gabriela Schaule
Department of Environmental IWW Rhenish-Westfalian Institute
Health for Water
Kuopio, Finland Department of Applied Microbiology
Mulheim, Germany
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Handbook for analytical methods and operational criteria for biofilm reactors
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Content
1 INTRODUCTION 5
2 STRUCTURE 5
3 MICROBIOLOGICAL METHODS 6
3.1 Enumeration of culturable microorganisms (water/biofilm) 6
3.4 Dehydrogenase activity with redox stain 5-cyano-2,3-ditolyl tetrazolium chloride (CTC)
(water/biofilm) 8
4 CHEMICAL METHODS 9
4.1 Measurement of adenosine triphosphate (ATP) 9
4.8 Assessment of nucleic acid damages by chlorination using fluorochrome staining (SYBR-
II or PI) and flow cytometry 21
4.10 Protocol use for biofilm extraction and dispatch samples to: 26
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1 Introduction
The objective of workpakage 2 is to provide monitoring systems for assessing biofilm
development in situ, on-line and non-destructively. Research will be organised on two
levels: one will aim to intercalibrate reactors for biofilm build-up, the second will aim
to develop biofilm monitoring devices. The main challenge is to establish devices
having both high sensitivity, rapid response potential, and an early warning capacity.
The second deliverable of workpackage 2 is to make a handbook for basic
methodologies. This handbook will describe the common protocols of all basic
analytical methods, which are used by partners participating to SAFER programme.
This information enables the exchange of information and comparison of different
methods.
The handbook contains information about methods used for characterisation of the
water , biofilm sampling and biofilm characterisation.
2 Structure
Within three chapters the different methods are listed starting with the microbiological
methods (1), followed by the chemical (2) and the physical methods (3) which are
used within the working groups of SAFER.
If for any of the methods an EN ISO Standard is available, this method will be the
basis and will be eventually modified by the partners. The modifications are listed.
The general structure of the method description:
1. Scope /principles
2. References
3. Definitions
4. Materials
5. Procedure
6. Expression of results
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Handbook for analytical methods and operational criteria for biofilm reactors
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3 Microbiological methods
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References
Hobbie, J.E., R.J. Daley, Jaspers, S. (1977): Use of Nucleopore filters for counting bacteria by
fluorescence microscopy. Appl. Environ. Microbiol. 33: 12225-1228.
ASTM Standard Test Method: Designation: F 1095 – 8 (Reapproved 1994). Rapid
enumeration of bacteria in Electronic-Grade Purified Water Systems by Direct Count
Epifluorescence Microscopy.
Materials
4´, 6-Diamidino-2-phenylindole (DAPI), black Nucleopore filter (pore size 0.2 µm),
non-fluorescent immersion oil.
All reagents should be filtered through a cellulose nitrate filter with the pore size of
0.2 µm.
Procedure
Place one black polycarbonate membrane filter (0.2 µm pore size, laser beamed) on
top of the sampling port, assuring that the shiny side of the filter is facing upwards.
Filter an aliquot of the sample and stop the filtration process immediately when the
sample is filtered through. Supplement with 1 mL DAPI (10 µg/mL) and add 1 mL
Triton X-100 (0.1 %). The final concentration should be 5 µg/mL. The incubation
time should be 15 to 20 minutes. Then the supernatant is filtered through and the
filter with the stained bacteria placed in a petri dish or any other box to let it air dry. If
the filter will be stored more than 2 days, add formaldehyde (1 % v/v) to the DAPI
solution.
The air dried filter is prepared for the microscope by embedding it in immersion oil on
the surface of a clean microscope slide; like a sandwich the filter is between the
immersion oil and a clean glass coverslip.
The enumeration of bacteria and other microorganismns are performed with a
magnification of at least 1000 fold in a epifluorescence microscope. All blue stained
cells are counted in randomly chosen microscopic viewing fields delineated by the
eyepiece micrometer. There should be 10-50 cells per viewing field. In minimum 300
bacteria should be counted or so many viewing fields that the coefficient of variation
of < 30% is obtained.
Material
• Black polycarbonate membrane filter (pore size 0,2 µm) and a filtration set
• Epifluorescent microscope and nonfluorescent immersion oil
• 5-cyano-2,3-ditolyl tetrazolium chloride (CTC, Polysciences). Reactant solution of
CTC in tubes can be kept frozen at 4ºC or have to be freshly prepared.
• 4´, 6-diamidino-2-phenylindole (DAPI).
Procedure for water samples
Add CTC solution to the water sample to a final concentration of 4 mM CTC and
incubate the mixture for 4 hours in the dark between 22 and 28ºC.
• Addition of nutrients (R2A is useful for drinking water samples).
• If there are many bacteria present the sample should be agitated to avoid anoxic
conditions.
Filter the sample after the incubation time through a black polycarbonate membrane
filter (pore size 0.2 µm) and wash slightly with water. Either the filter is now placed in
a box to be air dried or supllememnted with 1 mL of DAPI (5 µg/mL) to stain all
bacteria. DAPI will be filter through after 25 minutes. Then the filter is air dried.
The dried filter will be placed with nonfluorescent immersion oil on glass microscope
slides. The examination is performed at 1000 magnification using an epifluorescent
microscope. Count the red fluorescent cells as dehydrogenase active cells and the
blue ones (DAPI) as total cell number.
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4 Chemical methods
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Handbook for analytical methods and operational criteria for biofilm reactors
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Handbook for analytical methods and operational criteria for biofilm reactors
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Materials
• Incubation vessels - 100 ml volume borosilicate glass vials (larger volumes are
advisable) with caps.
• Hot water bath (65-70 °C)
• Continuously adjustable pipettes capable of delivering between 10 and 100 µL,
between 200 and 1000 µL and between 1000 and 5000 µL.
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thiosulfate solution added to each vial. Add 100µL of mineral salts solution to the
each vial. Cap vials and pasteurize in 65-70 °C water bath for 30 min.
Inoculation and incubation
Cool, inoculate with stock inoculum of P. fluorescens P-17 and Aquaspirillum sp.
NOX (final concentration of bacteria 500-1000/ml) by removing cap and using a
carbon-free pipet. Plastic, sterile tips for continuously adjustable pipets are suitable.
Inoculated samples are incubated at 15 °C in the dark for 9 days. If higher
temperature is used, maximum growth is reached earlier, which shortens the
incubation time (see below).
Enumeration of test bacteria
Bacterial concentrations are analysed on incubation days 7, 8 and 9. If higher
temperature is used, maximum is reached earlier (should be tested beforehand).
Shake vigorously the vials and make dilution series. Mechanical shaker (Vortex) may
be used to shake the dilutions. Plate at least 3 dilutions (10-2, 10-3, 10-4 and 10-5)
(dilutions depends on assumed AOC concentrations) on R2A agar. Incubate plates at
room temperature for 3 days and score the number of colonies of each strain.
P. fluorescens P-17 colonies appear on the plates first; they are 2 to 4 mm in
diameter with diffuse yellow pigmentation. Aquaspirillum sp. NOX colonies are small
(1 to 2 mm diameter) white dots. It may be necessary to count P. fluorescens and
Aquaspirillum sp. colonies at different dilutions. Sample vials on three separate days.
Count all colonies on selected plates containing 25 to 250 colonies of each bacterium
and compute colony counts (Swanson et al. 1992).
Determination of the yield of P. fluorescens P-17 and Aquaspirillum sp. NOX. The
growth yield of the test bacteria is determined using sodium acetate as a substrate
individually for P. fluorescens P-17 and Aquaspirillum sp. NOX. It is acceptable to
use the previously derived empirical yield values of 6.9 x 106 CFU P. fluorescens P-
17/µg acetate-C and 2.1 x 107 CFU Aquaspirillum sp. NOX/µg acetate-C at 15 °C.
Expression of results
Average the viable count results for the average density over 3-day period (or take a
maximum value) and calculate concentration of AOC as the product of the of the
viable counts and the inverse of the yield:
Result = µg AOC/L = [(P. fluorescens CFU/mL)(1/yield) + (Aquaspirillum sp. NOX
CFU/mL)(1/yield)] (1000mL/L)
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Procedure
Preparation of stock inoculum
Prepare turbid suspension of Ps. fluorescens P-17 by transferring colonies from R2A
agar into 2 to 3 mL filtered (0.2 µm), autoclaved sample. Use colonies not older than
one week. The autoclaved media water can be any water that supports growth of Ps.
fluorescens P-17. Before inoculation water is added of 150 µL/lmineral salts solution,
1000 µg acetate-C/L and pasteurised (30 min, 65°C).
Incubate at room temperature (≤ 25 °C) until the viable cell count reaches the
stationary phase. The stationary phase is reached when the viable cell count, as
measured by spread plates, reaches maximum value. Store stock cultures for not
more than 12 months at 5 °C.
dilution series. Mechanical shaker (Vortex) may be used to shake the dilutions. Plate
at least 3 dilutions (dilutions depend on assumed MAP concentrations) on R2A agar.
Incubate plates at room temperature for 3 days and score the number of colonies.
Count all colonies on selected plates containing 25 to 250 colonies of each bacterium
and compute colony counts.(Swanson et al., 1992).
Expression of results
The maximum microbial growth number (CFU/ml) is converted to phosphorus
concentration by using the yield factor. In determining of the yield factor, maximum
growth (cfu) of Ps. fluorescens is related to different concentrations of Na2HPO4. The
yield factor is derived from the slope of the line when cell growth is plotted against
PO4-P concentration (Lehtola et al., 1999). Also, previously derived empirical yield
value of 3.73 x 108 CFU Ps. fluorescens P-17/µg PO4-P can be used (Lehtola et al.,
1999)
The maximum plate counts are transformed with a conversion factor into the amount
of phosphorus:
µg MAP/L = (CFU/mL) x (1000 mL/L)
(Measured yield factor or 3.73 x 108 CFU/µg PO4-P*/L)
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Aluminium paper
Tube racks (Fisher, A 73 764 481)
Protective gloves
Vortexer
Procedure
The protocol is organized in 4 steps:
* Sampling water in aseptic conditions and chlorination neutralization.
* Bacterial staining with SYBR Green II or with propidium iodide (PI).
* Samples treatment analysis with flow cytometer.
* Treatment of data recorded by flow cytometry thanks to a software which allows to
translate files in flow cytometry language into files in ASCII.
Sampling
Sampling of water must be carefully done (i.e. aseptically) in order to prevent
contamination with dead or alive cells. Residual oxidant must be neutralized with
sodium thiosulfate.
Bacterial staining with SYBR-II or with PI
SYBR-II is a fluorochrome which stains efficiently nucleic acids. Its chemical formula
is covered by a patent and its mode of staining on nucleic acids is unknown. This
staining is done with a quantum yield higher than the other fluorochromes (Lebaron,
1998). SYBR-II is a membrane-permeant (Herrera et al., 2002) and has a low
intrinsic fluorescence, there is no need to destain to remove free dye (Haugland,
2002). This fluorochrome emits a green fluorescence (513 nm) when it is excited by a
flow cytometer argon laser at 488 nm, so the fluorescence is detected by the FL1
channel of flow cytometer FACSCalibur.
PI is a membrane-impermeant fluorochrome: it crosses only structurally damaged
membrane, so it is usually used as an indicator of membrane permeability. The
chemical structure of PI is a phenanthridine structure which allows binding to DNA by
intercalating between the bases with little or no sequence preference and with a
stoichiometry of one dye per 4-5 base pairs of DNA (Haugland, 2002). PI also binds
to RNA. PI emits a red fluorescence (617 nm) when it is excited by a flow cytometer
argon laser at 488 nm, so the fluorescence of PI is detected by the FL3 channel of
flow cytometer FACSCalibur.
One milliliter of sample in Falcon sterile tube is mixed with 0.5 µL of the SYBR-II
solution or with PI solution (final concentration 10 µg/mL). Samples are incubated
during 30 minutes in the dark, at room temperature (22±1°C). After staining step,
sample is immediately analysed by flow cytometer.
Counting of the total cell number and determination of the relative
fluorescence by flow cytometry
The counting of the fluorescent total cells is determined by combining staining with
SYBR Green II (or with PI) and flow cytometer (FACSCalibur, Becton Dickinson, San
Jose, California). Bacterial samples are analyzed by flow cytometer (FACSCalibur,
Becton Dickinson) with a theoretical flow rate of 40 µL/min and the signals are
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treated with a BDCellQuest software (Becton Dickinson). The real flow rate is
checked by the difference in weight of the analysed tube before and after analysis. In
order to not saturate detection systems, dilutions are necessary in order to have a
counting under 2000 events/s.
All analysis is done in two steps. First, analyse an unstained sample in order to
determine the background and the self-fluorescence of the sample. Second, repeat
the analysis with stained sample by fluorochrome.
Analysis of sample without fluorochrome (SYBR-II or PI)
One millilitre of the sample is introduced in a sterile tube adapted for flow cytometer
(Becton Dickinson). This sample is analyzed by flow cytometer and the different
settings of sensitivity and amplification of FSC and SSC parameters are adjusted in
order to placed background and self-fluorescence of the sample in the bottom left
square with as coordinates (x,y) (2; 2) of the cytogram FL1 = f (SSC) for SYBR-II
(Figure 1) (or FL3 = f (SSC) for PI) thanks to settings.
Background
SSC SSC
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The difference between before and after analysis weigh allows us to determine the
analysed volume per minute. Thanks to a determination of a real flow rate and the
number of fluorescent cells, the total number of bacteria per millilitre can be
determined.
Single cell analysis
Flow cytometers generally store data in specialized flow cytometry standard (FCS)
format, in which the intensities of each scatter or fluorescence parameter are
encoded in a specialized, compact binary format that cannot be displayed in a word
processor, or imported easily into other software.
Conversion to plain text ASCII puts the intensities values in plain text that can be
viewed and edited in a word processor or used in a spreadsheet or generic data
plotting software. "ASCII" designates a standard method of coding plain text or
numbers on computers. Most textual information on computers is stored in ASCII.
"Plaint text" files, conventionally named ending with .txt, contain only the text in
ASCII.
The conversion of FCS listmode data files to plain text ASCII is done by Median
Fluorescence Intensity (MFI) software. MFI is a software for analysis of flow
cytometry data, it is a DOS program, so it works only on PC and not on Macintosh.
MFI can be downloaded freely by Internet at this adress :
http ://www.umass.edu/microbio/mfi. The procedures of installing and operating MFI
program can be found at this adress :
http://www.umass.edu/microbio/mfi/install.htm#running.
Table 1 represents a concrete example of a MFI-generated ASCII list mode data
from FCS file.
Table1 : Example of ASCII list mode data from FCS file
Each line represents a single event. The numbers in the columns labelled FSC, SSC
and FL1 represent the scatter and fluorescence intensities for each event. If imported
into generic scientific plotting or spreadsheet software, the above plain text file can
be used as input to generic plotting software, mathematics or statistics software, or
spreedsheets.
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MFI software allows to improve the signal treatment of data recorded by flow
cytometry (fluorescence intensity mean of population of interest) in order to get single
cell analysis.
References
Haugland, R.P. (2002). Handbook of fluorescent probes and research products, 9th edition.
Eugene : Molecular Probes Inc., 679 pages.
Herrera G., A. Martinez, M. Blanco and J.E. O'Connor (2002). Assessment of Escherichia coli
B with enhanced permeability to fluorochromes for flow cytometric assays of bacterial cell
function. Cytometry 49 : 62-69.
Lebaron P., N. Parthuisot and P. Catala (1998). Comparison of blue nucleic acid dyes for flow
cytometric enumeration of bacteria in aquatic systems. Appl. Environ. Microbiol. 64 (5) : 1725-
1730.
Phe M.H., M. Dossot and J.C. Block (2004). Chlorination effect on the fluorescence of nucleic
acid staining dyes. Water Res. 38 : 3729-3737.
Phe M.H., M. Dossot, H. Guilloteau and J.C. Block. Use of nucleic acid staining dyes to
assess chlorinated drinking water bacteria injuries by flow cytometry. Water Res. (submitted).
Saby S., I. Sibille, L. Mathieu, J.L. Paquin and J.C. Block (1997). Influence of water
chlorination on the counting of bacteria with DAPI (4',6- diamidino-2-phenylindole). Appl.
Environ. Microbiol. 63 : 1564-1569.
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4.10 Protocol use for biofilm extraction and dispatch samples to:
Extraction of biofilm
Fill a glass flask exempt from organic matter with ultra-pure water (about 7 mL of
water per cm2 of coupon) acidified by HNO3 (pH < 2).
Place the coupon on the surface of the water (see figure below)
Ultrasonic probe
15 W –10 min
Glass vial
(20 mL)
Material (coupon)
(biofilm support)
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The main analytical methods and the test equipment used in the laboratory of
Microbiology and Chemistry are described in specific Standard Operating Procedures
(SOP). The competence of the personnel is provided by internal audits and
continuous appropriate education and training.
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grant from DGXII of the Commission of the EU) Part I has been adopted in MSCL
and it covers procedures that as far as possible quarantee:
• adherence of CABRI to international European or national regulations as
• well as to ethical and safety standards in the field of biotechnology;
• authenticity of biological materials;
• purity of cultures or absence of contaminants;
• quality-controlled processing of cultures;
• accuracy of data collected and supplied;
• punctuality and adherence to delivery standards.
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6.1 Propella (The common biofilm monitoring device for all partners)
Possible uses:
• Measurement of surface biological colonisation
• Corrosion
• Entartrement and deposits
• Salting out contaminants by materials
• Biological and chemical stability of water
• Surface and water disinfection testing
Fields of application
• Research studies
• Testing of materials
• In situ biofilm, corrosion measurements on water plants and
distribution networks
Introduction
Principles of PROPELLA®
The Propella® reactor provides an original and efficient solution to undertake tests
on a laboratory scale, and in particular:
• to control independently the hydraulic residence time and the speed of
water circulation
• to control hydraulic water flow which is indispensable to reproduce
transfers between solid and liquid phases
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The reactor can test real canalisation sections or only coupons of materials, with or
without continuous flow. The flow rate near the pipe is controlled by the rotation
speed of the propeller.
According to needs, sampling devices can be put on the studied pipe to measure
surface properties. These devices can be sampled without emptying or stopping the
reactor.
Except for the studied canalisation section, all materials in contact with water are of
inox 316l and Teflon to insure the chemical inertia of the system. These materials
can be adapted however to other needs.
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Characteristics
motor
coupons
biofilm
sampling
section of devices
material under
study vane
double
internal
cylinder
water for
thermoregulation
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Setup examples
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CONTAINING THE
SONICATION TO AVOID
INCREASE OF
TEMPERATURE
Repeat the sonication as above by placing the coupon in a new sterile flask
containing 25 ml of bacterial cell-free distilled water. If the count is less than 1% of
bacteria compared with the first sonication, you can estimate one sonication is
sufficient. Otherwise do two/three successive sonications.
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Scope
The RotoTorqueTM (Rotating Annular Reactor) (Characklis, 1990) is a useful device
for biofilm growth under defined conditions. It was modified by Griebe and Flemming
(1996) and later again by Schulte and Wingender (2000). The Rotating Annular
Reactor can be applied in research studies, in the testing of materials or cleaning
procedures and in in situ measurements in water plants and distribution networks.
References
Griebe, T., Flemming, H.-C. 2000. Rotating annular reactors for controlled growth of
biofilms. In: Flemming, H.-C., Szewzyk, U., Griebe, T. (Eds.), Biofilms, Lancaster,
Pennsylvania, Techonomic Publishing Company, pp. 23-40.
Schulte, S. 2003. Efficacy of hydrogen peroxide against biofilm bacteria. PhD theses
of the University of Duisburg-Essen.
Materials
The following list summarises experimental variables that are important in the
selection, design and construction of all biofilm devices when different research
questions will be addressed.
Physical parameters:
• Flow velocity + Shear stress
• Temperature
• Surface properties, composition and characteristics of the internal
materials
• Hydraulic residence time
Chemical parameters:
• Substrate composition and concentration
• Bioproducts in the biofilm matrix and bulk liquid phase
• Redox potential
• Inorganic ions
• Organic and inorganic particles
• Biological parameters:
• Microorganism type (algae, protozoa, bacteria, viruses, etc.)
• Defined or undefined culture
• Mixed or pure culture
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Engine
Influent
extractable
Screw for the extraction of
coupons test-surfaces
of coupons
(Coupons)
rotating
inner cylinder
with
recirculation
tubes
outer cylinder
with uptake
for the
outlet
coupons
Figure 4: Scheme of the Rotating Annular Reactor after Griebe and Flemming
(1996). In the figure there are shown two focus levels. The water containing
inner space is marked light blue
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Re = 11,846 x revolutions/min010002000300040005000600070000200400600revolutions/min
Procedure
Setup examples
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Diagram of the second stage model biofilm system with multiple assemblages of
coupons suspended from rigid titanium wire inserted through silicone rubber bungs in
the top ports. The weir system is used to maintain the volume at the required level.
Temperature, oxygen and pH probes are not shown.
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C
A
B
A
Figure 10. Photos of (a) biofilm monitoring system with (b) reservoir and (c)
inlet and sewerage.
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detector detector
Flow irection
Light Light
source source
ee e
Figure 11: Schematic view of a DTM device
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Biofilm
Substratum
EPS
Optical Fiber
Bacteria
Illuminated
biofilm area
IIluminating light
Backscattered
light
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Figure 13 shows the basic configuration of the sensor head. The distal end of a
single fibre, cut and polished perpendicularly to its optical axis, is used as test
surface.
WATER
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Water Sensors
Figure 15: Mechatronic Surface Sensor
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Scope
The development of the biofilms is monitored on a germanium crystal, which is
compatible with vibrational spectroscopic measurements, in a continuously drinking
water fed flow chamber included in an open circuit (Figure 16). To improve the
sensitivity and the delay of response, the crystal is firstly covered by a Luria Bertani
(5g/L) conditioning film, and then colonised by two hours exposure to Pseudomonas
fluorescens (Pf) suspension, as bacterium model frequently isolated in drinking
waters. Absorption bands for proteins (Amide II band) in the vibrational spectra are
chosen as probes, because these macromolecules are major constituents of bacteria
and biofilms.
ATR/FT-IR analysis
ATR/FT-IR spectra are measured between 4000 and 800 cm-1 on a Bruker Vector 22
spectrometer equipped with a KBr beam splitter and a DTGS (deuterated triglycine
sulphate) thermal detector. The resolution of the single beam spectra is 4 cm-1. The
number of bi-directional double-sided interferogram scans is 100, which corresponds
to a 1 min accumulation. The interferograms are apodised with the Blackman-Harris
3-Term function. No smoothing but a baseline correction is subsequently applied.
The ATR flow cell is a SPECAC cell (Eurolabo, ref. 11160) designed to enclose a
horizontal trapezoid crystal. The incidence angle of the ATR crystal is 45°, which
allows six internal reflections on the upper face in contact with the sample (figure 17).
The compartment of the spectrometer containing the flow cell is continuously purged
with dry and decarbonated air provided by a Balstom compressor for removing water
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Handbook for analytical methods and operational criteria for biofilm reactors
49
vapour and carbon dioxide. FT-IR measurements are made at room air-conditioned
temperature (21°C +/- 2 °C). Irradiance throughout the empty cell is about 11 % of
the full signal (without the ATR accessory). ATR spectra are shown with an
absorbance scale corresponding to log(Rreference/Rsample), where R is the internal
reflectance of the device. The ratio of the single beam ATR spectrum of the
conditioned film (A) + bulk species in the studied water (B) to the spectrum of (A) +
(B) + bacteria gave the absorbance scale spectrum of the bacteria attached on the
Ge crystal. When necessary, the contribution of water vapour due to variation in
relative humidity in the room was eliminated. The corresponding spectrum was
obtained separately by calculating the difference between spectra of "wet" and dry
air. Despite the absorbance scale used the ATR spectra is not strictly proportional to
the absorption coefficients as no further correction is applied.
IR sourcedetectorentranceexitGe crystalBiofilm
EVK1-CT-2002-00108 Surveillance and control of microbiological stability in drinking water distribution networks
Handbook for analytical methods and operational criteria for biofilm reactors
50
was homogenised with vortex, and staid at rest 3 hours before removing the glycerol
with a sterile Pasteur pipette. They kept frozen at -80°C.
Standardized cultures were obtained by growing the organisms at 28°C, under
stirring (350rpm) in Luria Bertani broth (LB Broth Miller, Difco, 244620) for two
consecutive periods (the first 24h and the second 10 hours). The cells were collected
at the end of their exponential phase of growth. The suspension was centrifuged and
washed two times with sterile saline solution (NaCl 90/00 ). Then, the pellet was
resuspended in NaCl 90/00 and a LB solution at 5g/L was inoculated to obtain a
bacterial suspension whose absorbance was about 0.31 at 620nm. After 1h, under
stirring (350rpm), at room temperature (21°C), this suspension was flowing into the
open circuit.
EVK1-CT-2002-00108 Surveillance and control of microbiological stability in drinking water distribution networks
Handbook for analytical methods and operational criteria for biofilm reactors