Handbook For Analytical Methods and

SAFER
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
(Version 1.0)
WP2. Tools for biofilm monitoring
Prepared by: Ilkka Miettinen National Public Health Institute Department of Environmental Health Kuopio, Finland Gabriela Schaule IWW Rhenish-Westfalian Institute for Water Department of Applied Microbiology Mulheim, Germany
DATE: 28th May, 2003
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
Content
1 INTRODUCTION 2 STRUCTURE 3 MICROBIOLOGICAL METHODS
4 4 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-2,3DITOLYL 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 4.1 TOTAL ORGANIC CARBON /DISSOLVED ORGANIC CARBON .............29
29
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 5.1 QUALITY CONTROL AND QUALITY ASSURANCE IN CR4 .................38 5.2 QUALITY CONTROL AND QUALITY ASSURANCE IN CR6 .................38 5.3 QUALITY CONTROL AND QUALITY ASSURANCE IN CR9 .................39 6 BIOFILM MONITORING DEVICES 40 38
6.1 PROPELLA (THE COMMON BIOFILM MONITORING DEVICE FOR ALL PARTNERS) ....................................................................................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 .......................................................................59 Capacitive sensors ................................................................................60 Electrochemical devices .......................................................................60
6.7 BIOFILM FORMATION MONITORING USING ATR-FTIR SENSOR [CR2] ...........................................................................................62 7 APPENDIX: CALIBRATION OF THE SONICATION PROBE 64
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 buildup, 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.
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 1. References 2. Definitions 3. Materials 4. Procedure 5. Expression of results
3 3.1
Microbiological methods 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 the original instructions from the PDF-file HPC ISO6222.pdf from the ftp-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 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 2C 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 low for spread plate. The membranes (diameter 47 mm) have a pore size of 0.2 m. The sample (more than 1 mL) will be filtered and the filter placed on the agar plate. Air bubbles under the filter should be avoided. Expression of results Results are expressed as the mean number of bacterial CFUs per mL of water sample
3.3
Total cell number (water /biofilm)
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-2phenylindole (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 but the disadvantage is the staining of noncellulare material. The total cell number of biofilms fixed to the surface will evaluated by the Confocal Laser Scanning Microscope after staining or if the biofilm is thin with the epiflourescence microscope like the filtered water sample. 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 m), non-fluorescent immersion oil. All reagents should be filtered through a cellulose nitrate filter with the pore size of 0.2 m. Procedure Place one black polycarbonate membrane filter (0.2 m 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 g/mL) and add 1 mL Triton X-100 (0.1 %).
The final
concentration should be 5 g/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 glas coverslip. The enumeration of bacteria and other microorganisms 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.
3.4
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, 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 manufacturers instructions. For Colilert, the sample size used is 100 ml. For further instructions see PDF-file: Colilert.pdf from the ftp-site of SAFER.
3.5
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. The oxidized substance 5-cyano-2,3-ditolyl tetrazolium chloride is noncolorless and nonfluorescent. It is water soluble and can pass the cell wall and will be intracellular reduced via electron transport system. The reduced molecules will form a water insoluble accumulation intracellularly that means a crystal which is fluorescent (520 nm) after excitation with fluorescent light. The counterstaining of the cells with DAPi will show alll cells in blue and the deghydrogenase active cells in red. Because cells which are starving do not have enough enzymatic activity 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 0.2 m black polycarbonate membrane filter and a filter set Epifluorescent microscope Nonfluorescent immersion oil 5-cyano-2,3-ditolyl tetrazolium chloride (CTC, Polysciences). Reactant solution of CTC in tubes can be kept frozen at 4C 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 28C. 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 m) 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 g/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.
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 inoculated on GVPC medium. One liter water samples are concentrated by membrane filtration (pore size 0.2 mm). The filter is cut into small pieces with sterile scissors and shaken in a mixer for 5 min with 5 mL of the original sample water and glass beads. After shaking, one portion of sample is further concentrated using centrifugation (6000 g, 10 min). Two decontamination methods are applied, i.e. acid wash (pH 2.2, 4 min, and heat pre-treatment at 50oC for 30 min. The differently treated portions of samples are inoculated on
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the media and the plates are incubated at 37oC for 10 days. Suspected legionella colonies are identified with growth and latex agglutination tests.
3.7
Isolation and culture of mycobacteria (water /biofilm)
Scope This method is applied for the isolation and culture of mycobacteria from water 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 some
decontamination methods and growth media for the isolation of mycobacteria from northern brook waters. Journal of Applied Microbiology. 1997; 82: 121127. Neumann M, Schulze-Rbbecke R, Hagenau C, Behringer K. Comparison of methods for isolation of mycobacteria from water. Applied and Environmental Microbiology 1997; 63:547-552. Schulze-Rbbecke, R., Janning, B., & Fischeder, R. (1992). Occurrence of mycobacteria 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 lipid analyses: comparison with biochemical tests and 16S rRNA sequencing. J Clin 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 from stream 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.
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- Decontamination denotes, in the connection of mycobacterial culture, the chemical destruction of other microbes that a sample may contain. Principles The quantitation of mycobacteria from environmental samples is usually based on culture. The concentration of mycobacteria in a water sample may be small. Therefore water samples must be concentrated for culture by filtration. From biofilm samples mycobacteria are detached into fluid before culture. As mycobacteria grow more slowly than most other microbes, the number of other microbes is usually reduced before culture by chemical decontamination. 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 caused by chemicals depends on the chemical used, on its concentration, and on the length of treatment. Therefore it is important to strictly adhere to treatment times and procedures given in the instructions. For the decontamination of water and biofilm samples, cetylpyridinium chloride (CPC) method is commonly applied. Some of the mycobacteria are potential pathogens. Therefore extra care is needed when handling them. All operations capable of causing aerosols should be carried out in a laminar flow cabinet or safety cabinet. Materials - Apparatus for weighing and filter sterilizing the reagents - Automatic pipettes (250 - 1000 L 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 m, 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
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- Tweezers - Volumetric flasks, sterile, 250 ml - Waste container Reagents, media and diluents Cetylpyridinium chloride monohydrate Sterile deionized water, bottled in portions of 30 ml Sterile 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 the substance. The flask is filled to the mark with sterile deionized water. The solution is sterilized by filtration (0.2 mm). The solution is kept in a screw-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 waterglycerol 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). Procedure Concentration of water samples for the culture of mycobacteria The funnels and other parts of the suction filtration apparatus in contact with the sample are rinsed with ethanol, flamed and cooled by rinsing them with sterile deionized water. A suitable amount (e.g. 6 ml) of sample water is pipetted into a sterile screwcapped bottle with glass beads.
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The filter (usually Ultipor N66, pore size 0.2 m, diameter 47 mm, Pall, UK) is disinfected by slowly immersing it for a few seconds into freshly boiled deionized water. The filter is placed on the filtration apparatus. The amount of sample to be filtered (e.g. 1000 - 2000 ml) is measured in the funnel with a sterile measuring cylinder and sucked through the filter. The funnel is rinsed with a small amount of sterile deionized water, which is also sucked through the filter. If the sample is turbid and/or difficult to filter, the amount to be filtered can be divided onto more than one filter. (See ISO 11731:1998). The filter is removed from the filtration apparatus with sterile tweezers in a sterile glass petri dish. Scissors and tweezers are dipped in ethanol, flamed and allowed to cool. The scissors and tweezers are used to cut the filter into small pieces in a sterile screw-capped bottle containing glass beads and the previously measured amount of sample water. The bottle is fixed onto the test tube shaker and shaken with full speed for 5 minutes. If more than one filter is used in the filtering of a sample, the shaking time is extended to 10 minutes. The concentrated samples are immediately decontaminated, or cultured as non-decontaminated. Detachment of the cells from biofilms From the pieces of biofilm sampler, the accumulated cells will be detached according to the instructions provided separately for each biofilm sampler. The biofilm is suspended into appropiate volume of sterile deionized water. Decontamination with cetylpyridinium chloride Five milliliters of the concentrated water sample or biofilm suspension is pipetted to a centrifuge tube. To drinking water samples, 0.5 mL of 0.01 % CPC solution and to biofilm samples 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 +4C (8600 x g). The supernatant is discarded into waste container.
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30 mL of sterile deionized water is added to each sediment. Each tube is mixed thoroughly and then centrifuged as above. The supernatant is again discarded into waste container. To each sediment is added e.g. 400 L of sterile deionized water. The sediment is mixed thoroughly. When necessary, sediment adhering to tube wall is carefully loosened with a disposable sterile culture stick. Additional dilutions of 10-1 and 10-2 can be made to sterile deionized water (e.g. from biofilm samples). Each sediment is cultured immediately after decontamination Culture of mycobacteria 50 L of the sediment is pipetted into a vial or petri dish of medium. The sample is spread evenly on the surface of medium. Likewise also undecontaminated sediment or a dilution of it can be cultured. The screw-capped vials are closed tightly. Petri dishes are closed with Parafilm and packed into plastic bags to prevent drying of the media during the lengthy incubation time necessary for the growth of mycobacteria. Vials are placed horizontally on suitable racks. Mycobacteria are usually incubated at 30 C. For the study of potentially pathogenic, thermotolerant mycobacterial species (e.g. Mycobacterium avium), 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 during the incubation. Each time the number and colony morphology of new colonies are noted. A Ziehl-Neelsen -staining is made from each colony with different colony morphology of each growth medium. Acid-fast colonies are noted as preliminary mycobacteria. Each colony can also be subcultured on e.g. Mycobacteria 7H11 - medium.
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Subcultures are incubated at the same temperature as the primary culture. Strains of preliminary mycobacteria are deep-frozen for a more detailed identification. Identification When necessary, the isolates can be identified by gas liquid chromatography analyzes of cellular fatty acids and alcohols (Torkko et al., 1998) and complementary tests for biochemical and growth characteristics (Torkko et al., 1998, Torkko et al., 2000). Sequencing for the 16S rDNA can be applied for the 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 from colonies of cultured cells. The method can also be applied for direct identification of mycobacteria from samples, eg. smears, sediments or tissue samples. 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 procedure usually stained fuchsian red, other microbes will be stained blue. The specificity of Ziehl-Neelsen staining for mycobacteria is based on the lipid rich cell wall of mycobacteria, which prevents the leaching of the stain out of the cell during the acid-alcohol rinse. In spite of the secondary stain with methylene blue mycobacteria remain red while other microbes are stained blue. Bacterial or fungal spores or some Actinobacteria may be partially acidEVK1-CT-2002-00108 Surveillance and control of microbiological stability in drinking water distribution networks Handbook for analytical methods and operational criteria for biofilm reactors
16
alcohol resistant and thus remain red. These can, however, usually be distinguished from mycobacteria by their size and shape. Some of the mycobacteria are potential pathogens. Therefore extra care is needed when handling them. Materials A rack for drying microscopic slides, e.g. a metal test tube rack Bunsen burner and matches Deionized water in a rinsing bottle Filter paper Fume cupboard or cabinet Glass bottle, e.g. 500 ml Large funnel Microscope, e.g. Olympus CH-2 Microscopic slides Plastic freezing boxes, 6 (0.5 l capacity) Protective gloves Sterile deionized water Sterile pasteur pipettes Sterile toothpics and culture sticks Staining bowls with cradles Timer Tray Reagents Carbolic 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 rinsed into a 2000 mLmeasuring flask and filled to the mark with deionized water. Caution! Phenol is irritating/toxic, protective gloves and preferably also goggles should be used when handling it! - Solutions A and B are combined together. The reagent is mixed 1 - 2 weeks in 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. Lffler methylene blue
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- 3.0 g of methylene blue is dissolved in 300 mLof ethanol. 1.0 g of KOH is dissolved in deionized water in a 1000 mLvolumetric flask and filled to the mark. The solutions are combined and kept in a dark glass bottle at room temperature, for max. 3 months. Control strains e.g.. Mycobacterium intracellulare ATCC 13950 (positive control) Escherichia coli ATCC 25922 (negative control) Procedure Sample preparation A portion of cultures to be studied and of control strains are spread in a drop of water on microscopic slides. The slides are allowed to air-dry. Thereafter the slides are fixed by passing through a Bunsen flame 6 times. Unfixed slides may be infective, so handling them 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 and dark place. Positive and negative controls should be included in each batch of staining and for each lot of carbolic fuchsine. The slides to be stained (samples and controls) are placed in a staining cradle to the staining bowl. Enough carbolic fuchsine is poured into the bowl to cover the slides. From this point on, the procedure is carried out in a fume cupboard and using protective gloves. The slides are allowed to stain overnight (approx. 16 hours). The slides are rinsed by inserting the staining cradle for a few times in a freezing box containing deionized water. The slides are washed by inserting the staining cradle in acid-alcohol rinse for 1 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 freshly filtered (through filter paper) Lffler methylene blue solution for 2 minutes The slides are rinsed by immersing the cradle for several times in a vessel containing deionized water. This procedure is repeated using another portion of deionized water. The slides are set on a tray covered with filter paper. Another piece of filter paper is put on top of the slides and pressed lightly to dry the slides.
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The slides are stored on the tray in room temperature, covered with a fresh piece of filter paper. Microscopic examination A drop of immersion oil is placed on the slide. The slides are examined with light microscope, using oil immersion objective with magnification of 100x. Mycobacteria appear as fuchsian red, often slightly curved rods, whose length can vary from nearly coccoid to long rods. They can also be in formations like a string of pearls. Expression of results The 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 forms Spores or some Actinobacteria may also be stained red. If the microbes on the slide are fuchsian red but not rods, the result is recorded as +, but the form of the cells must be noted.
3.9
Noroviruses (water/biofilms)
Scope Identification of noroviruses (common gastroentiritis virus) from water /biofilm samples. The test is mainly qualitative presence/absence test. References Kukkula M, Maunula L, Silvennoinen E and v. Bonsdorff C-H (1999). Outbreak of viral gastroenteritis due to drinking water contaminated by Norwalk-like viruses. J. Infect. Dis. 180:1771-1776. Maunula L., Piiparinen, H. and C.-H. v. Bonsdorff (1999). Confirmation of Norwalk-like virus amplicons after RT-PCR by microplate hybridization and direct sequencing. J. Virol. Methods, 83, 125 - 134.
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Vesanen M, Piiparinen H, Kallio A and Vaheri A (1996). Detection of herpes simplex virus DNA in cerobrospinal fluid samples using the polymerase chain reaction and microplate hybridization. J. Virol. Methods 59:1-11.
Procedure Isolation of viruses Water samples are concentrated before NLV RT-PCR test by filtering water samples (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 ml is achieved by using a Centricon-100 microconcentrator (Amicon, Beverley, USA). RNA extraction for RT-PCR Viral RNA is extracted using phenol containing Tripure reagent (Roche) using the following procedure: 100 L water concentrate is added into tubes containing 1 mLTripure reagent and the tubes are shaken vigorously. 200 L of chloroform is added, the shaking is repeated and the samples are incubated for 10 min at room temperature. The water phase is separated after 15 min centrifugation at 11 500 x g at 4C. The samples are precipitated by ethanol and Na-acetate, pH 6.5, with glycogen as a carrier. The RNA is transcribed into cDNA in a separate reaction from PCR using Expand reverse transcriptase (Roche) at 43C for 1h. Degenerate NVp110 primer at a concentration of 0.5 M is used for both genogroups in a total volume of 20 L in the presence of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 10 mM DTT, 875 M each dNTP, 2U RNAsin inhibitor/L (Promega, Madison, WI) and 10U/L of RT enzyme with 6 L extracted sample solution. After transcription the total reaction mixture (20 L) is added to a PCR mixture (final volume of 100 L) containing 10 mM TrisHCl, pH 8.3, 50 mM KCl, 2.8 mM Mg2Cl, 0.2 M of both primers and 0.025 U AmpliTaq Gold polymerase (Perkin Elmer)/L. The cDNA is amplified in 40 cycles of 94C 1 min, 50C 1 min 20 sec and 72C 1 min with preceeding heating step for 10 min at 94C and a final incubation at 72C for 15 min. Agarose gel electrophoresis
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The PCR products are electrophoresed in minigels consisting of 3% Metaphor agarose (FMC BioProducts, Rockland, ME) in TBE buffer (0.89 M Tris, 0.89 boric acid and 0.02 M EDTA, pH 8.0) at 100 V for about 1 h. After staining with 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. The probes are labeled using the 3'-DIG oligonucleotide tailing kit (Roche). The microplate hybridization is prepared according to Vesanen et al. (1996) using streptavidin-coated white microplates (Labsystems, Helsinki, Finland). 10 L of PCR sample is added per well in the presence of binding buffer (25 mM Tris-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 22C by shaking for 650 rpm in a microplate 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 with a microplate washer using washing buffer 1 (25 mM Tris, 125 mM NaCl, 20 mM MgCl2, 3% Tween 20). The digoxigenin-labeled probe (each probe used individually) in a final concentration of 0.25 pmol is added in hybridization buffer of 5 x SSC (1 x SSC = 0.15 M NaCl, 0.0015 M sodium citrate), 1 x Denhardt, 0.1 % SDS and incubated for 30 min at 40C by shaking at 650 rpm in a microplate incubator. The wells are washed six times with washing buffer 2 containing 0.05 x SSC and 0.3% Tween 20. Anti-digoxigenin antibody conjugates with alkaline phosphatase (5 mU/50L; 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 22C, 650 rpm. The wells are then washed six times with washing buffer 1. The substrate used, LumiPhos 538 (Lumigen, Inc., Southfield, MI), was added at 50L per well and after 30 min the 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 = mean value + 2 x standard deviation. The cut-off values are 105, 64 and 100 for probes G1x, G2a and G2b, respectively. A common value of 100 for all the probes is used for simplicity.
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Expression of result The test shows the presence/absense of norovirus genome in the test sample. The genotype of the norovirus can be identifyid. The semiquantitative result 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 instructions from 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 m membrane (eg. HAWG047S1, Millipore Ltd.). The membrane is placed in campylobacter enrichment broth. The broth includes basal broth (e. g. Bolton broth base), selective supplement (e. g. containing cephoperazone, vancomysin, trimethoprim and amphotericin B/natamysin) and lysed cattle or horse blood. Sensitivity might be improved by adding the selective supplement to the broth only after 4-8 hours pre-incubation. The membrane is incubated in the enrichment broth microaerobically at 371 C for 444 hours. After incubation, 10 L of the broth is plated with a sterile loop on the surface of solid mCCDA medium and incubated at 41,51 C for 444 hours before counting.
3.11 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 or 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 : pre-concentration of the sample
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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 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 L 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 m. 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 L of LUMACULT. Then 500 L 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 L 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 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
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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. The stages of bacterial ATP quantification: - concentrated sample on membrane + NRB + LUMACULT, - 100 L 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 L 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 = 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)
---------------------- x [added quantity of standard ATP] [I (RLU)]
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K = second result in RLU = RLU2 I= K-(C-B) = RLU corresponding to the added quantity of standard ATP Expression of results Depending on quantity of standard ATP
3.12 Extraction of extracellular polymeric substances (EPS) Scope The extracellular polymeric substances (EPS) are mainly composed of polysaccharides and proteins and smaller amounts of other substances such as 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 the polymeric matrix and the sorbed substances in the EPS from the cells. References Frolund, B., Palmgren, R., Keiding, K., Nielsen, P. H. (1996). Extraction of extracellular polymers from activated sludge using a cation exchange resin. Wat. Res. 30, 1749-1758. Wingender, J., Neu, T. R., Flemming, H. C. (1999). Microbial Extracellular Polymeric 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).
Procedure To extract the EPS first prepare the extraction buffer: 2 mM Na3PO4, 4 mM NaH2PO4, 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 volatile suspended solids (estimated by dry weight) and add 70 g Dowex resin per 1 g
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volatile solids. Stir the suspension with a propeller stirrer at 900 rpm for 2 hours at 0C (ice-water bath). Centrifuge the liquid suspension for 1 min at 12 000 g, fill the supernatant in another 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 the supernatant through membrane filters (cellulose acetate; 0,2 m diameter). The pellet can be discharged. The obtained EPS solution can be freeze dried or used directly for further biochemical analyses. The Dowex resin can be regenerated by washing several times with deionized water and finally regenerated with NaCl (100 g/L) solution in water.
3.13 Proteins (Lowry method) (water /biofilms) Scope Total protein can be quantified using a modified Lowry Kit (SIGMA P-5656) for Protein determination (Peterson's Modification of Micro-Lowry Method) with bovine serum albumin (BSA) as a standard. It is a colorimetric method based in the Folin reaction. The blue colour appearing in the assay is due to the reaction of protein with copper ion in alkaline solution called Biuret Reaction. The reduction of the phosphomolybdate-phosphotungstic acid in the Folin reagent by the aromatic amino acids in the treated protein is the reason therefore. This method is useful for proteins that are already in solution or that are soluble in dilute alkali. Reference Protocol of Modified Lowry Kit (Sigma Procedure No. P5656). Lowry, O. H., N. J., Rosenbrough, R. J., Randall (1951): Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem. 193: 265-275. Material Modified Lowry Kit (SIGMA P-5656) Vortex mixer Spectrophotometer. Procedure Optical 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 g/mL.
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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) Scope The 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.
Reference Protocol 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).
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- Prepare standard solutions of BSA in the concentration range of 0,2 to 0,9 mg/mL - Pipet in duplicate 100 mL 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. Reference Dubois 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
Procedure First the solutions for the calibration have to be prepared by dissolving glucose in deionized water in concentrations from 20 to 200 g/mL starting with 0 g/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.
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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 4.1
Chemical methods Total organic carbon /dissolved organic carbon
a. 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.
b. A modification of non-purgeable organic carbon (NPOC) analyses (water) [CR6]
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 m 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 %
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)

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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 oxalatecarbon 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 mg 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 mg 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, Proc. 16th 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.
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Swanson K.M.J., Busta F.F., Peterson E.H., Johnson M.G. 1992. Count methods. In 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 mL, between 200 and 1000 mL and between 1000 and 5000 mL. - 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,
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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 mm) 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, 65C) 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 mg 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 mL sodium thiosulfate solution added to each vial. Add 100mL 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. Use the following equation to calculate volume of inoculum: (bacteria in sample, CFU/mL) x (sample volume, ml)
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volume of inoculum = ---------------------------------------------CFU/mL stock inoculum 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/mg acetate-C and 2.1 x 107 CFU Aquaspirillum sp. NOX/mg acetate-C at 15 C. Expression of results Average the viable count results for the average density over 3-day period (or take a maximum value) and calculate concentration of AOC as the product of the of the viable counts and the inverse of the yield:
Result = mg AOC/L = [(P. fluorescens CFU/mL)(1/yield) + (Aquaspirillum sp. NOX CFU/mL)(1/yield)] (1000mL/L)
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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)
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 phosphatephosphorus 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 P17 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 mg 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.
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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. In 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 mL, between 200 and 1000 mL ,and between 1000 and 5000 mL. - 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 60C 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). 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.
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Procedure Preparation of stock inoculum Prepare turbid suspension of Ps. fluorescens P-17 by transferring colonies from R2A agar into 2 to 3 mL filtered (0.2 mm), 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 mL/lmineral salts solution, 1000 mg acetate-C/L and pasteurised (30 min, 65C). 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 ml sodium thiosulfate solution for 100 ml sample. 100 mL of mineral salts solution and 400 mL 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).
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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 dilution series. Mechanical shaker (Vortex) may be used to shake the dilutions. Plate at least 3 dilutions (dilutions depend on assumed MAP concentrations) on R2A agar. Incubate plates at room temperature for 3 days and score the number of colonies. Count all colonies on selected plates containing 25 to 250 colonies of each bacterium and compute colony counts.(Swanson et al., 1992). Expression of results The maximum microbial growth number (CFU/ml) is converted to phosphorus concentration by using the yield factor. In determining of the yield factor, maximum growth (cfu) of Ps. fluorescens is related to different concentrations of Na2HPO4. The yield factor is derived from the slope of the line when cell growth is plotted against PO4-P concentration (Lehtola et al., 1999). Also, previously derived empirical yield value of 3.73 x 108 CFU Ps. fluorescens P17/mg PO4-P can be used (Lehtola et al., 1999) The maximum plate counts are transformed with a conversion factor into the amount of phosphorus: mg MAP/L = (CFU/mL) x (1000 mL/L) (Measured yield factor or 3.73 x 108 CFU/mg PO4-P*/L)
4.5
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.
5 5.1
Quality control and quality assurance 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.
5.2
Quality control and quality assurance in CR6
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.
39
5.3
Quality control and quality assurance in CR9
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 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.
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.
41
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 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.
42
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.
43
CHARACTERISTICS
m water s u p p l
o r y
o ex i t
co
sect i f o n o material er u n d s t u d y
b fi l m i o sam p l i dev ces i vane
double i ernal t n cy l er i
water for t erm reg at h o u
il
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, 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
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 (60C), 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 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.
47
3.2
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 and in in situ measurements in water plants and distribution networks. References More detailed information about the reactor and its applications is described in Griebe and Flemming (2000): 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. 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. The variations of the Annular Reactor are described in brackets. Physical parameters: Flow velocity Shear stress Temperature Surface properties, composition and characteristics of the internal materials Hydraulic residence time Substrate composition and concentration Bioproducts in the biofilm matrix and bulk liquid phase Redox potential Inorganic ions Organic and inorganic particles Chemical parameters:
48
Biological parameters: Microorganism type (algae, protozoa, bacteria, viruses, etc.) Defined or undefined culture Mixed or pure culture
Engine
Influent extractable test-surfaces (Coupons)
Screw for the extraction of of coupons
rotating inner cylinder
with recirculation tubes
outer cylinder
with uptake
outlet
for
the
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
Table 1: Dimensions of the Rotating Annular Reactor Inner cylinder: Height Bore Exposed area (vertical) Exposed area (horizontal) Total area Outer cylinder: Height Bore Exposed area (vertical) Exposed area (horizontal) Total area Coupons: Number Width Length Exposed length Exposed area Total area (x 12) Percentage on the total area of the outer cylinder Recirculation tubes: Number Angle Length Bore Exposed area Total Rotating Annular Reactor: Volume mL ca. 650 dependent on the rotation speed 1565,3 23,68 2,76 cm cm cm 4 80 18,5 1,0 231,47 cm cm cm cm % 12 1,5 22,0 20,1 30,9 370,8 44,6 cm cm cm cm cm 20,6 11,30 731,1 100,3 831,4 unit cm cm cm cm cm 18,0 10,2 576,8 157,1 733,9
Exposed total area (without recirculation tubes) Percentage of the coupons on the total area Specific area
cm % cm/cm
Table 2: Measurements of the middle rotating velocities and Reynoldsnumbers in the Rotating Annular Reactor with different revolutions per minute Revolutions per min [U min-1] 50 200 600 ri calculated Horizontal velocity Vertical velocity Total velocity. Re [m s-1] 0,13 0,53 1,60 [m s-1] 0,10 0,44 1,29 [m s-1] 0,01 0,03 0,08 [m s-1] 0,10 0,45 1,29 514 2226 6439
ri: rotating velocity of the inner cylinder Re Reynolds number
7000 6000 5000 Reynolds-numbers 4000 3000 2000 1000 0 0 200 400 600
Re = 11,846 x revolutions/min
revolutions/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 121C or disinfected by biocides
51
with 200 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 PVC coupons that are colonised with biofilms 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 g/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
Figure 6 : Images of Rotating Annular Reactors (modification of Schulte and 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 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.
6.3
Flow cell reactor [CR7]
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

6.4
Pipeline biofilm collector [CR 6 and CR9]
Biofilm monitoring system udes 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 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 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
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.
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 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 source
Light source
Figure 9: Schematic view of a DTM device FOS and FluS are both fiber optic based devices. Optical fiber heads are implemented in the water system. Depending on the data operating software backscattered light or excited fluorescence from the biofilm can be detected and analysed. A schematic view of a FOS is shown in Figure 10.
Substratum
Biofilm EPS
Optical Fiber
Bacteria Illuminated biofilm area
IIluminating light Backscattered light
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 a piezoelectric ceramic to generate a short pulse that is "read" by other piezo in a different location. Through mathematic analysis of the obtained signal it will be possible to evaluate the thickness of the biofilm. This technique utilizes the direct 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 of plate metal defects, namely in the aerospace industry, and in soil consistence analysis in civil engineering. The monitor where the piezo sensors are located is a half-tube flow cell (see figure 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 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 a 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 piezo-electric sensors (a half-tube flow cell) and the capacitive sensor will be inserted on the flat surface of this flow cell.
Electrochemical devices The electrochemical device is based on voltammetry with platinum electrodes and can be used very successfully due to its sensitivity and selectivity. This technique is used to detect biofilm formation by water bacteria on the surface of large tip platinum electrodes. The working electrodes (WE) are platinum discs with 1 mm diameter. The electrodes are prepared by sealing a platinum wire into a glass tube and polishing the surface of the cross section with alumina powder, on a polishing cloth; the internal end of the platinum wire is sealed to a copper wire that provided the external contact. The reference electrode (RE) is a Metrohm silver/silver electrode (reference 6.0702.100) and all the data are reported here versus this reference. The auxiliary electrode (CE) is a platinum spiral.
61
Figure 12: Electrochemical device for biofilm analyses on platinum electrodes The electrochemical experiments are carried out using a potentiostat Autolab type PGSTAT 20, Ecochemie. The cyclic voltammetry experiments are carried out in a two-compartment, three-electrode cell at room temperature, as represented in the Figure 12. Each electrode is subjected to the electrochemical treatment by immersion in the solution of interest and recycling the potential between the appropriate limits. The voltammograms are recorded using the data acquisition program GPES 4.6. The technique used is repetitive cyclic voltammetry, where a triangular potential sweep is continuously applied to the working electrode and the current passing through the cell is recorded as a function of the applied potential (Pletcher, 1991). The potential profile is, therefore, a linear function of time and can be described as Eapp = Ei vt, where Eapp is the applied potential at a time t, EI is the initial potential and v is the scan rate in V/s. At a pre-set value of the scan rate is reversed and the potential is scanned to the initial value. This cycle can be repeated as many times as required.
62
The application of repetitive cyclic voltammetry is in itself a method of cleaning platinum electrodes and the appearance of a different voltammogram after the electrode has been in contact with a foulant is an indication of biofilm formation. This fact constitutes the basis of the detector of biofilm formation described here since the smallest deposit on the electrode surface changes the pattern observed when the platinum electrode surface is clean. In drinking water systems, due the high resistivity of the media (low ionic force), microelectrodes should be used instead. Different electrode configurations can be applied, according to the case under study (as microband microelectrodes). However, the most important feature is that, whatever the geometry, they are particularly suited for many analytical determinations, 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, which is compatible with vibrational spectroscopic measurements, in a continuously drinking water fed flow chamber (Figure 14). To improve the sensitivity and the delay of response, the crystal is colonised by one hours exposure to E. cloacae suspension, as a coliform model frequently isolated in noncompliance drinking waters. Absorption bands for proteins and polysaccharides in the vibrational spectra are chosen as probes, because these macromolecules are major constituents of bacteria and biofilms.
ATR/FT-IR analysis ATR/FT-IR spectra are measured between 4000 and 800 cm-1 on a Bruker Vector 22 spectrometer equipped with a KBr beam splitter and a DTGS (deuteriated triglycine sulphate) thermal detector. The resolution of the single beam spectra is 4 cm-1. The number of bi-directional double-sided interferogram 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 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. 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 vapour and carbon dioxide. FT-IR measurements are made at room air-conditioned temperature (20C 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 ZnSe 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.
entrance Biofilm
exit
ZnSe crystal IR source detector
Figure 15: Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) flow through cell Biofilm study in flow cell The 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 m filtered water (18 to 24 hours) to remove the ethanol (control by recording spectra) and to measure any deposits of organic molecules from the water sample, (iii) the bacterial suspension (1 hour) which allowed bacteria to
64
adhere to the surface of the crystal, (iv) the tested 0.2 m filtered water for 15 days. Ethanol, tested waters and bacterial suspension were passed through the ATR flow cell by using an up-stream Gilson Minipuls 3 pump with a flow of 30 mL h-1 (hydraulic residence time approximately 4 min). The infrared spectra of the hydrated dynamic biofilm were automatically collected every 15 minutes for the first hour and then every two hours.
Coliform bacteria suspension Enterobacter cloacae strain was isolated from a hydrous medium. The strain, conserved at 80C, was placed at 37C 1C for 24 hours before inoculation on CASO agar and incubated 24 hours at 37C 1C. A suspension of OD620
nm
from 0.2 to 0.4 (about 1 x 108 cells per mL) was
prepared in sterile glass flasks. Bacteria were recovered by centrifugation (10000 g; 10 min; 20C). The supernatant was removed and then the pellet was resuspended in 0.2 m filtered (Millipore, ref. 106329) tested water and washed by centrifugation (10000 g; 10 min; 20C) to eliminate the residual organic carbon from the nutritive medium. The pellet was homogenised in sterile drinking water (autoclaved, 121C; 15 min), and then the final suspension, whose concentration was about 1 x 108 cells per mL, was stored at 4 to 10C for up to two hours. 7 APPENDIX: Calibration of the sonication probe

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Surveillance and control of microbiological stability in drinking water distribution networks

WP2. Tools for biofilm monitoring

DATE: 28th May, 2003

1 INTRODUCTION 2 STRUCTURE 3 MICROBIOLOGICAL METHODS

3.1 ENUMERATION OF CULTURABLE MICROORGANISMS (WATER/BIOFILM)..............................................................................5 3.2

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

Microbiological methods Enumeration of culturable microorganisms (water/biofilm)

b. Number of colony forming units on R2A nutrient agar

Total cell number (water /biofilm)

1 mL DAPI (10 g/mL) and add 1 mL Triton X-100 (0.1 %).

Coliform bacteria and E. coli (water and biofilm)

Isolation and culture of mycobacteria (water /biofilm)

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

Ziehl-Neelsen staining for mycobacteria

---------------------- x [added quantity of standard ATP] [I (RLU)]

Chemical methods Total organic carbon /dissolved organic carbon

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

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)

Microbially available phosphorus (MAP) (water)

Quality control and quality assurance in CR6

Quality control and quality assurance in CR9

Biofilm monitoring devices

Propella (The common biofilm monitoring device for all partners)

b fi l m i o sam p l i dev ces i vane

water for t erm reg at h o u

Figure 2: Photos of Propella

Rotating Annular Reactor (modified RotoTorqueTM) [CR4]

Influent extractable test-surfaces (Coupons)

Screw for the extraction of of coupons

rotating inner cylinder

with recirculation tubes

ri: rotating velocity of the inner cylinder Re Reynolds number

Biofilm generator [CR5]

The schematic representation of the biofilm generator is given on Figure 7.

Flow cell reactor [CR7]

Figure 8: Linear flow through reactor for biofilm

Pipeline biofilm collector [CR 6 and CR9]

Figure 13. Schematic drawing of biofilm monitoring system in CR9

DTM, FOS, and FluS sensors [CR4[

Bacteria Illuminated biofilm area

IIluminating light Backscattered light

Figure 10: Schematic view of a FOS

Electrochemical, nanovibration, and capacitive monitors (CR 7)

Biofilm formation monitoring using ATR-FTIR sensor [CR2]

ZnSe crystal IR source detector

from 0.2 to 0.4 (about 1 x 108 cells per mL) was

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