Handbook for Analytical Methods And

EVK1-CT-2002-00108 Surveillance and control of microbiological stability in drinking water distribution networksHandbook for analytical methods and operational criteria for biofilm reactors

SAFER

EVK1-CT-2002-00108

Surveillance and control of microbiologicalstability in drinking water distribution networks

Handbook for analytical methods andoperational criteria for biofilm reactors

(Version 1.0)

WP2. Tools for biofilm monitoring

Prepared by:Ilkka MiettinenNational Public Health InstituteDepartment of EnvironmentalHealthKuopio, Finland

Gabriela SchauleIWW Rhenish-Westfalian Institutefor WaterDepartment of AppliedMicrobiologyMulheim, Germany

DATE: 28th May, 2003


Content

1 INTRODUCTION 4

2 STRUCTURE 4

3 MICROBIOLOGICAL METHODS 5

3.1 ENUMERATION OF CULTURABLE MICROORGANISMS(WATER/BIOFILM)..............................................................................5

3.2 B. NUMBER OF COLONY FORMING UNITS ON R2A NUTRIENT AGAR..5

3.3 TOTAL CELL NUMBER (WATER /BIOFILM).......................................6

3.4 COLIFORM BACTERIA AND E. COLI (WATER AND BIOFILM) ...............7

3.5 DEHYDROGENASE ACTIVITY WITH REDOX STAIN 5-CYANO-2,3-DITOLYL TETRAZOLIUM CHLORIDE (CTC) (WATER/BIOFILM) ...................8

3.6 LEGIONELLA (WATER/BIOFILMS)..................................................9

3.7 ISOLATION AND CULTURE OF MYCOBACTERIA (WATER /BIOFILM) ...10

3.8 ZIEHL-NEELSEN STAINING FOR MYCOBACTERIA ..........................15

3.9 NOROVIRUSES (WATER/BIOFILMS).............................................18

3.10 THERMOPHILIC CAMPYLOBACTERS (WATER /BIOFILMS) ................21

3.11 MEASUREMENT OF ADENOSINE TRIPHOSPHATE (ATP) ................21

3.12 EXTRACTION OF EXTRACELLULAR POLYMERIC SUBSTANCES (EPS)24

3.13 PROTEINS (LOWRY METHOD) (WATER /BIOFILMS) .......................25

3.14 PROTEINS (BRADFORD METHOD) .............................................26

3.15 CARBOHYDRATES (WATER /BIOFILMS) .......................................27

4 CHEMICAL METHODS 29

4.1 TOTAL ORGANIC CARBON /DISSOLVED ORGANIC CARBON.............29


3

4.2 ASSIMILABLE ORGANIC CARBON (AOC) (WATER)........................29

4.3 TOTAL PHOSPHORUS...............................................................34

4.4 MICROBIALLY AVAILABLE PHOSPHORUS (MAP) (WATER) .............34

4.5 TOTAL NITROGEN....................................................................37

5 QUALITY CONTROL AND QUALITY ASSURANCE 38

5.1 QUALITY CONTROL AND QUALITY ASSURANCE IN CR4 .................38



6 BIOFILM MONITORING DEVICES 40

6.1 PROPELLA (THE COMMON BIOFILM MONITORING DEVICE FOR ALLPARTNERS) ....................................................................................40

6.2 BIOFILM GENERATOR [CR5].....................................................52

6.3 FLOW CELL REACTOR [CR7] ....................................................53

6.4 PIPELINE BIOFILM COLLECTOR [CR 6 AND CR9].........................54

6.5 DTM, FOS, AND FLUS SENSORS [CR4[...................................57

6.6 ELECTROCHEMICAL, NANOVIBRATION, AND CAPACITIVE MONITORS(CR 7)..........................................................................................59

Piezo sensors – monitor .......................................................................59Capacitive sensors ................................................................................60Electrochemical devices .......................................................................60

6.7 BIOFILM FORMATION MONITORING USING ATR-FTIR SENSOR[CR2] ...........................................................................................62

7 APPENDIX: CALIBRATION OF THE SONICATION PROBE 64


1 Introduction

The objective of workpakage 2 is to provide monitoring systems for assessingbiofilm development in situ, on-line and non-destructively. Research will beorganised on two levels: one will aim to intercalibrate reactors for biofilm build-up, the second will aim to develop biofilm monitoring devices. The mainchallenge is to establish devices having both high sensitivity, rapid responsepotential, and an early warning capacity. The second deliverable ofworkpackage 2 is to make a handbook for basic methodologies. Thishandbook will describe the common protocols of all basic analytical methods,which are used by partners participating to SAFER programme. Thisinformation enables the exchange of information and comparison of differentmethods.

The handbook contains information about methods used for characterisationof the water , biofilm sampling and biofilm characterisation.

2 Structure

Within three chapters the different methods are listed starting with themicrobiological methods (1), followed by the chemical (2) and the physicalmethods (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 bethe basis and will be eventually modified by the partners. The modificationsare listed.

The general structure of the method description:

1. Scope /principles

1. References

2. Definitions

3. Materials

4. Procedure

5. Expression of results


3 Microbiological methods

3.1 Enumeration of culturable microorganisms (water/biofilm)

a. European Standard: EN ISO 6222 (Water quality – Enumeration ofculturable micro-organisms – Colony count by inoculation in a nutrient agarculture medium).

See the original instructions from the PDF-file “ HPC ISO6222.pdf” from theftp-site of SAFER.

3.2 b. Number of colony forming units on R2A nutrient agar

Scope

The bacterial colony forming units (CFU) are enumerated by spread platemethod for heterotrophic plate counts.

Reference

Reasoner DJ and Geldreich EE (1985). A new medium for the enumerationand subculture of bacteria from potable water. Appl. Environ. Microb. 49: 1-7.

Procedure

Heterotrophic plate counts (HPC) are estimated by spread plating methodusing 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 at22 ± 2�C before the colony forming units (CFU) are counted by eye.

Membrane filter method will be used if the cell density in the sample is too lowfor spread plate. The membranes (diameter 47 mm) have a pore size of 0.2µm. The sample (more than 1 mL) will be filtered and the filter placed on theagar plate. Air bubbles under the filter should be avoided.

Expression of results

Results are expressed as the mean number of bacterial CFUs per mL ofwater sample


6

3.3 Total cell number (water /biofilm)

Scope

This method describes a procedure for counting all bacteria in water andhomogenised and/or diluted biofilm samples using the dye 4´, 6-diamidino-2-phenylindole (DAPI). There are other fluorochromes which can be used forthe same purpose e.g. Acidine Orange and Syto 9. Acridine Orange is the dyewhich is often used by the semiconductor industry to estimate the total cellnumber in ultra pure water. The procedure follows in this case the ASTMStandard Test Method Designation F 1095-8. The advantage of AcridineOrange is that it will show bright cells but the disadvantage is the staining ofnoncellulare material.

The total cell number of biofilms fixed to the surface will evaluated by theConfocal Laser Scanning Microscope after staining or if the biofilm is thin withthe epiflourescence microscope like the filtered water sample.

References

Hobbie, J.E., R.J. Daley, Jaspers, S. (1977): Use of Nucleopore filters forcounting 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 byDirect Count Epifluorescence Microscopy.

Materials

4´, 6-Diamidino-2-phenylindole (DAPI), black Nucleopore filter (pore size 0.2µm), non-fluorescent immersion oil.

All reagents should be filtered through a cellulose nitrate filter with the poresize of 0.2 µm.

Procedure

Place one black polycarbonate membrane filter (0.2 µm pore size, laserbeamed) on top of the sampling port, assuring that the shiny side of the filteris facing upwards. Filter an aliquot of the sample and stop the filtrationprocess immediately when the sample is filtered through. Supplement with


7

1 mL DAPI (10 µg/mL) and add 1 mL Triton X-100 (0.1 %). The finalconcentration should be 5 µg/mL. The incubation time should be 15 to 20minutes. Then the supernatant is filtered through and the filter with the stainedbacteria placed in a petri dish or any other box to let it air dry. If the filter willbe 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 inimmersion oil on the surface of a clean microscope slide; like a sandwich thefilter is between the immersion oil and a clean glas coverslip.

The enumeration of bacteria and other microorganisms are performed with amagnification of at least 1000 fold in a epifluorescence microscope. All bluestained cells are counted in randomly chosen microscopic viewing fieldsdelineated by the eyepiece micrometer. There should be 10-50 cells perviewing field. In minimum 300 bacteria should be counted or so many viewingfields that the coefficient of variation of < 30% is obtained.

3.4 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, andmEndo LES) can be used for E. coli and coliform counting.

See instructions of chromogenic agars from ftp-site of SAFER: “Harlequin coliagar.pdf / Oxoid coli agar.pdf / Cromocult agar.pdf / Tergitol agar.pdf”.

Also Colilert Quantitray (IDEXX) method can be used if considered necessaryfor E.coli/coliform counting. The colilert analyses follows the manufacturer´sinstructions. For Colilert, the sample size used is 100 ml. For furtherinstructions see PDF-file: “Colilert.pdf” from the ftp-site of SAFER.


3.5 Dehydrogenase activity with redox stain 5-cyano-2,3-ditolyltetrazolium chloride (CTC) (water/biofilm)

Scope

The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) can be appliedfor direct epifluorescent microscopic counting of dehydrogenase active(metabolically active) bacteria. The oxidized substance 5-cyano-2,3-ditolyltetrazolium chloride is noncolorless and nonfluorescent. It is water soluble andcan pass the cell wall and will be intracellular reduced via electron transportsystem. The reduced molecules will form a water insoluble accumulationintracellularly that means a crystal which is fluorescent (520 nm) afterexcitation with fluorescent light. The counterstaining of the cells with DAPi willshow alll cells in blue and the deghydrogenase active cells in red. Becausecells which are starving do not have enough enzymatic activity nutrients mightbe added during the incubation time with CTC to activate the cells.

References

Rodriguez GG, Phipps D, Ishiguro K, Ridgway HF (1992), Use of afluorescent 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-ditolyltetrazolium chloride for quantifying planctonic and sessile respiring bacteria indrinking water. Appl. Envir. Microbiology 59: 3850-3857.

Material

0.2 µm black polycarbonate membrane filter and a filter set

Epifluorescent microscope

Nonfluorescent immersion oil

5-cyano-2,3-ditolyl tetrazolium chloride (CTC, Polysciences). Reactantsolution of CTC in tubes can be kept frozen at 4ºC or have to be freshlyprepared.

4´, 6-diamidino-2-phenylindole (DAPI).


9

Procedure for water samples

Add CTC solution to the water sample to a final concentration of 4 mM CTCand 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 avoidanoxic conditions.

Filter the sample after the incubation time through a black polycarbonatemembrane filter (pore size 0.2 µm) and wash slightly with water. Either thefilter is now placed in a box to be air dried or supllememnted with 1 mL ofDAPI (5 µg/mL) to stain all bacteria. DAPI will be filter through after 25minutes. Then the filter is air dried.

The dried filter will be placed with nonfluorescent immersion oil on glassmicroscope slides. The examination is performed at 1000 magnification usingan epifluorescent microscope. Count the red fluorescent cells asdehydrogenase active cells and the blue ones (DAPI) as total cell number.

3.6 Legionella (water/biofilms)

See the original instructions from the PDF-file “ Legionella ISO11731.pdf”from the ftp-site of SAFER.

Modification (UKU [CR6], Finland)

Culture of samples for legionellae is done according to a standard method(ISO 11731, Water quality –detection and enumeration of Legionella, 1998)using GVPC and BCYE media. Nonconcentrated water is directly inoculatedon GVPC medium. One liter water samples are concentrated by membranefiltration (pore size 0.2 �m). The filter is cut into small pieces with sterilescissors and shaken in a mixer for 5 min with 5 mL of the original samplewater and glass beads. After shaking, one portion of sample is furtherconcentrated using centrifugation (6000 g, 10 min). Two decontaminationmethods are applied, i.e. acid wash (pH 2.2, 4 min, and heat pre-treatment at50oC for 30 min. The differently treated portions of samples are inoculated on


10

the media and the plates are incubated at 37oC for 10 days. Suspectedlegionella colonies are identified with growth and latex agglutination tests.

3.7 Isolation and culture of mycobacteria (water /biofilm)

ScopeThis method is applied for the isolation and culture of mycobacteria fromwater and biofilm samples.References

Difco. Dehydrated culture media and reagents for microbiology. Detroit,Michigan, USA. 1984. 10th edition 567-569.

Iivanainen E, Martikainen PJ, Katila M-L. Comparison of somedecontamination methods and growth media for the isolation of mycobacteriafrom northern brook waters. Journal of Applied Microbiology. 1997; 82: 121-127.

Neumann M, Schulze-Röbbecke R, Hagenau C, Behringer K. Comparison ofmethods for isolation of mycobacteria from water. Applied and EnvironmentalMicrobiology 1997; 63:547-552.

Schulze-Röbbecke, R., Janning, B., & Fischeder, R. (1992). Occurrence ofmycobacteria in biofilm samples. Tubercle and Lung Disease, 73, 141-144.

Torkko P, Suutari M, Suomalainen S, Paulin L, Larsson L, M-L Katila. 1998.Separation among species of Mycobacteriaum terrae complex by lipidanalyses: comparison with biochemical tests and 16S rRNA sequencing. JClin Microbiol 36:499-505.

Torkko P., Suomalainen S., Iivanainen E., Suutari M., Tortoli E., Paulin L. &Katila M.-L. Mycobacterium xenopi and related organisms isolated fromstream waters in Finland and the description of Mycobacterium botniense sp.nov. Int. J. Syst. Evol Microbiol. 2000; 50: 282-289.

Water quality - detection and enumeration of Legionella. ISO 11731:1998

Definitions- Mycobacteria are acid-fast, slowly growing bacteria.


11

- Decontamination denotes, in the connection of mycobacterial culture, thechemical destruction of other microbes that a sample may contain.

PrinciplesThe quantitation of mycobacteria from environmental samples is usuallybased on culture. The concentration of mycobacteria in a water sample maybe small. Therefore water samples must be concentrated for culture byfiltration. From biofilm samples mycobacteria are detached into fluid beforeculture.As mycobacteria grow more slowly than most other microbes, the number ofother microbes is usually reduced before culture by chemicaldecontamination. This is possible because, due to their lipid rich surface,mycobacteria are more tolerant to many chemicals than other microbes are.This tolerance, however, is only relative. The injury to mycobacteria causedby chemicals depends on the chemical used, on its concentration, and on thelength of treatment. Therefore it is important to strictly adhere to treatmenttimes and procedures given in the instructions. For the decontamination ofwater and biofilm samples, cetylpyridinium chloride (CPC) method iscommonly applied.Some of the mycobacteria are potential pathogens. Therefore extra care isneeded when handling them. All operations capable of causing aerosolsshould be carried out in a laminar flow cabinet or safety cabinet.

Materials- Apparatus for weighing and filter sterilizing the reagents- Automatic pipettes (250 - 1000 µL and 1 - 5 ml) and sterile disposable

pipette tips- Beaker, e.g. 250 ml- Bunsen burner and tripod- Denatured ethanol (A12t)- Filters (e.g. Ultipor N66, pore size 0.2 µm, diameter 47 mm, Pall, UK)- Refrigerated centrifuge and rotor (e.g. Sorvall RC-5C and SS-34, duPont,

Wilmington, DE, USA)- Scissors- Sterile deionized water- Sterile glass petri dishes- Sterile measuring cylinders- Sterile plastic disposable culture sticks- Sterile screw-capped bottles for reagents- Sterile screw-capped bottles (e.g. 100 ml) with glass beads- Sterile, screw-capped plastic centrifuge tubes (capacity 38 ml)- Suction filtration apparatus- Test tube shaker (e.g. Vortex, Scientific Industries, Bohemia, NY, USA)- Timer


12

- Tweezers- Volumetric flasks, sterile, 250 ml- Waste container

Reagents, media and diluentsCetylpyridinium chloride monohydrateSterile deionized water, bottled in portions of 30 mlSterile deionized water

CPC 0.01%0.026 g of cetylpyridinium chloride monohydrate is weighed into a sterile 250

mLvolumetric flask and sterile deionized water is added to dissolve thesubstance. The flask is filled to the mark with sterile deionized water.The solution is sterilized by filtration (0.2 �m). The solution is kept in ascrew-capped bottle at room temperature.

Mycobacteria 7H11 - medium- 5 mLof glycerol is added to 900 mLof deionized water. 21.0 g of

Mycobacteria 7H11 Agar Medium (Difco) is dissolved into the water-glycerol mixture with heating.

- The medium is sterilized at 121 °C for 15 minutes.- The medium is allowed to cool to 50-55 °C. 100 mLof Middlebrook OADC

enrichment (Difco) is aseptically added to the medium.- The medium is dispensed into petri dishes, or into screw-capped vials. The

vials are allowed to solidify as slants.- The media are stored in a refrigerator, covered with aluminium foil to prevent

deterioration by light, and are used within one month of preparation.- Also egg media with formulations of different pH or carbon source may be

used for the culture, see Iivanainen et al. (1997).

ProcedureConcentration of water samples for the culture of mycobacteria

The funnels and other parts of the suction filtration apparatus in contact withthe sample are rinsed with ethanol, flamed and cooled by rinsing them withsterile deionized water.

A suitable amount (e.g. 6 ml) of sample water is pipetted into a sterile screw-capped bottle with glass beads.


13

The filter (usually Ultipor N66, pore size 0.2 µm, diameter 47 mm, Pall, UK) isdisinfected by slowly immersing it for a few seconds into freshly boileddeionized water.

The filter is placed on the filtration apparatus. The amount of sample to befiltered (e.g. 1000 - 2000 ml) is measured in the funnel with a sterilemeasuring cylinder and sucked through the filter. The funnel is rinsed with asmall amount of sterile deionized water, which is also sucked through thefilter. If the sample is turbid and/or difficult to filter, the amount to be filteredcan be divided onto more than one filter. (See ISO 11731:1998).

The filter is removed from the filtration apparatus with sterile tweezers in asterile glass petri dish. Scissors and tweezers are dipped in ethanol, flamedand allowed to cool. The scissors and tweezers are used to cut the filter intosmall pieces in a sterile screw-capped bottle containing glass beads and thepreviously measured amount of sample water.

The bottle is fixed onto the test tube shaker and shaken with full speed for 5minutes. If more than one filter is used in the filtering of a sample, the shakingtime is extended to 10 minutes.

The concentrated samples are immediately decontaminated, or cultured asnon-decontaminated.

Detachment of the cells from biofilms

From the pieces of biofilm sampler, the accumulated cells will be detachedaccording to the instructions provided separately for each biofilm sampler.The biofilm is suspended into appropiate volume of sterile deionized water.

Decontamination with cetylpyridinium chlorideFive milliliters of the concentrated water sample or biofilm suspension ispipetted to a centrifuge tube.To drinking water samples, 0.5 mL of 0.01 % CPC solution and to biofilmsamples 5 mL of 0.01 % CPC solution is added.The samples are left to stand at room temperature for 5 minutes.The samples are centrifuged for 15 minutes at +4�C (8600 x g).The supernatant is discarded into waste container.


14

30 mL of sterile deionized water is added to each sediment. Each tube ismixed thoroughly and then centrifuged as above.The supernatant is again discarded into waste container.

To each sediment is added e.g. 400 µL of sterile deionized water. Thesediment is mixed thoroughly. When necessary, sediment adhering to tubewall is carefully loosened with a disposable sterile culture stick. Additionaldilutions of 10-1 and 10-2 can be made to sterile deionized water (e.g. frombiofilm samples).

Each sediment is cultured immediately after decontamination

Culture of mycobacteria

50 µL of the sediment is pipetted into a vial or petri dish of medium. Thesample is spread evenly on the surface of medium. Likewise alsoundecontaminated sediment or a dilution of it can be cultured.

The screw-capped vials are closed tightly. Petri dishes are closed withParafilm and packed into plastic bags to prevent drying of the media duringthe lengthy incubation time necessary for the growth of mycobacteria. Vialsare placed horizontally on suitable racks.

Mycobacteria are usually incubated at 30 °C. For the study of potentiallypathogenic, thermotolerant mycobacterial species (e.g. Mycobacteriumavium), also parallel incubation temperatures of 36 °C or 42 °C are used.

Mycobacteria are incubated for at least 8 weeks.

During the incubation time the vials or dishes are examined e.g. weekly duringthe incubation. Each time the number and colony morphology of new coloniesare noted.

A Ziehl-Neelsen -staining is made from each colony with different colonymorphology of each growth medium. Acid-fast colonies are noted aspreliminary mycobacteria. Each colony can also be subcultured on e.g.Mycobacteria 7H11 - medium.


15

Subcultures are incubated at the same temperature as the primary culture.

Strains of preliminary mycobacteria are deep-frozen for a more detailedidentification.

IdentificationWhen necessary, the isolates can be identified by gas liquid chromatographyanalyzes of cellular fatty acids and alcohols (Torkko et al., 1998) andcomplementary tests for biochemical and growth characteristics (Torkko et al.,1998, Torkko et al., 2000). Sequencing for the 16S rDNA can be applied forthe isolates unidentifiable by the methods described above (Torkko et al.,2000).

3.8 Ziehl-Neelsen staining for mycobacteria

Scope

This method is used for the primary identification of mycobacteria fromcolonies of cultured cells. The method can also be applied for directidentification of mycobacteria from samples, eg. smears, sediments or tissuesamples.

References

Bartholomew JW. Stains for microorganisms in smears. In: Clark G ed.Staining procedures. Baltimore: Williams & Wilkins, 1981:375-440.

Definitions

Ziehl-Neelsen staining is used for the primary identification of mycobacteria.Mycobacteria, which are resistant to acid and alcohol, are in this procedureusually stained fuchsian red, other microbes will be stained blue.

The specificity of Ziehl-Neelsen staining for mycobacteria is based on the lipidrich cell wall of mycobacteria, which prevents the leaching of the stain out ofthe cell during the acid-alcohol rinse. In spite of the secondary stain withmethylene blue mycobacteria remain red while other microbes are stainedblue. Bacterial or fungal spores or some Actinobacteria may be partially acid-


16

alcohol resistant and thus remain red. These can, however, usually bedistinguished from mycobacteria by their size and shape.

Some of the mycobacteria are potential pathogens. Therefore extra care isneeded when handling them.

MaterialsA rack for drying microscopic slides, e.g. a metal test tube rackBunsen burner and matchesDeionized water in a rinsing bottleFilter paperFume cupboard or cabinetGlass bottle, e.g. 500 mlLarge funnelMicroscope, e.g. Olympus CH-2Microscopic slidesPlastic freezing boxes, 6 (0.5 l capacity)Protective glovesSterile deionized waterSterile pasteur pipettesSterile toothpics and culture sticksStaining bowls with cradlesTimerTray

ReagentsCarbolic fuchsine- Solution A: 6.0 g fuchsine is dissolved in 200 mLof ethanol. The solution is

kept at 37� C overnight.- Solution B: 90.0 g of solid phenol is dissolved in about 10 mLof warm,

deionized water on a water bath of about 45� C. The solution is rinsedinto a 2000 mLmeasuring flask and filled to the mark with deionizedwater. Caution! Phenol is irritating/toxic, protective gloves and preferablyalso goggles should be used when handling it!

- Solutions A and B are combined together. The reagent is mixed 1 - 2 weeksin advance of use and is kept in a dark glass bottle at room temperature,for max. 2 months.

Acid-alcohol- 60 mLof 37% HCl is mixed with 1940 mLof ethanol. The solution is kept at

room temperature for max. 6 months.

Löffler methylene blue


17

- 3.0 g of methylene blue is dissolved in 300 mLof ethanol. 1.0 g of KOH isdissolved in deionized water in a 1000 mLvolumetric flask and filled tothe mark. The solutions are combined and kept in a dark glass bottle atroom temperature, for max. 3 months.

Control strainse.g.. Mycobacterium intracellulare ATCC 13950 (positive control)Escherichia coli ATCC 25922 (negative control)

ProcedureSample preparation

A portion of cultures to be studied and of control strains are spread in a dropof water on microscopic slides.The slides are allowed to air-dry. Thereafter the slides are fixed by passingthrough a Bunsen flame 6 times. Unfixed slides may be infective, so handlingthem should be avoided, and slides should be fixed as soon as possible.Control slides can be made in advance. They should be stored in a dry anddark place. Positive and negative controls should be included in each batch ofstaining and for each lot of carbolic fuchsine.The slides to be stained (samples and controls) are placed in a staining cradleto the staining bowl.Enough carbolic fuchsine is poured into the bowl to cover the slides. From thispoint on, the procedure is carried out in a fume cupboard and using protectivegloves.The slides are allowed to stain overnight (approx. 16 hours).The slides are rinsed by inserting the staining cradle for a few times in afreezing box containing deionized water.The slides are washed by inserting the staining cradle in acid-alcohol rinse for1 minute.The slides are rinsed by flushing with deionized water from the rinsing bottle.The slides are after-stained by inserting the cradle into a bowl of freshlyfiltered (through filter paper) Löffler methylene blue solution for 2 minutesThe slides are rinsed by immersing the cradle for several times in a vesselcontaining deionized water. This procedure is repeated using another portionof deionized water.The slides are set on a tray covered with filter paper. Another piece of filterpaper is put on top of the slides and pressed lightly to dry the slides.


18

The slides are stored on the tray in room temperature, covered with a freshpiece of filter paper.

Microscopic examinationA drop of immersion oil is placed on the slide. The slides are examined withlight microscope, using oil immersion objective with magnification of 100x.Mycobacteria appear as fuchsian red, often slightly curved rods, whose lengthcan vary from nearly coccoid to long rods. They can also be in formations likea string of pearls.

Expression of resultsThe results are recorded as follows:+ = fuchsian red rods (probable mycobacteria)� = fuchsian red rods (even a few cells) and blue cells (probably badly

stained mycobacteria)- = totally blue microbes of varying formsSpores or some Actinobacteria may also be stained red. If the microbes onthe slide are fuchsian red but not rods, the result is recorded as +, but theform of the cells must be noted.

3.9 Noroviruses (water/biofilms)

ScopeIdentification of noroviruses (common gastroentiritis virus) from water /biofilmsamples. The test is mainly qualitative presence/absence test.References

Kukkula M, Maunula L, Silvennoinen E and v. Bonsdorff C-H (1999). Outbreakof viral gastroenteritis due to drinking water contaminated by Norwalk-likeviruses. J. Infect. Dis. 180:1771-1776.

Maunula L., Piiparinen, H. and C.-H. v. Bonsdorff (1999). Confirmation ofNorwalk-like virus amplicons after RT-PCR by microplate hybridization anddirect sequencing. J. Virol. Methods, 83, 125 - 134.


19

Vesanen M, Piiparinen H, Kallio A and Vaheri A (1996). Detection of herpessimplex virus DNA in cerobrospinal fluid samples using the polymerase chainreaction and microplate hybridization. J. Virol. Methods 59:1-11.

Procedure

Isolation of viruses

Water samples are concentrated before NLV RT-PCR test by filtering watersamples (1 liter) through a positively charged membrane (AMF-Cuno,Zetapor, Meriden). Water sample is prefiltered through a fiberglass filter.Viruses are eluted from the membrane with 50 nM glycine-NaOH, pH 9.5,containing 1% beef extract. Further concentration to 100 �l is achieved byusing a Centricon-100 microconcentrator (Amicon, Beverley, USA).

RNA extraction for RT-PCR

Viral RNA is extracted using phenol containing Tripure reagent (Roche) usingthe following procedure: 100 µL water concentrate is added into tubescontaining 1 mLTripure reagent and the tubes are shaken vigorously. 200 µLof chloroform is added, the shaking is repeated and the samples areincubated for 10 min at room temperature. The water phase is separated after15 min centrifugation at 11 500 x g at 4°C. The samples are precipitated byethanol and Na-acetate, pH 6.5, with glycogen as a carrier.

The RNA is transcribed into cDNA in a separate reaction from PCR usingExpand reverse transcriptase (Roche) at 43°C for 1h. Degenerate NVp110primer at a concentration of 0.5 µM is used for both genogroups in a totalvolume of 20 µL in the presence of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5mM MgCl2, 0.001% gelatin, 10 mM DTT, 875 µM each dNTP, 2U RNAsin

inhibitor/µL (Promega, Madison, WI) and 10U/µL of RT enzyme with 6 µLextracted sample solution. After transcription the total reaction mixture (20 µL)is added to a PCR mixture (final volume of 100 µL) containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.8 mM Mg2Cl, 0.2 µM of both primers and 0.025 U

AmpliTaq Gold polymerase (Perkin Elmer)/µL. The cDNA is amplified in 40cycles of 94°C 1 min, 50°C 1 min 20 sec and 72°C 1 min with preceedingheating step for 10 min at 94°C and a final incubation at 72°C for 15 min.

Agarose gel electrophoresis


20

The PCR products are electrophoresed in minigels consisting of 3% Metaphoragarose (FMC BioProducts, Rockland, ME) in TBE buffer (0.89 M Tris, 0.89boric acid and 0.02 M EDTA, pH 8.0) at 100 V for about 1 h. After stainingwith ethidium bromide, DNA is located using UV light (Maunula et al., 1999).

Microplate hybridization

The probes used in this procedure are aligned sequences of strains. Theprobes are labeled using the 3'-DIG oligonucleotide tailing kit (Roche). Themicroplate hybridization is prepared according to Vesanen et al. (1996) usingstreptavidin-coated white microplates (Labsystems, Helsinki, Finland). 10 µLof PCR sample is added per well in the presence of binding buffer (25 mMTris-HCl, pH 7.5, 125 mM NaCl, 5 mM EDTA, 5x Denhardt's (1x Denhardt's =0.2% bovine serum albumin, BSA, 0.2% Ficoll, 0.2% polyvinylpyrrolidone),0.1% Tween 20, and incubated for 30 min at 22°C by shaking for 650 rpm in amicroplate incubator. The strands of DNA are separated with 100 mM NaOH,300 mM NaCl eluting buffer for 1 min. The wells are washed three times witha microplate washer using washing buffer 1 (25 mM Tris, 125 mM NaCl, 20mM MgCl2, 3% Tween 20). The digoxigenin-labeled probe (each probe used

individually) in a final concentration of 0.25 pmol is added in hybridizationbuffer of 5 x SSC (1 x SSC = 0.15 M NaCl, 0.0015 M sodium citrate), 1 xDenhardt, 0.1 % SDS and incubated for 30 min at 40°C by shaking at 650 rpmin a microplate incubator. The wells are washed six times with washing buffer2 containing 0.05 x SSC and 0.3% Tween 20. Anti-digoxigenin antibodyconjugates with alkaline phosphatase (5 mU/50µL; Roche) in conjugate buffer(25 mM Tris-HCl, pH 7.5, 125 mM NaCl, 20 mM MgCl2, 0.3% Tween 20, 1%

BSA) is added and incubated for 30 min at 22°C, 650 rpm. The wells are thenwashed six times with washing buffer 1. The substrate used, LumiPhos 538(Lumigen, Inc., Southfield, MI), was added at 50µL per well and after 30 minthe RLU values (relative light units) are measured by a luminometric reader(Luminoscan, Labsystems).

The cut-off values for each probe are determined with gel-negative samples(140-164 samples tested in 22 assays) with formula: cut-off value = meanvalue + 2 x standard deviation. The cut-off values are 105, 64 and 100 forprobes G1x, G2a and G2b, respectively. A common value of 100 for all theprobes is used for simplicity.


21

Expression of resultThe test shows the presence/absense of norovirus genome in the testsample. The genotype of the norovirus can be identifyid. The semiquantitativeresult can be obtained using dilution series of the sample.

3.10 Thermophilic campylobacters (water /biofilms)

The method is a modification from ISO/CD 17995. See original instructionsfrom the PDF-file “Campylobacters ISO17995.pdf” from the SAFER ftp-site.

A modification of the thermophilic campylobacter method [CR6]:

1000 mL (or more) of water is filtered through 0,45 µm membrane (eg.HAWG047S1, Millipore Ltd.). The membrane is placed in campylobacterenrichment broth. The broth includes basal broth (e. g. Bolton broth base),selective supplement (e. g. containing cephoperazone, vancomysin, trimetho-prim and amphotericin B/natamysin) and lysed cattle or horse blood.Sensitivity might be improved by adding the selective supplement to the brothonly after 4-8 hours pre-incubation. The membrane is incubated in theenrichment broth microaerobically at 37�1 �C for 44�4 hours. After incubation,10 µL of the broth is plated with a sterile loop on the surface of solid mCCDAmedium and incubated at 41,5�1 �C for 44�4 hours before counting.

3.11 Measurement of adenosine triphosphate (ATP)

ScopeThe survival of a cell necessitates a continual energy input to allow theaccomplishment of all the metabolic activities. One of the energisingmolecules commonly determined is a nucleotide which is a marker of thebacterial biomass : adenosine triphosphate or ATP. By hydrolysis of the 3phosphate bonds, ATP can release some energy, which is directly usable bythe cell. The measurement of ATP is carried out in 4 successive stagesdescribed below :

- pre-concentration of the sample


22

- extraction of the cellular ATP- enzymatic determination by bioluminescence- ATP quantification and expression of results.

The measurement of ATP is carried out with an apparatus and products fromthe LUMAC company. For all the stages, followed protocols are thoseprescribed by this company.

ProcedurePre-concentration of samples to be analysed

The method used is a membrane filtration with a moderate pressure in a viewto limit the bacterial stress. The sample is first filtered on a cellulosicmembrane and then rinsed with an demineralised, sterile and apyrogenicwater. Bacteria retained by the membrane then suffer a reactivation phaseaccording to the LUMAC protocol : input on the membrane of 500 µL ofpeptoned bubble without ATP (LUMACULT, ref. 9233-1) during a contacttime equal to 15 minutes.In our experiments, samples (between 8 and 20 mLin volume) were filteredon a sterile membrane in cellulose acetate with a porosity of 0.45 µm. Thepre-concentration was applied both on the immersion waters in contact withtested materials and on the sonication product of the biofilm present onmaterials.

ATP extractionAmong many extraction products used for the determination of the ATP, we

choose a detergent commercialised by LUMAC : the NRB� for which thechemical composition is always a manufacturing secret.

The membrane is first soaked in 500 µL of LUMACULT. Then 500 µL ofextraction product (NRB) is added and mixed by soft agitation during 30seconds. The determination of the ATP is then carried out with 200 µL of thismixture. A negative check sample is included, replacing the sample by 8 to10 mLof demineralised, sterile and apyrogenic water.

Enzymatic measurement of ATPThe most common quantification method of the bacterial ATP is based on anenzymatic reaction and on bioluminescence detection. The ATP determ-ination method is based on the use of the Luciferase (an enzyme extracted


23

and purified from the glow-worm (Photinus pyralis) and its substrate namedLuciferine.

In presence of Mg++ ions, oxygen and ATP and with the luciferase as acatatyst, the luciferine is oxidized. The reaction is endergonic: a consumptionof ATP molecules occurs with production of photons. Their emission isproportional to the quantity of the consumed ATP: they are quantified by abioluminometer (LUMAC, ref. M2500). In our experiments, the ATPdetermination was implemented with a commercial kit from LUMAC.

The photometer produces some Relative Light Unit (RLU) during theenzymatic reaction. The conversion of these RLU into ATP concentration inthe sample is obtained by proportioned additions of standard ATP.Proportioned additions consist of an initial measurement of ATP on thesample to be analyse. Then a known quantity of ATP is introduced in thesame measurement cell, and a second measurement of ATP is carried out.The bioluminometer contains a data colection program: all the reaction timesare first stored and proportioned additions are allowed.The stages of bacterial ATP quantification:

- concentrated sample on membrane + NRB + LUMACULT,- 100 µL of the enzymatic complex Luciferine – Luciferase (LUMIT PM) are

automatically added by an integrated pump system,- integration of photons produced for 10 seconds,- results of the quantity of ATP in the sample in RLU (RLU1),- addition of 20 µL of standard ATP (LUMAC) with a know concentration,- integration for 10 seconds,- results of the quantity of total ATP in RLU (sample ATP and standard ATP)

in the analysed volume (RLU2).the quantity of ATP present in the sample is calculated as below :

[C (RLU) - B (RLU)]ATP = ---------------------- x [added quantity of standard ATP]

[I (RLU)]where,

B = RLU corresponding to the background noise of the apparatus C-B = RLU corresponding to the quantity of ATP in the sample (first

result = RLU1)


24

K = second result in RLU = RLU2

I= K-(C-B) = RLU corresponding to the added quantity of standardATP

Expression of resultsDepending on quantity of standard ATP

3.12 Extraction of extracellular polymeric substances (EPS)

ScopeThe extracellular polymeric substances (EPS) are mainly composed ofpolysaccharides and proteins and smaller amounts of other substances suchas DNA, lipids and humic substances. In order to quantify the total protein,polysaccharide content as well as other extracellular substances in the EPS,the biofilm has to be submitted to an extraction procedure to separate thepolymeric matrix and the sorbed substances in the EPS from the cells.

ReferencesFrolund, B., Palmgren, R., Keiding, K., Nielsen, P. H. (1996). Extraction ofextracellular polymers from activated sludge using a cation exchange resin.Wat. Res. 30, 1749-1758.Wingender, J., Neu, T. R., Flemming, H. C. (1999). Microbial ExtracellularPolymeric Substances. J. Wingender, T. T. Neu und H. C. Flemming. Berlin,Springer (ISBN 3-540-65720-7).

Material- pH meter- Propeller Stirrer- Na3PO4, NaH2PO4, NaCl, KCl- Dowex resin (50X8, Na+ form, 20-50 mesh, Fluka 44445).

ProcedureTo extract the EPS first prepare the extraction buffer: 2 mM Na3PO4, 4 mMNaH2PO4, 9 mM NaCl, 1 mM KCl, pH = 7. Then wash the Dowex resin(50X8, Na+ form, 20-50 mesh, Fluka 44445) with buffer several times.Dilute the biofilm sample to a content of approximately 8 g/L volatilesuspended solids (estimated by dry weight) and add 70 g Dowex resin per 1 g


25

volatile solids. Stir the suspension with a propeller stirrer at 900 rpm for 2hours at 0ºC (ice-water bath).Centrifuge the liquid suspension for 1 min at 12 000 g, fill the supernatant inanother vial, keep the pellet so that the resin can be regenerated later.Centrifuge the supernatant twice for 15 min at 12 000 g and filtrate thesupernatant through membrane filters (cellulose acetate; 0,2 µm diameter).The pellet can be discharged. The obtained EPS solution can be freeze driedor used directly for further biochemical analyses. The Dowex resin can beregenerated by washing several times with deionized water and finallyregenerated with NaCl (100 g/L) solution in water.

3.13 Proteins (Lowry method) (water /biofilms)

ScopeTotal protein can be quantified using a modified Lowry Kit (SIGMA P-5656) forProtein determination (Peterson's Modification of Micro-Lowry Method) withbovine serum albumin (BSA) as a standard. It is a colorimetric method basedin the Folin reaction. The blue colour appearing in the assay is due to thereaction of protein with copper ion in alkaline solution called Biuret Reaction.The reduction of the phosphomolybdate-phosphotungstic acid in the Folinreagent by the aromatic amino acids in the treated protein is the reasontherefore. This method is useful for proteins that are already in solution or thatare soluble in dilute alkali.ReferenceProtocol of Modified Lowry Kit (Sigma Procedure No. P5656).Lowry, O. H., N. J., Rosenbrough, R. J., Randall (1951): Protein Measurementwith the Folin Phenol Reagent. J. Biol. Chem. 193: 265-275.

MaterialModified Lowry Kit (SIGMA P-5656)Vortex mixerSpectrophotometer.

ProcedureOptical densities are measured at 750 nm and the protein concentration is

determined in relation to bovine serum albumin which is used as a standard.

The calibration is made by solutions of BSA in water with concentrations in

the range from 0 to 60 µg/mL.


26

First prepare standard solutions of BSA in the concentration range of 0 to 60

mg/L. Add 0,5 mL of Lowry reagent to 0,5 ml of each sample which has been

pipetted in test tubes. Stir carefully (avoid foaming) and incubate the mixture

for 20 min (exactly) at room temperature. Then add 0,25 mL of Folin-Ciocalteu

phenol reagent and stir gently. Incubate the mixture for 30 min at room

temperature in the dark. Measure in triplicate the absorbance at 750 nm of

each sample.

3.14 Proteins (Bradford Method)

ScopeThe total solubilized protein content can be determined using the Bio-Rad

Protein Assay, based on the method of Bradford. It involves the addition of an

acidic dye (Coomassie brilliant blue) to protein solution and subsequent

measurement of optical density at 595 nm. Comparing to a standard curve

obtained with bovine serum albumin (BSA), a relative measurement of protein

concentration in samples can be assessed.

ReferenceProtocol of Bio-Rad Protein Assay

Materials- Bio-Rad Protein Assay- Vortex mixer- Incubator- Spectophotometer- Filter Whatman #1

Procedure- Dilute the suspensions of biofilm 2x, 5x and 10x adding filtered deionized

water.

- Add 2,3 mL of 3 M NaOH for each mL of diluted suspensions.

- Hydrolyze the mixture for 1 hour at room temperature.

- Neutralize the mixtures with 3 N HCl.

- Dilute 5x the Coomassie brilliant blue dye and filter through Whatman #1

membrane (this solution is stable for 2 weeks, if kept at room

temperature).


27

- Prepare standard solutions of BSA in the concentration range of 0,2 to 0,9

mg/mL

- Pipet in duplicate 100 �L of each standard protein solution, the sample

suspension and filtered dwater to 10 mL tubes.

- Add 5 mL of diluted dye reagent to each tube and vortex.

- Incubate at room temperature for 5 min.

- Measurethe absorbance at 595 nm.

3.15 Carbohydrates (water /biofilms)

Carbohydrates are determined by the phenol-sulfuric acid assay using

glucose as a standard. Other standards as e.g. ribose could be used but then

the results are not comparable with the results obtained by using the glucose

calibration standard.

ReferenceDubois M., Gilles K. A., Hamilton J. K., Rebers P. A., Smith F. (1956).

Colorimetric method for determination of sugars and related substances, Anal.

Chem., 28: 350-356.

Material- Spectrophotometer- Vortex mixer- Glucose- Phenol (5 % w/v in deionized water)- Sulphuric acid (conc.)- Water bath

ProcedureFirst the solutions for the calibration have to be prepared by dissolving

glucose in deionized water in concentrations from 20 to 200 µg/mL starting

with 0 µg/mL. From each solution 0,5 mL have to be poured in a test tube with

a lid.

Then 0.5 mL of each samples or diluted samples have to be pipetted as well

in test tubes. Then add quickly 0,5 mL of 5 % phenol, mix the solutions with

the Vortex and add 2,5 mL of concentrated sulphuric and vortex again.


28

The mixture has to be incubated for 10 min (exactly) at 30 °C in a water bath,

then for 5 min at room temperature to cool down. Measure in triplicate the

absorbency at 490 nm of each sample.


4 Chemical methods

4.1 Total organic carbon /dissolved organic carbon

a. European Standard CEN 1484 : “Water analysis - Guidelines for thedetermination of total organic carbon (TOC) and dissolved organic carbon(DOC). See PDF-file “TOC DOC CEN1484.pdf” from the SAFER tfp-site.

b. A modification of non-purgeable organic carbon (NPOC) analyses(water) [CR6]

Non-purgeable organic carbon (NPOC) is analysed by a method which is amodification of a CEN 1484 “ Water determination. Guidelines for analyses oftotal organic carbon (TOC) and dissolved organic carbon (DOC)” standard.

Non-purgeable organic carbon (NPOC) is determined from water sampleswhich are acidified (pH 3) with hydrochloric acid and purged with nitrogen gas(10 minutes) before the analyses. The content of organic carbon is analysedby 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 andno 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 %

4.2 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 (CR6 and CR9)


30

ScopeThe AOC bioassay using Pseudomonas fluorescens P-17 and Aquaspirillumsp. NOX involves growth to a maximum density of a small inoculum in a batchculture of pasteurized test water. Pasteurization inactivates native microflora.The test organisms are enumerated by spread plate method for heterotrophicplate counts and the density of viable cells is converted to AOCconcentrations 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 isassumed to be the maximum number of organisms that can be supported bythe nutrients in the sample and the yield on acetate carbon is assumed toequal the yield on naturally occurring AOC (van der Kooij 1982, Kaplan andBott 1989). The underlying assumption of the AOC bioassay is that thebioassay organism(s) represent the physiological capabilities of thedistribution system microflora. In some waters (e.g humic waters) inorganicnutrients regulate bacterial growth (Miettinen et al., 1999). Thus, to ensurethat carbon is limiting bacterial growth, enough of inorganic nutrients areadded in sample of test water.

In theory, concentrations of less than 1 �g C/L can be detected. In practice,organic carbon contamination during glassware preparation and samplehandling imposes a limit of detection of approximately 5 to 10 �g AOC/L. Highconcentration of metals (esp. Al, Cu) is toxic for strain P. fluorescens, whichmakes this procedure unsuitable for waters containing these metals.

References

Kaplan L.A., Bott T.L. 1989. Measurement of assimilable organic carbon inwater distribution systems by a simplified bioassay technique. In Advances inWater Analysis and Treatment, Proc. 16th Annu. AWWA Water QualityTechnology Conf., Nov. 13-17, 1988, St. Louis, Mo., p. 475. American WaterWorks Assoc., Denver, Colo.

Miettinen I.T., Vartiainen T. and Martikainen P.J. 1999. Determination ofassimilable 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 andsubculture of bacteria from potable water. Appl. Environ. Microbiol. 49:1-7.


31

Swanson K.M.J., Busta F.F., Peterson E.H., Johnson M.G. 1992. Countmethods. In Compendium of methods for the microbiological examination offoods. Vanderzant C., Splittstoesser D.F., eds. APHA, Washington, 75-95.

Van der Kooij D., Visser A., Oranje J.P. 1982. Multiplication of fluorescentpseudomonads at low substrate concentrations in tap water. Antonie vanLeeuwenhoek 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

�L, between 200 and 1000 �L and between 1000 and 5000 �L.- Eppendorf vials or glass tubes for dilution series.- Glass test tubes- 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,


32

cap with foil, and heat to 550 �C for 6 h or 250 �C for 8 h. Use same cleaningprocedure for all glassware.

ProcedurePreparation of stock inoculum

Prepare turbid suspension of P. fluorescens P-17 and Aquaspirillum sp. NOXby transferring colonies from R2A agar plates into 2 to 3 mL of filtered (poresize 0.2 �m) and autoclaved sample to obtain a final concentration ofapproximately 108 cfu/ml (e.g. it corresponds to 0.10 absorbance of microbemixture 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 waterthat supports growth of P. fluorescens P-17 can be used. Before inoculation,water is added 1/1000 final dilution of mineral salts solution and finalconcentration of 1000 �g acetate C/L.

Incubate at room temperature (� 25 �C) until the viable cell count reaches thestationary phase. The stationary phase is reached when the viable cell count,measured by colony forming units (spread plate method), reaches itsmaximum value. Store stock cultures not more than 12 months at 5 �C. Afterthe stationary phase is reached, make a viable count of the culture (spreadplate) to determine the appropriate volume of inoculum to be added to eachbioassay vessel.

Preparation of incubation waterCollect 500 mL sample in an organic-free vessel and pour into two parallel100 ml vials. Neutralize samples containing disinfectant residuals with 100 �Lsodium thiosulfate solution added to each vial. Add 100�L of mineral saltssolution to the each vial. Cap vials and pasteurize in 65-70 �C water bath for30 min.

Inoculation and incubationCool, inoculate with stock inoculum of P. fluorescens P-17 and Aquaspirillumsp. NOX (final concentration of bacteria 500-1000/ml) by removing cap andusing a carbon-free pipet. Plastic, sterile tips for continuously adjustablepipets are suitable. Use the following equation to calculate volume ofinoculum:

(bacteria in sample, CFU/mL) x (sample volume, ml)


33

volume of inoculum = ---------------------------------------------- CFU/mL stock inoculum

Inoculated samples are incubated at 15 �C in the dark for 9 days. If highertemperature is used, maximum growth is reached earlier, which shortens theincubation time (see below).

Enumeration of test bacteriaBacterial concentrations are analysed on incubation days 7, 8 and 9. If highertemperature is used, maximum is reached earlier (should be testedbeforehand). Shake vigorously the vials and make dilution series. Mechanicalshaker (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 thenumber of colonies of each strain.P. fluorescens P-17 colonies appear on the plates first; they are 2 to 4 mm indiameter with diffuse yellow pigmentation. Aquaspirillum sp. NOX colonies aresmall (1 to 2 mm diameter) white dots. It may be necessary to count P.fluorescens and Aquaspirillum sp. colonies at different dilutions. Sample vialson three separate days. Count all colonies on selected plates containing 25 to250 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 asubstrate individually for P. fluorescens P-17 and Aquaspirillum sp. NOX. It isacceptable to use the previously derived empirical yield values of 6.9 x 106

CFU P. fluorescens P-17/�g acetate-C and 2.1 x 107 CFU Aquaspirillum sp.NOX/�g acetate-C at 15 �C.Expression of results

Average the viable count results for the average density over 3-day period (ortake a maximum value) and calculate concentration of AOC as the product ofthe of the viable counts and the inverse of the yield:

Result = �g AOC/L = [(P. fluorescens CFU/mL)(1/yield) + (Aquaspirillum sp. NOXCFU/mL)(1/yield)] (1000mL/L)


34

4.3 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.

4.4 Microbially available phosphorus (MAP) (water)

ScopeThe MAP bioassay using Pseudomonas fluorescens P-17 involves growth toa maximum density of a small inoculum in a batch culture of pasteurized testwater. Pasteurization inactivates native microflora. The test organism isenumerated by spread plate method for heterotrophic plate counts and thedensity of viable cells is converted to MAP concentrations by an empiricallyderived yield factor for the growth of Ps. fluorescens P-17 on phosphate-phosphorus as standard. The number of organisms at stationary phase isassumed to be the maximum number of organisms that can be supported bythe nutrients in the sample. The yield on phosphate-phosphorus (PO4-P) isassumed to be equal the yield on naturally occurring MAP. Ps. fluorescens P-17 has phosphatase activity. The underlying assumptions of the MAPbioassay are that the carbon and inorganic nutrients, with the exception ofphosphorus, are present in excess, i.e., that phosphorus is limiting (Lehtola etal., 1999). Concentrations 0.08-10 �g 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 newsensitive bioassay for determination of microbially available phosphorus inwater. Appl. Environ. Microbiol. 65:2032.


35

Reasoner D.J., Geldreich E.E. 1985. A new medium for the enumeration andsubculture of bacteria from potable water. Appl. Environ. Microbiol. 49:1.

Swanson K.M.J., Busta F.F., Peterson E.H., Johnson M.G. 1992. Countmethods. In Compendium of methods for the microbiological examination offoods. 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

�L, between 200 and 1000 �L ,and between 1000 and 5000 �L.- 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 orpasteurize at 60�C for 35 min. Store at 5 �C. Solution may be held for upto 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 gCaCl2.2H2O, 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).

Preparation of incubation vessels:Wash vials with phosphate-free detergent, rinse with hot water, immerse in0.1 N HCl for 2 h, and rinse with deionized water three times, dry, cap withfoil, and sterilize e.g. by heat treatment. Use same cleaning procedure for allglassware.


36

ProcedurePreparation of stock inoculum

Prepare turbid suspension of Ps. fluorescens P-17 by transferring coloniesfrom R2A agar into 2 to 3 mL filtered (0.2 �m), autoclaved sample. Usecolonies not older than one week. The autoclaved media water can be anywater that supports growth of Ps. fluorescens P-17. Before inoculation wateris added of 150 �L/lmineral salts solution, 1000 �g acetate-C/L andpasteurised (30 min, 65�C).

Incubate at room temperature (� 25 �C) until the viable cell count reaches thestationary phase. The stationary phase is reached when the viable cell count,as measured by spread plates, reaches maximum value. Store stock culturesfor not more than 12 months at 5 �C.

Preparation of sample waterCollect water samples in an glass vessels and pour 100 mL into Erlenmeyervessels. Use two parallel vials/vessels for MAP measurement. Neutralizesamples containing disinfectant residuals with 50 �l sodium thiosulfatesolution for 100 ml sample. 100 �L of mineral salts solution and 400 �L ofsodium acetate solution are added to the each vial. Cap vials and pasteurizein 65-70 �C water bath for 30 min.

Inoculation and incubationCooled waters are inoculate with approximately 1000 colony forming units(CFU)/mL (usually 2 drops of stock inoculum by pasteur pipette, concentrationshould be tested beforehand) of Ps. fluorescens P-17. Use the followingequation 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 highertemperature is used, maximum growth is reached earlier, which shortens theincubation time (see below).


37

Enumeration of test bacteriaBacterial concentrations are analysed on incubation days 4-8 (starting from 4th

day and continuing until 8th day). If higher temperature is used, maximum isreached earlier (3-7 days, should be tested beforehand). Shake vigorously thevials and make dilution series. Mechanical shaker (Vortex) may be used toshake the dilutions. Plate at least 3 dilutions (dilutions depend on assumedMAP concentrations) on R2A agar. Incubate plates at room temperature for 3days and score the number of colonies. Count all colonies on selected platescontaining 25 to 250 colonies of each bacterium and compute colonycounts.(Swanson et al., 1992).

Expression of resultsThe maximum microbial growth number (CFU/ml) is converted to phosphorusconcentration by using the yield factor. In determining of the yield factor,maximum growth (cfu) of Ps. fluorescens is related to different concentrationsof Na2HPO4. The yield factor is derived from the slope of the line when cellgrowth 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/�g PO4-P can be used (Lehtola et al., 1999)

The maximum plate counts are transformed with a conversion factor into theamount of phosphorus:

�g MAP/L = (CFU/mL) x (1000 mL/L)

(Measured yield factor or 3.73 x 108 CFU/�g PO4-P*/L)

4.5 Total nitrogen

European Standard: ENV 12260 (Water quality – Determination of boundnitrogen (TNb) – following oxidization of nitrogen oxides).

See the original instructions from the PDF-file “ Nitrogen EN 12260.pdf” from

the ftp-site of SAFER.


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 ofMicrobiology and Chemistry are described in specific Standard OperatingProcedures (SOP). The competence of the personnel is provided by internalaudits 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 basedon the Principles of GLP and the standard EN ISO/IEC 17025. TheDepartment has common Standard Operating Procedures, which areestablish by the Management and Quality Assurance Unit. Internal auditsconcern the common procedures of the Department and the main processesof the laboratories.

The main analytical methods and the test equipment used in the laboratory ofEnvironmental Microbiology are described in specific SOPs (so called SOPMB). The competence of the personnel is provided by continuous appropriateeducation and training.


39


International Depository Authority Microbial Strain Collection of Latvia (MSCL)in practice follows the principles listed in "Guidelines for the Establishmentand 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 inmicrobiological laboratories. CABRI QUALITY GUIDELINES (DemonstrationProject ERBBIO4-CT96-0231, co-funded by grant from DGXII of theCommission of the EU) Part I has been adopted in MSCL and it coversprocedures that as far as possible quarantee:-adherence of CABRI to international European or national regulations aswell 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 andworkshops organized by ECCO( European Culture collection organization) aswell by appropriate education.


6 Biofilm monitoring devices

6.1 Propella (The common biofilm monitoring device for all partners)

PROPELLA� THE DYNAMIC CONCEPT TO STUDY INTERACTIONSBETWEEN 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/orexposed surfaces (corrosion, scaling) is explained by the interaction betweenthe liquid and material phases.


41

It is difficult to predict the intensity of these water-material interactions and inmany cases it is necessary to expose the material to water in order toevaluate the compatibility of the two products.

Contrary to static tests, Propella® was built to simulate in the laboratory apiece of pipe transporting liquid such as potable water, thermal waters, etc.

PRINCIPLES OF PROPELLA®

The Propella® reactor provides an original and efficient solution to undertaketests on a laboratory scale, and in particular:

� to control independently the hydraulic residence time and the speedof water circulation

� to control hydraulic water flow which is indispensable to reproducetransfers between solid and liquid phases

� to study water characteristics and exposed surfaces

Propella® is a perfectly mixed reactor, in which the liquid is pushed by apropulsive propeller through an internal tube (see the illustration below). Theliquid flows along the canalisation section studied, as in a real pipe. It is easyto impose a defined Reynolds number by fixing the circulation rate in the pipe.The hydraulic residence time is inversely proportional to the alimentation flowof 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 bythe rotation speed of the propeller.

According to needs, sampling devices can be put on the studied pipe tomeasure surface properties. These devices can be sampled without emptyingor stopping the reactor.


42

Except for the studied canalisation section, all materials in contact with waterare of inox 316l and Teflon to insure the chemical inertia of the system. Thesematerials can be adapted however to other needs.

The reactor allows, among others :

� to model disinfectant use in drinking water distribution networks, and theinfluence 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, andalso to model bacterial growth with or without disinfectant.

The reactor permits :

� to modify and/or maintain the fluid characteristics by introducing reactiveinto the reactor ;

� to modify and/or maintain a defined agitation characterised by a Reynoldsnumber, and also a direction flow ;

� to quantify microbial deposits on pipewalls and sample a part of thecolonised 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 internalthermoregulation system.


43

CHARACTERISTICS

m o t or

b i ofi l msam p l idev ices

water fort herm oreg u lat i o n

vane

doublei n ternalcy l i ner

sect i o n ofmaterial u n ders t u d y

water s u p p l y ex i t

co u p o

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


44

SETUP EXAMPLES

Figure 2: Photos of Propella

Propella© reactor, dimensions

Internal cylinder : height 460 mm, internal diameter 44,0 mm, externaldiameter 72,5 mm (thickness of material = 14,25 mm)

External cylinder : height 500 mm, external diameter 110,0 mm, internaldiameter 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 orequal detergent, concentration 1%) and then careful brushing by hand

� rinsing thoroughly with tap water


45

� 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...

Sampling of biofilm from a PropellaProcedure is described in a separate file on the ftp-site of SAFER“Propella_sampling.doc (3.6 MB)

Biofilm removal protocolPVC coupons that are colonized with biofilms are taken from the samplingdevices without discontinuing the water flow within the distribution system.They are then placed in sterile flasks containing 25 ml of bacterial cell-freedistilled water. Less than 30 minutes later, the biofilm is dispersed by a gentlesonication (2 min. ultrasounds at 2 W, 20 KHz; Bioblock Scientific Vibra cell,model 72401; probe model 72403, Bioblock, USA, diameter 3mm). The probeis placed 1 cm above coupon, inside bacterial cell-free distilled water. Thebacterial content of the resulting suspensions must be analysed within onehour.


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 flaskcontaining 25 ml of bacterial cell-free distilled water. If the count is less than1% of bacteria compared with the first sonication, you can estimate onesonication is sufficient. Otherwise do two/three successive sonications.


47

3.2 Rotating Annular Reactor (modified RotoTorqueTM) [CR4]

ScopeThe RotoTorqueTM (Rotating Annular Reactor) (Characklis, 1990) is a usefuldevice for biofilm growth under defined conditions. It was modified by Griebeand Flemming (1996) and later again by Schulte and Wingender (2000). TheRotating Annular Reactor can be applied in research studies, in the testing ofmaterials and in in situ measurements in water plants and distributionnetworks.

References

More detailed information about the reactor and its applications is described inGriebe and Flemming (2000):

Griebe, T., Flemming, H.-C. 2000 . Rotating annular reactors forcontrolled growth of biofilms. In: Flemming, H.-C., Szewzyk, U., Griebe, T.(Eds.), Biofilms, Lancaster, Pennsylvania, Techonomic Publishing Company,pp. 23-40.

MaterialsThe following list summarises experimental variables that are important in theselection, design and construction of all biofilm devices when differentresearch questions will be addressed. The variations of the Annular Reactorare described in brackets.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


48

� Biological parameters:� Microorganism type (algae, protozoa, bacteria, viruses, etc.)� Defined or undefined culture� Mixed or pure culture

Figure 4: Scheme of the Rotating Annular Reactor after Griebe andFlemming (1996). In the figure there are shown two focus levels. Thewater containing inner space is marked light blue

Influent

Engine

extractabletest-surfaces(Coupons)

rotatinginner cylinder

outlet

withrecirculationtubes

Screw for the extraction ofof coupons

outer cylinder

with uptake

for the


Table 1: Dimensions of the Rotating Annular Reactor

Inner cylinder: unitHeight cm 18,0Bore cm 10,2Exposed area (vertical) cm² 576,8Exposed area (horizontal) cm² 157,1Total area cm² 733,9Outer cylinder:Height cm 20,6Bore cm 11,30Exposed area (vertical) cm² 731,1Exposed area (horizontal) cm² 100,3Total area cm² 831,4Coupons:Number 12Width 1,5Length cm 22,0Exposed length cm 20,1Exposed area cm² 30,9Total area (x 12) cm² 370,8Percentage on the total area of the outer cylinder % 44,6Recirculation tubes:Number 4Angle ° 80Length cm 18,5Bore cm 1,0Exposed area cm² 231,47Total Rotating Annular Reactor:Volume mL ca. 650

dependent on therotation speed

Exposed total area (without recirculation tubes) cm² 1565,3Percentage of the coupons on the total area % 23,68Specific area cm²/cm³ 2,76


Table 2: Measurements of the middle rotating velocities and Reynolds-numbers in the Rotating Annular Reactor with different revolutions perminute

Revolutionsper min

ri calculated Horizontal velocity Vertical velocity Total velocity. Re

[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 2226600 1,60 1,29 0,08 1,29 6439

ri: rotating velocity of the inner cylinder

Re Reynolds number

Re = 11,846 x revolutions/min

0

1000

2000

3000

4000

5000

6000

7000

0 200 400 600

revolutions/min

Figure 5: Reynolds-number of the annular reactor as a function ofrotating velocity.

Procedure

Sterilisation and operation of the Rotating Annular Reactor Prior to the experiments the reactor system including all tubes is eithersterilised by autoclaving for 20 minutes at 121°C or disinfected by biocides

Reynolds-numbers


51

with 200 mg/L hydrogen peroxide for 2 hours at a rotation speed of the innercylinder of 400 rpm. The sterile Rotating Annular Reactor is then fed with drinking water flowingwith 50 mL/h corresponding to a residence time of 12 hours.

Sampling the biofilm from the test-surfacesPVC coupons that are colonised with biofilms are taken from the samplingdevices without discontinuing the water flow within the distribution system.They are analysed for bacterial density directly by using epifluorescencemicroscopy. 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 bacterialcells must be counted at 1.000 x magnification on the coupon.For indirect enumeration the biofilm bacteria are scraped mechanically with arazor blade and disaggregated on a vortex for 3 minutes. With this bacterialsuspension the total cell number (see chapter total cell number) and theheterotrophic plate count (see chapter enumeration of culturablemicroorganisms) are performed.

Setup examples

Figure 6 : Images of Rotating Annular Reactors (modification of Schulteand Wingender, 2000) under operation conditions


6.2 Biofilm generator [CR5]

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 assemblagesof coupons suspended from rigid titanium wire inserted through siliconerubber bungs in the top ports. The weir system is used to maintain the volumeat the required level. Temperature, oxygen and pH probes are not shown.


6.3 Flow cell reactor [CR7]

The biofilms are formed on several adhesion slides placed within flow cellreactors (which have a semi-circular cross section), where drinking water canflow under different hydrodynamic conditions. The adhesion slides, which canbe made in different sizes and of different materials, are glued to rectangularpieces of PMMA properly fitted in the apertures of the flow cell. Theequipment is connected to a side stream in a drinking water system or asimulated drinking water system, as schematically represented in the Figure8.

Figure 8: Linear flow through reactor for biofilm


6.4 Pipeline biofilm collector [CR 6 and CR9]

Biofilm monitoring system udes in CR9The biofilm monitoring systems consist for Pipeline biofilm collector andPropella and a plug flow reservoir. The system is connected directly to thewater supply system of Riga. Water from the water system water is collectedin the reservoir that ensures constant pressure and flow in the biofilm reactors(Figures 12 and 13).The same device will be used as the reference biofilm samplers in CR6(Finland) and CR9 (Latvia). The differences in the devices/test systemsbetween CR6 and CR9 are described in the text.

Maintenance

Biofilm collectors are 10 cm long PVC pipes, which are connected togetherone after another with ball valves and stainless steel loops. Cleaning of thepipeline system parts follows the Propella cleaning procedure. The pipelinesystem is installed on stainless steel frame. The amount of the PVC pipes inone biofilm collector will vary in different tests: 7 samplings with 3 replicatesequals to 21 subsamples. Water flow through the pipes will be adjusted to 500ml/min (= 0.1 m/s) using flow meters/valves. Before sampling, ball valves atboth sides of a PVC pipe are closed and the pipe samples full of water areremoved and replaced with new pipes. Valves are opened and water flow isconnected back after the sampling.

Biofilm sampling

Part of water (1.5 ml) is removed from the detached pipe and a spoonful ofsterile glass beads are inserted into the pipe. To detach biofilm from the innerside of the pipe, the pipe-valve system containing water and glass is vortexedfor 20 minutes. This water-biofilm sample is combined with the water removedbefore 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 vortexedwater-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


55

water flows first into a small glass vessel, where it is pumped with peristaltic

pump into the device.

The figures presenting the CR6 Pipeline system which is also shown in the

ftp-site of SAFER.

Figure 13. Schematic drawing of biofilm monitoring system in CR9


Figure 14. Photos of (a) biofilm monitoring system with (b) reservoir and(c) inlet and sewerage.

B

A

C


6.5 DTM, FOS, and FluS sensors [CR4[

DTM is an optical measurement system consisting of two measurement cells– pairs of optical windows (Figure 9). One pair is continuously cleaned inorder to prevent biofilm building. The fouling effect on the non-cleaned cellcan be directly measured if the signal of the cell with clean surface issubtracted.

Figure 9: Schematic view of a DTM device

FOS and FluS are both fiber optic based devices. Optical fiber heads areimplemented in the water system. Depending on the data operating softwarebackscattered light or excited fluorescence from the biofilm can be detectedand analysed. A schematic view of a FOS is shown in Figure 10.

Biofilm

Bacteria

IIluminating light

Backscatteredlight

Optical Fiber

Illuminatedbiofilm area

EPS

Substratum

detector

Lightsource

detector

Lightsource

Flow irection


58

Figure 10: Schematic view of a FOS


6.6 Electrochemical, nanovibration, and capacitive monitors (CR 7)

Piezo sensors – monitor

The basic idea behind this monitor is to utilise the vibration produced by apiezoelectric ceramic to generate a short pulse that is "read" by other piezo ina different location. Through mathematic analysis of the obtained signal it willbe possible to evaluate the thickness of the biofilm. This technique utilizes thedirect and converse electromechanical properties of piezoelectric materials,allowing simultaneous actuation and sensing, usually called -smart structures

Until now, the piezoceramics have been successfully used in the diagnosis ofplate metal defects, namely in the aerospace industry, and in soil consistenceanalysis in civil engineering.

The monitor where the piezo sensors are located is a half-tube flow cell (seefigure 11) and the sensors are glued to the flat surface of this flow cell.

Reading Water Piezosensors sensor

Nano-vibration producer

Figure 11: Piezo-sensors monitor


60

Capacitive sensors

Capacitive sensors are analogous, non-contact devices. A capacitive bridge isbuilt from internal capacitance and the capacitances created by the proximityof the object (the biofilm) to be measured. The dielectric is directly related tothe distance/medium in front of the sensor plate; ultra-precise electronicsconvert the capacitance information into a analog signal. Very precise sensorscan be made, up to de resolution of 0.01 nanometer.The monitor is identical to the one containing the piezo-electric sensors (ahalf-tube flow cell) and the capacitive sensor will be inserted on the flatsurface of this flow cell.

Electrochemical devices

The electrochemical device is based on voltammetry with platinum electrodesand can be used very successfully due to its sensitivity and selectivity. Thistechnique is used to detect biofilm formation by water bacteria on the surfaceof large tip platinum electrodes.

The working electrodes (WE) are platinum discs with 1 mm diameter. Theelectrodes are prepared by sealing a platinum wire into a glass tube andpolishing the surface of the cross section with alumina powder, on a polishingcloth; the internal end of the platinum wire is sealed to a copper wire thatprovided the external contact. The reference electrode (RE) is a Metrohmsilver/silver electrode (reference 6.0702.100) and all the data are reportedhere versus this reference. The auxiliary electrode (CE) is a platinum spiral.


61

Figure 12: Electrochemical device for biofilm analyses on platinumelectrodes

The electrochemical experiments are carried out using a potentiostat Autolabtype PGSTAT 20, Ecochemie. The cyclic voltammetry experiments are carriedout in a two-compartment, three-electrode cell at room temperature, asrepresented in the Figure 12.

Each electrode is subjected to the electrochemical treatment by immersion inthe solution of interest and recycling the potential between the appropriatelimits. The voltammograms are recorded using the data acquisition programGPES 4.6.

The technique used is repetitive cyclic voltammetry, where a triangularpotential sweep is continuously applied to the working electrode and thecurrent passing through the cell is recorded as a function of the appliedpotential (Pletcher, 1991). The potential profile is, therefore, a linear functionof time and can be described as Eapp = Ei ± vt, where Eapp is the appliedpotential at a time t, EI is the initial potential and v is the scan rate in V/s. At apre-set value of the scan rate is reversed and the potential is scanned to theinitial value. This cycle can be repeated as many times as required.


62

The application of repetitive cyclic voltammetry is in itself a method ofcleaning platinum electrodes and the appearance of a different voltammogramafter the electrode has been in contact with a foulant is an indication of biofilmformation. This fact constitutes the basis of the detector of biofilm formationdescribed here since the smallest deposit on the electrode surface changesthe pattern observed when the platinum electrode surface is clean.

In drinking water systems, due the high resistivity of the media (low ionicforce), microelectrodes should be used instead. Different electrodeconfigurations can be applied, according to the case under study (asmicroband microelectrodes). However, the most important feature is that,whatever the geometry, they are particularly suited for many analyticaldeterminations, and are largely used to detect biological compounds

6.7 Biofilm formation monitoring using ATR-FTIR sensor [CR2]

Scope

The development of the biofilms is monitored on a zinc selenide crystal, whichis compatible with vibrational spectroscopic measurements, in a continuouslydrinking water fed flow chamber (Figure 14). To improve the sensitivity andthe delay of response, the crystal is colonised by one hour´s exposure to E.cloacae suspension, as a coliform model frequently isolated in non-compliance drinking waters. Absorption bands for proteins and poly-saccharides in the vibrational spectra are chosen as probes, because thesemacromolecules 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 BrukerVector 22 spectrometer equipped with a KBr beam splitter and a DTGS(deuteriated triglycine sulphate) thermal detector. The resolution of the singlebeam spectra is 4 cm-1. The number of bi-directional double-sidedinterferogram scans was 100, which corresponds to a 1 min accumulation.The interferograms were apodised with the Blackman-Harris 3-Term function.No smoothing and no baseline correction were subsequently applied.


63

The ATR flow cell is a SPECAC cell (Eurolabo, ref. 11160) designed toenclose a horizontal trapezoid crystal. The incidence angle of the ATR crystalis 45°, which allows six internal reflections on the upper face in contact withthe sample. The compartment of the spectrometer containing the flow cell iscontinuously purged with dry and decarbonated air provided by a Balstomcompressor for removing water vapour and carbon dioxide. FT-IRmeasurements are made at room air-conditioned temperature (20°C � 2 °C).Irradiance throughout the empty cell is about 11 % of the full signal (withoutthe ATR accessory). ATR spectra are shown with an absorbance scalecorresponding to log(Rreference/Rsample), where R is the internal reflectance ofthe 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 theZnSe crystal. When necessary, the contribution of water vapour due tovariation in relative humidity in the room was eliminated. The correspondingspectrum was obtained separately by calculating the difference betweenspectra of "wet" and dry air. Despite the absorbance scale used the ATRspectra is not strictly proportional to the absorption coefficients as no furthercorrection is applied.

IR sourcedetector

entrance exit

ZnSe crystal

Biofilm

Figure 15: Attenuated Total Reflectance Fourier Transform Infrared(ATR-FTIR) flow through cell

Biofilm study in flow cellThe flow cell (figure 12) was successively and continuously supplied with (i)ethanol (70 % for 3 to 6 hours) to clean the system, (ii) the tested 0.2 µmfiltered water (18 to 24 hours) to remove the ethanol (control by recordingspectra) and to measure any deposits of organic molecules from the watersample, (iii) the bacterial suspension (1 hour) which allowed bacteria to


64

adhere to the surface of the crystal, (iv) the tested 0.2 µm filtered water for 15days. Ethanol, tested waters and bacterial suspension were passed throughthe ATR flow cell by using an up-stream Gilson Minipuls 3 pump with a flow of30 mL h-1 (hydraulic residence time approximately 4 min). The infraredspectra of the hydrated dynamic biofilm were automatically collected every 15minutes for the first hour and then every two hours.

Coliform bacteria suspensionEnterobacter cloacae strain was isolated from a hydrous medium. The strain,conserved at – 80°C, was placed at 37°C � 1°C for 24 hours beforeinoculation on CASO agar and incubated 24 hours at 37°C � 1°C. Asuspension of OD620 nm from 0.2 to 0.4 (about 1 x 108 cells per mL) wasprepared in sterile glass flasks. Bacteria were recovered by centrifugation(10000 g; 10 min; 20°C). The supernatant was removed and then the pelletwas resuspended in 0.2 µm filtered (Millipore, ref. 106329) tested water andwashed by centrifugation (10000 g; 10 min; 20°C) to eliminate the residualorganic carbon from the nutritive medium. The pellet was homogenised insterile drinking water (autoclaved, 121°C; 15 min), and then the finalsuspension, whose concentration was about 1 x 108 cells per mL, was storedat 4 to 10°C for up to two hours.

7 APPENDIX: Calibration of the sonication probe

Date post:	30-Oct-2014
Category:	Documents
Upload:	kareem-ismail
View:	46 times
Download:	9 times

Handbook for Analytical Methods And

Documents