Handbook SAFER V02light

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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
2
SAFER
EVK1-CT-2002-00108
Surveillance and control of microbiological

stability in drinking water distribution networks
Revised
Handbook for analytical methods and

operational criteria for biofilm reactors (Version
2.0)
WP2. Tools for biofilm monitoring
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
DATE: 13h November, 2005
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Content
1 INTRODUCTION 5
2 STRUCTURE 5
3 MICROBIOLOGICAL METHODS 6
3.1 Enumeration of culturable microorganisms (water/biofilm) 6
3.2 Total cell number 6
3.3 Coliform bacteria and E. coli (water and biofilm) 7
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
4.2 Total organic carbon /dissolved organic carbon 11
4.3 Modification of non-purgeable organic carbon (NPOC) analyses (water) 12
4.4 Assimilable organic carbon (AOC) (water) 13
4.5 Total phosphorus 16
4.6 Microbially available phosphorus (MAP) (water) 17
4.7 Total nitrogen 20
4.8 Assessment of nucleic acid damages by chlorination using fluorochrome staining (SYBR-
II or PI) and flow cytometry 21
4.9 Amino acid 26
4.10 Protocol use for biofilm extraction and dispatch samples to: 26
5 QUALITY CONTROL AND QUALITY ASSURANCE 27

5.1 Quality control and quality assurance in CR4 27
6 BIOFILM MONITORING DEVICES 29

6.1 Propella (The common biofilm monitoring device for all partners) 29
6.2 Rotating Annular Reactor (modified RotoTorqueTM) [CR4] 34
6.3 Biofilm generator 39

4
6.4 Flow cell reactor 40
6.5 Pipeline biofilm collector 41
6.6 Differential turgidity measurement 43
6.7 Fibre optic devices (FOS, and FluS sensors) 44
6.8 Electrochemical, nanovibration, and capacitive monitors 47

6.8.1 Mechatronic Surface Sensor - MSS 47
6.8.2 Capacitive sensors 47
6.9 Biofilm formation monitoring using ATR-FTIR sensor 48
5
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
6
3 Microbiological methods
3.1 Enumeration of culturable microorganisms (water/biofilm)

A) European Standard: EN ISO 6222 (Water quality – Enumeration of culturable
micro-organisms – Colony count by inoculation in a nutrient agar culture medium).
See original instructions from the pdf-file “HPC ISO6222.pdf” from SAFER ftp-site.
B) Number of colony forming units on R2A nutrient agar
Scope
The bacterial colony forming units (CFU) are enumerated by spread plate method for
heterotrophic plate counts.
Reference
Reasoner DJ and Geldreich EE (1985). A new medium for the enumeration and subculture of
bacteria from potable water. Appl. Environ. Microb. 49: 1-7.
Procedure
Heterotrophic plate counts (HPC) are estimated by spread plating method using 0.1
mL or 1 mL sample. The medium is R2A-agar (Difco, USA) (Reasoner and Geldreich
1985). R2A-agar plates are incubated for 7 days at 22 ± 2°C before the colony
forming units (CFU) are counted by eye.
Membrane filter method will be used if the cell density of the sample is too low for
direct spread plate. The membrane filters have a diameter of 47 mm, a pore size of
0.2 µm. The sample up from 10 mL will be filtered and the membrane filter placed on
one R2A agar plate. Be careful: air bubbles under the filter should be avoided.
Expression of results
Results are expressed as the mean number of bacterial CFU per mL of water sample
3.2 Total cell number

Scope
This method describes a procedure for counting all bacteria in water and
homogenised and/or diluted biofilm samples using the dye 4´, 6-diamidino-2-
phenylindole (DAPI). There are other fluorochromes which can be used for the same
purpose e.g. Acidine Orange and Syto 9. Acridine Orange is the dye which is often
used by the semiconductor industry to estimate the total cell number in ultra pure
water. The procedure follows in this case the ASTM Standard Test Method
Designation F 1095-8. The advantage of Acridine Orange is that it will show bright
cells with the disadvantage that the stain is much more unspecific to noncellulare
material.
The total cell number of biofilms can be evaluated directly without removing the
biofilm by using the Confocal Laser Scanning Microscope. Staining will be performed
the same way like water samples (filter method). Counting is performed with
epiflourescence microscope.
7
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.
3.3 Coliform bacteria and E. coli (water and biofilm)

See the original instructions from the PDF-file “ E.coli colforms ISO9308.pdf” from the
ftp-site of SAFER.
Alternative methods
Two alternative membrane filtration media (chromogenic Harlequin, and mEndo LES)
can be used for E. coli and coliform counting.
See instructions of chromogenic agars from ftp-site of SAFER: “Harlequin coli
agar.pdf / Oxoid coli agar.pdf / Cromocult agar.pdf / Tergitol agar.pdf”.
Also Colilert Quantitray (IDEXX) method can be used if considered necessary for
E.coli/coliform counting. The colilert analyses follows the manufacturer´s instructions.
For Colilert, the sample size used is 100 ml. For further instructions see PDF-file:
“Colilert.pdf” from the ftp-site of SAFER.
8
3.4 Dehydrogenase activity with redox stain 5-cyano-2,3-ditolyl

tetrazolium chloride (CTC) (water/biofilm)
Scope
The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) can be applied for direct
epifluorescent microscopic counting of dehydrogenase active (metabolically active)
bacteria. CTC is noncolorless and nonfluorescent. It is water soluble and can pass
solubilised in water the cell wall and will be intracellular reduced via electron
transport system. Inside the cell CTC will be reduced via electron transfer. The
reduced form of tetrazolium salts are called formazan. "CTC-formazan" molecules
are not water soluble. They accumulate intracellularly and build up o type of crystall
which is fluorescent (maximum at 520 nm) after excitation with fluorescent light (465
nm). It is possible to counterstain bacterial cells with DAPI after the incubation
procedure with CTC.
Bacteria with a low level of dehydrogenase might not be able to form a large crystal.
To get a information about the number of potentially active cells, nutrients might be
added during the incubation time with CTC to activate the cells.
References
Rodriguez GG, Phipps D, Ishiguro K, Ridgway HF (1992), Use of a fluorescent redox probe
for direct visualisation of actively respiring bacteria, Appl. Environ. Microbiol. 58, 6, 1801-8
Schaule G., Flemming H-C, Ridgway HF (1993). Use of 5-Cyano-2,3-ditolyl tetrazolium
chloride for quantifying planctonic and sessile respiring bacteria in drinking water. Appl. Envir.
Microbiology 59: 3850-3857.
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.
9
4 Chemical methods
4.1 Measurement of adenosine triphosphate (ATP)

Scope
The survival of a cell necessitates a continual energy input to allow the
accomplishment of all the metabolic activities. One of the energising molecules
commonly determined is a nucleotide which is a marker of the bacterial biomass :
adenosine triphosphate (ATP). By hydrolysis of the 3 phosphate bonds, ATP can
release some energy, which is directly usable by the cell. The measurement of ATP
is carried out in 4 successive stages described below :
1. pre-concentration of the sample
2. extraction of the cellular ATP
3. enzymatic determination by bioluminescence
4. ATP quantification and expression of results.
The measurement of ATP is carried out with an apparatus and products from the
LUMAC company. For all the stages, followed protocols are those prescribed by this
company.
Procedure
Pre-concentration of samples to be analysed
The method used is a membrane filtration with a moderate pressure in a view to limit
the bacterial stress. The sample is first filtered on a cellulosic membrane and then
rinsed with an demineralised, sterile and apyrogenic water. Bacteria retained by the
membrane then suffer a reactivation phase according to the LUMAC protocol: input
on the membrane of 500 µL of peptoned bubble without ATP (LUMACULT, ref. 9233-
1) during a contact time equal to 15 minutes.
In our experiments, samples (between 8 and 20 mLin volume) were filtered on a
sterile membrane in cellulose acetate with a porosity of 0.45 µm. The pre-
concentration was applied both on the immersion waters in contact with tested
materials and on the sonication product of the biofilm present on materials.
ATP extraction
Among many extraction products used for the determination of the ATP, we choose a
detergent commercialised by LUMAC : the NRB for which the chemical
composition is always a manufacturing secret.
The membrane is first soaked in 500 µL of LUMACULT. Then 500 µL of extraction
product (NRB) is added and mixed by soft agitation during 30 seconds. The
determination of the ATP is then carried out with 200 µL of this mixture. A negative
check sample is included, replacing the sample by 8 to 10 mLof demineralised,
sterile and apyrogenic water.
Enzymatic measurement of ATP
10
The most common quantification method of the bacterial ATP is based on an

enzymatic reaction and on bioluminescence detection. The ATP determination
method is based on the use of the Luciferase (an enzyme extracted and purified from
the glow-worm (Photinus pyralis) and its substrate named Luciferine.
In presence of Mg++ ions, oxygen and ATP and with the luciferase as a catatyst, the
luciferine is oxidized. The reaction is endergonic: a consumption of ATP molecules
occurs with production of photons. Their emission is proportional to the quantity of
the consumed ATP: they are quantified by a bioluminometer (LUMAC, ref. M2500). In
our experiments, the ATP determination was implemented with a commercial kit from
LUMAC.
The photometer produces some Relative Light Unit (RLU) during the enzymatic
reaction. The conversion of these RLU into ATP concentration in the sample is
obtained by proportioned additions of standard ATP.
Proportioned additions consist of an initial measurement of ATP on the sample to be
analyse. Then a known quantity of ATP is introduced in the same measurement cell,
and a second measurement of ATP is carried out. The bioluminometer contains a
data colection program: all the reaction times are first stored and proportioned
additions are allowed.
Stages of bacterial ATP quantification:
1. concentrated sample on membrane + NRB + LUMACULT,
2. 100 µL of the enzymatic complex Luciferine – Luciferase (LUMIT PM) are
automatically added by an integrated pump system,
3. integration of photons produced for 10 seconds,
4. results of the quantity of ATP in the sample in RLU (RLU1),
5. addition of 20 µL of standard ATP (LUMAC) with a know concentration,
6. integration for 10 seconds,
7. results of the quantity of total ATP in RLU (sample ATP and standard ATP) in the
analysed volume (RLU2).
As ng ATP per unit.
11
4.2 Total organic carbon /dissolved organic carbon

European Standard CEN 1484 : “Water analysis - Guidelines for the determination of
total organic carbon (TOC) and dissolved organic carbon (DOC). See PDF-file “TOC
DOC CEN1484.pdf” from the SAFER tfp-site.
12
4.3 Modification of non-purgeable organic carbon (NPOC) analyses

(water)
Non-purgeable organic carbon (NPOC) is analysed by a method which is a
modification of a CEN 1484 “ Water determination. Guidelines for analyses of total
organic carbon (TOC) and dissolved organic carbon (DOC)” standard.
Non-purgeable organic carbon (NPOC) is determined from water samples which are
acidified (pH 3) with hydrochloric acid and purged with nitrogen gas (10 minutes)
before the analyses. The content of organic carbon is analysed by Shimadzu TOC-
5000/5050 -analyzer. The combustion temperature is +680 °C.
Dissolved organic carbon (DOC) is analysed in a similar way as NPOC, except that
the water samples are prefiltered with 0,22 µm syringe filters and no nitrogen purging
is used.
Detection limit is 0,3 mg/L. Method is accredited.
Uncertainty of quantitative determination for different concentrations:
< 10 mg/l 15 % and > 10 mg/l 10 %
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4.4 Assimilable organic carbon (AOC) (water)

a. The Dutch standard method NEN 6271. See the original description « AOC NEN
6271.pdf-file » from the ftp-site of SAFER.
b. AOC method modification
Scope
The AOC bioassay using Pseudomonas fluorescens P-17 and Aquaspirillum sp.
NOX involves growth to a maximum density of a small inoculum in a batch culture of
pasteurized test water. Pasteurization inactivates native microflora. The test
organisms are enumerated by spread plate method for heterotrophic plate counts
and the density of viable cells is converted to AOC concentrations by an empirically
derived yield factor for the growth of P. fluorescens P-17 on acetate-carbon and
Aquaspirillum sp. NOX on oxalate-carbon as standards. The number of organisms at
stationary phase is assumed to be the maximum number of organisms that can be
supported by the nutrients in the sample and the yield on acetate carbon is assumed
to equal the yield on naturally occurring AOC (van der Kooij 1982, Kaplan and Bott
1989). The underlying assumption of the AOC bioassay is that the bioassay
organism(s) represent the physiological capabilities of the distribution system
microflora. In some waters (e.g humic waters) inorganic nutrients regulate bacterial
growth (Miettinen et al., 1999). Thus, to ensure that carbon is limiting bacterial
growth, enough of inorganic nutrients are added in sample of test water.
In theory, concentrations of less than 1 µg C/L can be detected. In practice, organic
carbon contamination during glassware preparation and sample handling imposes a
limit of detection of approximately 5 to 10 µg AOC/L. High concentration of metals
(esp. Al, Cu) is toxic for strain P. fluorescens, which makes this procedure unsuitable
for waters containing these metals.
References
Kaplan L.A., Bott T.L. 1989. Measurement of assimilable organic carbon in water distribution
systems by a simplified bioassay technique. In Advances in Water Analysis and Treatment,
th
Proc. 16 Annu. AWWA Water Quality Technology Conf., Nov. 13-17, 1988, St. Louis, Mo., p.
475. American Water Works Assoc., Denver, Colo.
Miettinen I.T., Vartiainen T. and Martikainen P.J. 1999. Determination of assimilable organic
carbon in humus-rich drinking waters. Water Res. 33 (10): 2277-2282.
Reasoner D.J., Geldreich E.E. 1985. A new medium for the enumeration and subculture of
bacteria from potable water. Appl. Environ. Microbiol. 49:1-7.
Swanson K.M.J., Busta F.F., Peterson E.H., Johnson M.G. 1992. Count methods. I n
Compendium of methods for the microbiological examination of foods. Vanderzant C.,
Splittstoesser D.F., eds. APHA, Washington, 75-95.
Van der Kooij D., Visser A., Oranje J.P. 1982. Multiplication of fluorescent pseudomonads at
low substrate concentrations in tap water. Antonie van Leeuwenhoek 48:229-243.
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.
14
• Eppendorf vials or glass tubes for dilution series.

• Glass test tubes and Vortex mixer.
• Petri dishes (disposable, plastic).
Reagents
• Sodium acetate stock solution, 400 mg acetate-C/L. Dissolve 2.267 g
CH3COONa.3H2O (or 1.71 g CH3COONa) in 1 L organic-carbon free, deionized
water. Transfer to 100-mL vials, cap and autoclave. Store at 5 °C. Solution may
be held for up to 12 months.
• Sodium thiosulfate solution. Dissolve 30 g Na2S2O3 in 1 L deionized water.
Transfer to 100 mL vials, cap and autoclave.
• Buffered water.
•
R2A agar (Reasoner and Geldreich 1985)
• Organic-free water.
• Mineral salts solution. Dissolve 4.55 g (NH4)2SO4, 0.2 g KH2PO4, 0.1 g
MgSO4.7H2O, 0.1 g CaCl2.2H2O, 0.2 g NaCl in 1 L organic-free water. Transfer to
100-mL vials, cap and autoclave.
• Cultures of test strains Pseudomonas fluorescens P-17 (ATCC 49642) and
Aquaspirillum sp. NOX (ATCC 49643).
Preparation of incubation vessels
Wash 100-mL vials with phosphate-free detergent, rinse with hot water, immerse in
0.1 N HCl for 2 h, and rinse with deionized water three times, dry, cap with foil, and
heat to 550 °C for 6 h or 250 °C for 8 h. Use same cleaning procedure for all
glassware.
Procedure
Preparation of stock inoculum
Prepare turbid suspension of P. fluorescens P-17 and Aquaspirillum sp. NOX by
transferring colonies from R2A agar plates into 2 to 3 mL of filtered (pore size
0.2 µm) and autoclaved sample to obtain a final concentration of approximately 108
cfu/ml (e.g. it corresponds to 0.10 absorbance of microbe mixture at 420 nm). The
microbe mixture is diluted with autoclaved water 10-3 - 10-4 and inoculated into
pasteurised (30 min, 65°C) water. Any fresh water that supports growth of P.
fluorescens P-17 can be used. Before inoculation, water is added 1/1000 final dilution
of mineral salts solution and final concentration of 1000 µg acetate C/L.
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,
measured by colony forming units (spread plate method), reaches its maximum
value. Store stock cultures not more than 12 months at 5 °C. After the stationary
phase is reached, make a viable count of the culture (spread plate) to determine the
appropriate volume of inoculum to be added to each bioassay vessel.
Preparation of incubation water
Collect 500 mL sample in an organic-free vessel and pour into two parallel 100 ml
vials. Neutralize samples containing disinfectant residuals with 100 µL sodium
15
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.
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)
16
4.5 Total phosphorus

ISO DIS 6878 (Water quality – Determination of phosphorus – Ammonium
molybdate spectrometric method) - See the original instructions from the PDF-file “
ISO_DIS 6879 determination of phosphorus.pdf” from the ftp-site of SAFER.
17
4.6 Microbially available phosphorus (MAP) (water)

Scope
The MAP bioassay using Pseudomonas fluorescens P-17 involves growth to a
maximum density of a small inoculum in a batch culture of pasteurized test water.
Pasteurization inactivates native microflora. The test organism is enumerated by
spread plate method for heterotrophic plate counts and the density of viable cells is
converted to MAP concentrations by an empirically derived yield factor for the growth
of Ps. fluorescens P-17 on phosphate-phosphorus as standard. The number of
organisms at stationary phase is assumed to be the maximum number of organisms
that can be supported by the nutrients in the sample. The yield on phosphate-
phosphorus (PO4-P) is assumed to be equal the yield on naturally occurring MAP.
Ps. fluorescens P-17 has phosphatase activity. The underlying assumptions of the
MAP bioassay are that the carbon and inorganic nutrients, with the exception of
phosphorus, are present in excess, i.e., that phosphorus is limiting (Lehtola et al.,
1999). Concentrations 0.08-10 µg MAP/L can be detected (Lehtola et al., 1999).
High concentration of metals (esp. Al, Cu) are toxic for strain Ps. fluorescens, which
makes this procedure unsuitable for waters containig these metals.
References
Lehtola M.J., Miettinen I.T., Vartiainen T., Martikainen P.J. 1999. A new sensitive bioassay for
determination of microbially available phosphorus in water. Appl. Environ. Microbiol. 65:2032.
Reasoner D.J., Geldreich E.E. 1985. A new medium for the enumeration and subculture of
bacteria from potable water. Appl. Environ. Microbiol. 49:1.
Swanson K.M.J., Busta F.F., Peterson E.H., Johnson M.G. 1992. Count methods. I n
Compendium of methods for the microbiological examination of foods. Vanderzant C.,
Splittstoesser D.F., eds. APHA, Washington, 75-95.
Materials
- Incubation vessels - borosilicate glass vials (volume at least 100 ml, 250-500 ml is
recommend because of more effective shaking) 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.
- Eppendorf vials or glass tubes for dilution series
- glass test tubes
- Vortex mixer
- Petri dishes (plastic)
- Sodium acetate stock solution, 400 mg acetate-C/L. Dissolve 2.267 g
CH3COONa.3H2O (or 1.71 g CH3COONa) in 1 L organic-carbon free, deionized
water. Transfer to 100-mL vials, cap and autoclave or pasteurize at 60°C for 35
min. Store at 5 °C. Solution may be held for up to 6 months.
- Sodium thiosulfate solution. Dissolve 30 g Na2S2O3 in 1 L deionized water. Transfer
to 100 mL vials, cap and autoclave.
- Buffered water.
- R2A agar (Reasoner and Geldreich 1985)
- phosphorus-free water. Alternatively, use HPLC-grade bottled water.
- Mineral salts solution. Dissolve 0.48 g NH4NO3, 0.1 g MgSO4.7H2O, 0.1 g
CaCl2.2H2O, 0.1 g NaCl, 0,1 g KCl in 1 L phosphorus-free water. Transfer to
100-mL vials, cap and autoclave.
- Culture of test strain Pseudomonas fluorescens P-17 (ATCC 49642).
18
Preparation of incubation vessels:

Wash vials with phosphate-free detergent, rinse with hot water, immerse in 0.1 N HCl
for 2 h, and rinse with deionized water three times, dry, cap with foil, and sterilize e.g.
by heat treatment. Use same cleaning procedure for all glassware.
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.
Preparation of sample water

Collect water samples in an glass vessels and pour 100 mL into Erlenmeyer vessels.
Use two parallel vials/vessels for MAP measurement. Neutralize samples containing
disinfectant residuals with 50 µl sodium thiosulfate solution for 100 ml sample. 100
µL of mineral salts solution and 400 µL of sodium acetate solution are added to the
each vial. Cap vials and pasteurize in 65-70 °C water bath for 30 min.
Inoculation and incubation

Cooled waters are inoculate with approximately 1000 colony forming units (CFU)/mL
(usually 2 drops of stock inoculum by pasteur pipette, concentration should be tested
beforehand) of Ps. fluorescens P-17. Use the following equation to calculate volume
of the inoculum:
(1000 CFU/mL) x (100 mL/vial)
volume of inoculum = ----------------------------------------------
CFU/mL stock inoculum
Inoculated samples are incubated at 15 °C in the dark for 8 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 4-8 (starting from 4th day
and continuing until 8th day). If higher temperature is used, maximum is reached
earlier (3-7 days, should be tested beforehand). Shake vigorously the vials and make
19
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).
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)
20
4.7 Total nitrogen

European Standard: ENV 12260 (Water quality – Determination of bound nitrogen
(TNb) – following oxidization of nitrogen oxides).
See the original instructions from the PDF-file “ Nitrogen EN 12260.pdf” from the ftp-
site of SAFER.
21
4.8 Assessment of nucleic acid damages by chlorination using

fluorochrome staining (SYBR-II or PI) and flow cytometry
Objectives
We aim to develop a sensitive and rapid method to assess the intracellular injuries
caused by disinfectants such as chlorine. The method combines cell staining with a
fluorochrome which efficiency depends on the nucleic acid integrity, and flow
cytometry for an objective quantification of the fluorescence changes. This
deliverable describes the procedure. The method gives a relative results which allow
to assess the intracellular injuries before and after bacterial chlorination.
Background
The bacteriological quality control of drinking water based on culture methods has
two important drawbacks: firstly, it is time-consuming method because bacteria
colonies on nutritive agar medium require from 1 to almost 15 days of incubation to
be conveniently observed. This prevents any use of such methods for an on-line
follow-up of the water bacteriological quality. Secondly, culture methods lead to an
important underestimation of the viable bacteria number: only a fraction of the
bacteria colony is cultivable (Roszak and Colwell, 1987). This fraction does not
represent the whole viable bacteria number, and more importantly underestimation is
worsened by the oxidant stress induced by disinfectants (hypochlorite, chloramine).
An alternative process to culture methods for controlling the disinfection efficiency
could use fluorescent dyes, which nucleic acid staining efficiency can be affected by
several processes, including chlorine disinfection. Many fluorescent probes are
commercially available and allow a good staining of all bacteria and rapid counting by
flow cytometry.
The main difficulty to apply fluorescence assay to the water quality disinfection after
chlorination is then to find a nucleic acid fluorescent probe that can (i) be a good
marker of damaged cells (or uninjured ones) as previously reported by Saby et al.
(1997) and Phe et al. (2004), (ii) be excited at 488 nm, the mostly used argon-laser
blue line in flow cytometers and (iii) lead to quantitative results. Besides, the water
quality control requires a quantitative result, and a high sensitivity, which means that
the probe should stain bacteria with very high complex rate constant and high
fluorescent quantum yield after excitation at 488 nm.
Here, we have selected SYBR Green II RNA Gel Stain (SYBR-II) and as propidium
iodide (PI) for quantitative staining of nucleic acids damages by chlorine. We have
defined the concentration of fluorochrome to be used, and the best contact time for
staining bacteria. At last, we have documented the way to count fluorescent events
and to measure fluorescence with flow cytometer.
Materials and reagents
SYBR Green II RNA gel stain (Molecular Probes, S-7586)
Propidium Iodide (Sigma, P-4170)
12×75 mm plastic Falcon tube (Becton Dickinson- 352054)
22
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
23
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.
FSC Background FL1

Background
Background
SSC SSC
Figure 1: Example of cytogram of a sample without SYBR-II.

Analysis of stained sample with SYBR-II or with PI
The first aim is to localize and to discriminate the bacterial population on background
using different parameters which are on the one hand, the forward scatter (FSC) and
the other hand the side scatter (SSC). It is this second parameter which is measured
because flow cytometer has a better sensitivity for this parameter than for FSC. The
bacterial sample is isolated using the graphical palette of the BDCellQuest software
(Becton Dickinson).
A new acquisition is initiated and the display mode carries out under histogram
reporting on the X axis either FL1 (for SYBR-II staining) or FL3 (for PI staining) or
SSC, and in the ordinate axis the number of counted events.
The acquisition time depends of the number of cells counted and the discrimination
of the bacterial populations. A zonation is realized around the population of interest
on the cytogram FL1 = f (SSC) for SYBR-II staining or FL3 = f (SSC) for PI staining.
24
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
Channel FSC SSC FL1

0 0 0 929
1 0 0 1
2 0 2 14
3 0 438 1
4 0 77 2
... ... ... ...
56 99 229 0
57 109 264 0
58 95 233 0
... ... ... ...
69 210 49 0
70 230 59 0
71 183 39 0
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.
25
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.
26
4.9 Amino acid
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)
Acidified (HNO3, pH<2)

Ultrapure water biofilm
(7 mL per cm2 of coupon)
• Extract biofilm with a ultrasound probe (∅ 3mm, 15 W during 10 minutes with

50% of rate of activation; apparatus : Sonifier II BRANSON model or similar)
• Extract the biofilm of two coupons by sample if it is possible
• Dispatch of sample
• Use glass flasks exempt of organic matter for expedition of sample. The
volume of flask must be small to decrease risk of contaminations. Flasks must
be closed with Teflon caps or caps covered with Teflon.
• Acidify the samples of water ( ultra pure HNO3, pH < 2).
• Store samples at 4°C and send in icebox via express transport.
27
5 Quality control and quality assurance
5.1 Quality control and quality assurance in CR4

The Laboratory of Microbiology and Chemistry has status of accredited testing
laboratory permit by DAR (the German Accreditation Service). The Laboratory of
Microbiology and Chemistry complies with the standard EN ISO/IEC 17025 and thus
will also operate in accordance with ISO 9001 and ISO 9002.
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.

The quality control system of KTL/ Department of Environmental Health is designed
to satisfy the internal managerial needs. Two official international quality standards
are used in the organisation. The Laboratory of Chemistry has status of accredited
testing laboratory permit by FINAS (the Finnish Accreditation Service). Laboratory of
Chemistry complies with the standard EN ISO/IEC 17025 and thus will also operate
in accordance with ISO 9001 and ISO 9002. The laboratory of Toxicology complies
with the OECD Principles of Good Laboratory Practice. The Toxicity Testing Unit is
approved in the national GLP compliance Program and inspected on a regular basis.
The quality system of Department of Environmental Health is created based on the

Principles of GLP and the standard EN ISO/IEC 17025. The Department has
common Standard Operating Procedures, which are establish by the Management
and Quality Assurance Unit. Internal audits concern the common procedures of the
Department and the main processes of the laboratories.
The main analytical methods and the test equipment used in the laboratory of
Environmental Microbiology are described in specific SOPs (so called SOP MB).
The competence of the personnel is provided by continuous appropriate education
and training.

International Depository Authority Microbial Strain Collection of Latvia (MSCL) in
practice follows the principles listed in "Guidelines for the Establishment and
Operation of Collections of Cultures of Microorganisms" /2nd Edition , June 1999;
Revised by the World Federation for Culture Collections (WFCC)/ with the purpose of
promoting high standards of scientific service in microbiological laboratories. CABRI
QUALITY GUIDELINES (Demonstration Project ERBBIO4-CT96-0231, co-funded by
28
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.
The competence of the personnel is provided by annual Training courses and

workshops organized by ECCO( European Culture collection organization) as well by
appropriate education.
29
6 Biofilm monitoring devices
6.1 Propella (The common biofilm monitoring device for all partners)
PROPELLA® THE DYNAMIC CONCEPT TO STUDY INTERACTIONS

BETWEEN WATER AND MATERIALS
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
In drinking water distribution networks, as in numerous industrial processes, the

degradation of water quality (biological, chemical contamination) and/or exposed
surfaces (corrosion, scaling) is explained by the interaction between the liquid and
material phases.
It is difficult to predict the intensity of these water-material interactions and in many
cases it is necessary to expose the material to water in order to evaluate the
compatibility of the two products.
Contrary to static tests, Propella® was built to simulate in the laboratory a piece of
pipe transporting liquid such as potable water, thermal waters, etc.
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
30
• to study water characteristics and exposed surfaces
Propella® is a perfectly mixed reactor, in which the liquid is pushed by a propulsive

propeller through an internal tube (see the illustration below). The liquid flows along
the canalisation section studied, as in a real pipe. It is easy to impose a defined
Reynolds number by fixing the circulation rate in the pipe. The hydraulic residence
time is inversely proportional to the alimentation flow of the reactor.
How the PROPELLA functions
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.
The reactor allows, among others :

• to model disinfectant use in drinking water distribution networks, and the influence
of pH, temperature, laminar or turbulent flow, bacterial deposits (biofilm), pipe
material, etc.
• to study the salting out of mineral and/or organics by the pipe material, and also to
model bacterial growth with or without disinfectant.
The reactor permits :

• to modify and/or maintain the fluid characteristics by introducing reactive into the
reactor ;
• to modify and/or maintain a defined agitation characterised by a Reynolds
number, and also a direction flow ;
• to quantify microbial deposits on pipewalls and sample a part of the colonised
surface of the reactor in contact with water ;
• to test different materials used in real drinking water distribution networks ;
• to work at different temperatures by the presence of an internal thermoregulation
system.
31
Characteristics
motor
water supply exit
coupons
biofilm
sampling
section of devices
material under
study vane
double
internal
cylinder
water for
thermoregulation
Figure 1: Scheme of Propella
• Surface/volume relation identical to a real pipe

• Volume : approx. 2.23 L (generally ∅ 100 x 500 mm)
• Flow variable speed from 0.05 to 0.5 m/s (typically 0.2 m/s)
• Inox or glass pipes available to study specifically water
• Material : inox 316L and Teflon (except studied pipe)
• Up to 20 sampling devices per pipe
• Possibility to connect reactors in series
32
Setup examples
Figure 2: Pictures of Propella
Propella© reactor, dimensions

Internal cylinder : height 460 mm, internal diameter 44,0 mm, external diameter 72,5
mm (thickness of material = 14,25 mm)
External cylinder : height 500 mm, external diameter 110,0 mm, internal diameter
93,4 mm, thickness of material = 8,3 mm
Normal procedure to clean PVC/PEHD coupons

• 3 hours soaking in a detergent solution (Aquet, Polylabo, reference 64528; a non-
ionic, neutral pH, no-phosphates and biodegradable detergent or equal
detergent, concentration 1%) and then careful brushing by hand
• rinsing thoroughly with tap water
• steeping in a chlorine solution (20 mg/L) during 15 minutes
• rinsing two times with distilled sterile water without bacterial cells
• drying in an oven (60°C), then storing in a sterile place
• then ready to use...
Normal procedure to clean Propella reactor

• De-assemble completely the Propella reactor
• Only for the external envelope (PVC or PEHD one): 1 hour in a chlorine solution
(100 mg Cl2/L) and then careful brushing
• Rinse thoroughly with tap water
• Rinse two times with distilled sterile water without bacterial cells.
• Wait until the propella reactor is completely dry
• Re-assemble Propella reactor, then ready to use...
33
Sampling of biofilm from a Propella

Procedure is described in a separate file on the ftp-site of SAFER
“Propella_sampling.doc (3.6 MB)
Biofilm removal protocol

PVC coupons that are colonized with biofilms are taken from the sampling devices
without discontinuing the water flow within the distribution system. They are then
placed in sterile flasks containing 25 ml of bacterial cell-free distilled water. Less than
30 minutes later, the biofilm is dispersed by a gentle sonication (2 min. ultrasounds at
2 W, 20 KHz; Bioblock Scientific Vibra cell, model 72401; probe model 72403,
Bioblock, USA, diameter 3mm). The probe is placed 1 cm above coupon, inside
bacterial cell-free distilled water. The bacterial content of the resulting suspensions
must be analysed within one hour.
DON'T FORGET TO PLACE
THE STERILE FLASK
CONTAINING THE
COUPON IN ICE DURING
SONICATION TO AVOID
INCREASE OF
TEMPERATURE
Figure 3: Dispersing the biofilm from Propella coupons
To determine sonication efficiency:
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.
34
6.2 Rotating Annular Reactor (modified RotoTorqueTM) [CR4]
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
35
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
36
Table 1: Dimensions of the Rotating Annular Reactor
Inner cylinder: unit

Height cm 18,0
Bore cm 10,2
Exposed area (vertical) cm_ 576,8
Exposed area (horizontal) cm_ 157,1
Total area cm_ 733,9
Outer cylinder:
Height cm 20,6
Bore cm 11,30
Exposed area (vertical) cm_ 731,1
Exposed area (horizontal) cm_ 100,3
Total area cm_ 831,4
Coupons:
Number 12
Width 1,5
Length cm 22,0
Exposed length cm 20,1
Exposed area cm_ 30,9
Total area (x 12) cm_ 370,8
Percentage on the total area of the outer cylinder % 44,6
Recirculation tubes:
Number 4
Angle ° 80
Length cm 18,5
Bore cm 1,0
Exposed area cm_ 231,47
Total Rotating Annular Reactor:
Volume mL ca. 650
dependent on the
rotation speed
Exposed total area (without recirculation tubes) cm_ 1565,3
Percentage of the coupons on the total area % 23,68
Specific area cm_/cm_ 2,76
Table 2: Measurements of the middle rotating velocities and Reynolds-

numbers in the Rotating Annular Reactor with different revolutions per minute
Revolutions ri calculated Horizontal velocity Vertical velocity Total velocity. Re
per min
[U min-1] [m s-1] [m s-1] [m s-1] [m s-1] -

50 0,13 0,10 0,01 0,10 514
200 0,53 0,44 0,03 0,45 2226
600 1,60 1,29 0,08 1,29 6439
ri: rotating velocity of the inner cylinder
Re Reynolds number
37
Re = 11,846 x revolutions/min010002000300040005000600070000200400600revolutions/min
Figure 5: Reynolds-number of the annular reactor as a function of rotating

velocity.
Procedure
Sterilisation and operation of the Rotating Annular Reactor

Prior to the experiments the reactor system including all tubes is either sterilised by
autoclaving for 20 minutes at 121°C or disinfected by biocides with 1000 mg/L
hydrogen peroxide for 2 hours at a rotation speed of the inner cylinder of 400 rpm.
The sterile Rotating Annular Reactor is then fed with drinking water flowing with
50 mL/h corresponding to a residence time of 12 hours.
Sampling the biofilm from the test-surfaces
Coupons can be made out of different materials (PVC, stainless steel, copper, ...)
When they are colonised with biofilms they are taken from the sampling devices
without discontinuing the water flow within the distribution system. They are analysed
for bacterial density directly by using epifluorescence microscopy. The biofilm is
stained with 4´, 6-diamidino-2-phenylindole (DAPI) having a final concentration of 5
µg/mL. On each slide, at least 300 bacterial cells must be counted at 1.000 x
magnification on the coupon.
For indirect enumeration the biofilm bacteria are scraped mechanically with a razor
blade and disaggregated on a vortex for 3 minutes. With this bacterial suspension the
total cell number (see chapter total cell number) and the heterotrophic plate count
(see chapter enumeration of culturable microorganisms) are performed.
Setup examples
38
Figure 6 : Images of Rotating Annular Reactors (modification of Schulte and

Wingender, 2000) under operation conditions
39
6.3 Biofilm generator

The schematic representation of the biofilm generator is given on Figure 7.
Figure 7: Chemostat lab biofilm generator
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.
40
6.4 Flow cell reactor

The biofilms are formed on several adhesion slides placed within flow cell reactors
(which have a semi-circular cross section), where drinking water can flow under
different hydrodynamic conditions. The adhesion slides, which can be made in
different sizes and of different materials, are glued to rectangular pieces of PMMA
properly fitted in the apertures of the flow cell. The equipment is connected to a side
stream in a drinking water system or a simulated drinking water system, as
schematically represented in the Figure 8.
Figure 8: Linear flow through reactor for biofilm
41
6.5 Pipeline biofilm collector
Biofilm monitoring system used in CR9

The biofilm monitoring systems consist for Pipeline biofilm collector and Propella and
a plug flow reservoir. The system is connected directly to the water supply system of
Riga. Water from the water system water is collected in the reservoir that ensures
constant pressure and flow in the biofilm reactors (Figures 9 and 10).
The same device will be used as the reference biofilm samplers in CR6 (Finland) and
CR9 (Latvia). The differences in the devices/test systems between CR6 and CR9 are
described in the text.
Maintenance
Biofilm collectors are 10 cm long PVC pipes, which are connected together one after
another with ball valves and stainless steel loops. Cleaning of the pipeline system
parts follows the Propella cleaning procedure. The pipeline system is installed on
stainless steel frame. The amount of the PVC pipes in one biofilm collector will vary
in different tests: 7 samplings with 3 replicates equals to 21 subsamples. Water flow
through the pipes will be adjusted to 500 ml/min (= 0.1 m/s) using flow meters/valves.
Before sampling, ball valves at both sides of a PVC pipe are closed and the pipe
samples full of water are removed and replaced with new pipes. Valves are opened
and water flow is connected back after the sampling.
Biofilm sampling
Part of water (1.5 ml) is removed from the detached pipe and a spoonful of sterile
glass beads are inserted into the pipe. To detach biofilm from the inner side of the
pipe, the pipe-valve system containing water and glass is vortexed for 20 minutes.
This water-biofilm sample is combined with the water removed before vortexing.
Finally the pipe is rinsed with sterile deionized water (5 ml) (CR9: pipe is rinsed twice
= total 10 mL) which is combined with the vortexed water-biofilm sample.
CR6: the test system CR6 uses tap water of Kuopio city as feed water. Pipeline
device is a system connected directly to the tap water system In Propella tap water
flows first into a small glass vessel, where it is pumped with peristaltic pump into the
device.
42
Figure 9. Schematic drawing of biofilm monitoring system in CR9
C
A
B
A
Figure 10. Photos of (a) biofilm monitoring system with (b) reservoir and (c)
inlet and sewerage.
43
6.6 Differential turgidity measurement

DTM is an optical measurement system consisting of two measurement cells – pairs
of optical windows (Figure 11). One pair is continuously cleaned in order to prevent
biofilm building. The fouling effect on the non-cleaned cell can be directly measured if
the signal of the cell with clean surface is subtracted.
detector detector
Flow irection
Light Light
source source
ee e
Figure 11: Schematic view of a DTM device
44
6.7 Fibre optic devices (FOS, and FluS sensors)

FOS and FluS are both fiber optic based devices. Optical fiber heads are
implemented in the water system.
Biofilm
Substratum
EPS
Optical Fiber
Bacteria
Illuminated
biofilm area
IIluminating light
Backscattered
light
Figure 12: Schematic view of a FOS
The Fibre Optical Device (FOS)

The principle of the fibre optical device is based on the determination of the local
concentration of light scattering particles depositing on a tip of an optical fibre. The
backscattered light quantified and the signal is related to the amount of material. This
signal is essential a sum parameter composed of the contributions of all light scattering
materials. However, it can be specified by calibrating with microorganisms and by
changing of the system by addition of nutrients or increase of shear forces; this will
influence the signal in a predictive way. The concentration is determined by the
scattering signal.
The measuring head
Light from a monochromatic or quasichromatic source is transmitted through optical
fibres to the system in question. The backscattered light is transmitted by a parallel
fibre to a detector. In a water system, the sensor is integrated evenly into the inner
surface and experiencing the same flow conditions as the rest of the internal wall.
Therefore, it can be considered as representative for the situation. Material
depositing on the surface will contribute the strongest signal of backscattered light
while particles in the water phase give a much lower signal. This has been
demonstrated in the calibration experiments carried out in WP 1. The diameter of the
measuring fibre is about 0,2 mm. Although it is made of glass, it has been observed
that the deposit forming on it is not distinguishable from that forming on other
surfaces such as stainless steel.
45
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
Integrated in SURFACE BIOFILM
Figure 13 Schematical depiction of the configuration of the sensor head. The

device is integrated into the surface which is monitored for deposition of
material
The FOS has been calibrated and the results were presented at one of the cluster
meetings which are documented in the protocols. The algorithm for the software has
been changed in order to allow distinguishing between biotic and abiotic material on
the basis of selection of specific wavelengths of the reflected light. A chip and a
sender will be implemented and the data can be transferred via satellite to remote
operators.
During the work performed in WP 1, the sensor was implemented into an Annular
Reactor (arrows in figure 14):
46
Figure 14: 3 FOS implemented in an Annular reactor
47
6.8 Electrochemical, nanovibration, and capacitive monitors
6.8.1 Mechatronic Surface Sensor - MSS

The basic idea behind this monitor is to utilise the nanovibrations to evaluate the
amount of the biofilm through mathematic analysis of the signal. This technique
utilizes the direct and converse electromechanical properties of smart materials,
allowing simultaneous actuation and sensing.
Until now, this technique has been successfully used in the diagnosis of plate metal
defects, namely in the aerospace industry, and in soil consistence analysis in civil
engineering. The monitor is a half-tube flow cell (see figure 15) and the sensors are
on the outer flat surface of this flow cell.
Water Sensors
Figure 15: Mechatronic Surface Sensor
6.8.2 Capacitive sensors

Capacitive sensors are analogous, non-contact devices. A capacitive bridge is built
from internal capacitance and the capacitances created by the proximity of the object
(the biofilm) to be measured. The dielectric is directly related to the distance/medium
in front of the sensor plate; ultra-precise electronics convert the capacitance
information into an analog signal. Very precise sensors can be made, up to de
resolution of 0.01 nanometer.
The monitor is identical to the one containing the mechatronics surface sensors (a
half-tube flow cell) and the capacitive sensor will be inserted on the flat surface of this
flow cell.
48
6.9 Biofilm formation monitoring using ATR-FTIR sensor
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.
Peristaltic Trash flask

pump
Sample flask ATR flow cell
Figure 16: Experimental device for the infrared biosensor
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
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
Figure 17: Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR)

flow through cell
Biofilm study in flow cell

The flow cell (figure 2) is successively and continuously supplied with (i) ethanol (75
% for 4 hours) to clean the system, (ii) non pyrogenic sterile water (1 hour at 40
ml/min and 10 hours at 0.7 ml/min) to remove the ethanol (control by recording
spectra) (iii) Luria Bertani (LB) nutritive medium to deposit a conditioned organic film
(during 5 to 10 hours), (iv) the bacterial suspension (2 hours) which allowed bacteria
to adhere to the surface of the crystal, (v) the tested 0.2 µm filtered water for 24
hours. Ethanol, bacterial suspension and water samples are passed through the ATR
flow cell by using an up-stream Gilson Minipuls 3 pump with a flow of 44 mL h-1
(hydraulic residence time approximately 4 min). The infrared spectra of the hydrated
dynamic biofilm are automatically collected every 15 minutes for the first hour and
then every hour.
Pseudomonas fluorescens bacteria suspension
Pseudomonas fluorescens (Pf) strain was purchased at Institut Pasteur (CIP6913,
Paris). Stock cultures were prepared with cryogenic balls in glycerol: a 24h-culture of
Pf realised on R2A (Difco, 218263) agar plate, identified by API 20NE strip (number
0147555). Then, the bacteria were scraped, and suspended in the cryogenic tube. It
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.

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-1-

Surveillance and control of microbiological

Handbook for analytical methods and

WP2. Tools for biofilm monitoring

DATE: 13h November, 2005

3.2 Total cell number 6

3.3 Coliform bacteria and E. coli (water and biofilm) 7

4.2 Total organic carbon /dissolved organic carbon 11

4.3 Modification of non-purgeable organic carbon (NPOC) analyses (water) 12

4.4 Assimilable organic carbon (AOC) (water) 13

4.5 Total phosphorus 16

4.6 Microbially available phosphorus (MAP) (water) 17

4.7 Total nitrogen 20

4.9 Amino acid 26

5 QUALITY CONTROL AND QUALITY ASSURANCE 27

5.2 Quality control and quality assurance in CR6 27

5.3 Quality control and quality assurance in CR9 27

6 BIOFILM MONITORING DEVICES 29

6.2 Rotating Annular Reactor (modified RotoTorqueTM) [CR4] 34

6.3 Biofilm generator 39

6.4 Flow cell reactor 40

6.5 Pipeline biofilm collector 41

6.6 Differential turgidity measurement 43

6.7 Fibre optic devices (FOS, and FluS sensors) 44

6.8 Electrochemical, nanovibration, and capacitive monitors 47

6.9 Biofilm formation monitoring using ATR-FTIR sensor 48

3.1 Enumeration of culturable microorganisms (water/biofilm)

3.2 Total cell number

3.3 Coliform bacteria and E. coli (water and biofilm)

3.4 Dehydrogenase activity with redox stain 5-cyano-2,3-ditolyl

4.1 Measurement of adenosine triphosphate (ATP)

The most common quantification method of the bacterial ATP is based on an

4.2 Total organic carbon /dissolved organic carbon

4.3 Modification of non-purgeable organic carbon (NPOC) analyses

4.4 Assimilable organic carbon (AOC) (water)

• Eppendorf vials or glass tubes for dilution series.

4.5 Total phosphorus

4.6 Microbially available phosphorus (MAP) (water)

Preparation of incubation vessels:

Preparation of sample water

Inoculation and incubation

Inoculated samples are incubated at 15 °C in the dark for 8 days. If higher

Enumeration of test bacteria

4.7 Total nitrogen

4.8 Assessment of nucleic acid damages by chlorination using

FSC Background FL1

Figure 1: Example of cytogram of a sample without SYBR-II.

Channel FSC SSC FL1

4.9 Amino acid

Acidified (HNO3, pH<2)

• Extract biofilm with a ultrasound probe (∅ 3mm, 15 W during 10 minutes with

5 Quality control and quality assurance

5.1 Quality control and quality assurance in CR4

5.2 Quality control and quality assurance in CR6

The quality system of Department of Environmental Health is created based on the

5.3 Quality control and quality assurance in CR9

The competence of the personnel is provided by annual Training courses and

6 Biofilm monitoring devices

PROPELLA® THE DYNAMIC CONCEPT TO STUDY INTERACTIONS

In drinking water distribution networks, as in numerous industrial processes, the

• to study water characteristics and exposed surfaces

Propella® is a perfectly mixed reactor, in which the liquid is pushed by a propulsive

How the PROPELLA functions