Water quality - Guidance standard on the enumeration of phytoplankton using inverted microscopy (Utermöhl technique)

The procedure described in this European Standard is based on the standard settling technique as defined by Utermöhl in 1958 [31]. It describes a general procedure for the estimation of abundance and taxonomic composition of marine and freshwater phytoplankton by using inverted light microscopy and sedimentation chambers, including the preceding steps of preservation and storage. Emphasis is placed on optimizing the procedure for the preparation of the microscopic sample. Many of the general principles of the approach described may also be applied to other techniques of enumerating algae (or other entities) using a (conventional) microscope, some of which are described in Annex E. This guidance standard does not cover field collection of samples or the analysis of picoplankton, quantitative analysis of free-floating mats of Cyanobacteria or specific preparation techniques for diatoms.

Wasserbeschaffenheit - Anleitung für die Zählung von Phytoplankton mittels der Umkehrmikroskopie (Utermöhl-Technik)

Diese Europäischen Norm legt ein Verfahren für das Standard-Sedimentationsverfahren fest, das auf dem von Utermöhl 1958 beschriebenen Test [31] beruht. Es beschreibt ein allgemeines Verfahren für die Abschätzung der Abundanz (Häufigkeit) und taxonomischen Zusammensetzung des marinen und limnischen Phytoplanktons unter Verwendung der Durchlichtumkehrmikroskopie und von Sedimentationskammern, einschließlich der vorausgehenden Schritte der Konservierung und Lagerung der Proben. Der Schwerpunkt liegt auf der Optimierung des Verfahrens für die Vorbereitung der zu mikroskopierenden Probe. Viele der allgemeinen Prinzipien können auch auf andere Techniken der Algenzählung (oder anderer Organismen) mit einem (konventionellen) Mikroskop angewandt werden; einige sind im Anhang E beschrieben. Diese Anleitung behandelt weder die Feldprobenahme noch die Analyse des Picoplanktons, noch die quantitative Analyse frei treibender Matten von Cyanobakterien (Blaualgen) noch spezifische Präparationstechniken für Kieselalgen.

Qualité de l'eau - Norme guide pour le dénombrement du phytoplancton par microscopie inversée (méthode Utermöhl)

Le mode opératoire décrit dans la présente Norme européenne est fondé sur la technique de sédimentation classique telle que définie par Utermöhl en 1958 [31]. Il s’agit d’un mode opératoire général pour l’estimation de l’abondance et de la composition taxinomique du phytoplancton marin et d’eau douce à l’aide d’un microscope inversé et de chambres de sédimentation, y compris les étapes préalables de conservation et de stockage. L’accent est mis sur l’optimisation du mode opératoire pour la préparation de l’échantillon microscopique. Un grand nombre de principes généraux de l’approche décrite, dont certains sont mentionnés dans l’Annexe E, peuvent aussi s’appliquer à d’autres techniques de dénombrement d’algues (ou d’autres entités) à l’aide d’un microscope (classique). La présente norme guide ne concerne pas la collecte d’échantillons sur le terrain, l’analyse du picoplancton, l’analyse quantitative d’amas flottants de cyanobactéries, ou les techniques spécifiques pour la préparation des diatomées.

Kakovost vode - Smerni standard za štetje fitoplanktona z invertno mikroskopijo (postopek po Utermöhlu)

General Information

Status
Withdrawn
Publication Date
29-Nov-2006
Withdrawal Date
27-Feb-2007
Current Stage
9093 - Decision to confirm - Review Enquiry
Completion Date
12-Jul-2022

Buy Standard

Standard
EN 15204:2007
English language
42 pages
sale 10% off
Preview
sale 10% off
Preview
e-Library read for
1 day

Standards Content (Sample)

6,67(16/29(16.,IHEUXDU67$1'$5'.DNRYRVWYRGH6PHUQLVWDQGDUG]DãWHWMHILWRSODQNWRQD]LQYHUWQRPLNURVNRSLMRSRVWRSHNSR8WHUP|KOX:DWHUTXDOLW\*XLGDQFHVWDQGDUGRQWKHHQXPHUDWLRQRISK\WRSODQNWRQXVLQJLQYHUWHGPLFURVFRS\8WHUP|KOWHFKQLTXH !"#$%&'( )&!*- . 5HIHUHQþQDãWHYLOND6,67(1HQIUGH,&6

EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 15204August 2006ICS 13.060.70 English VersionWater quality - Guidance standard on the enumeration ofphytoplankton using inverted microscopy (Utermöhl technique)Qualité de l'eau - Norme guide pour l'analyse de routine del'abondance et de la composition du phytoplancton parmicroscopie inversée (méthode d'Utermöhl)Wasserbeschaffenheit - Anleitung für die Zählung vonPhytoplankton mittels der Umkehrmikroskopie (Utermöhl-Technik)This European Standard was approved by CEN on 14 July 2006.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Central Secretariat or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36

B-1050 Brussels© 2006 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15204:2006: E

EN 15204:2006 (E) 2 Contents Page Foreword..............................................................................................................................................................3 Introduction.........................................................................................................................................................4 1 Scope......................................................................................................................................................5 2 Normative references............................................................................................................................5 3 Terms and definitions...........................................................................................................................5 4 Principle..................................................................................................................................................7 5 Equipment and preservatives...............................................................................................................7 6 Sample processing................................................................................................................................9 7 Counting procedure.............................................................................................................................12 8 Quantitative validation........................................................................................................................18 9 Measurement uncertainty...................................................................................................................19 Annex A (informative)

Optical characteristics of inverted microscopes.....................................................21 Annex B (informative)

Sample treatment........................................................................................................23 Annex C (informative)

Phytoplankton analysis strategies............................................................................27 Annex D (informative)

Identification................................................................................................................30 Annex E (informative)

Use of conventional compound microscopes.........................................................31 Annex F (informative)

Statistical procedure...................................................................................................34 Bibliography......................................................................................................................................................40

Figures Figure 1 — Random distribution of particles (note the open spaces).......................................................12 Figure 2 — Example of rule for counting cells on the edge of the field. Algae objects crossing both the top and left hand side of grid are not counted whilst those crossing the bottom and right hand side of grid are counted...........................................................................................................................14 Figure F.1 — Illustration of collecting algal data for a Run-test.................................................................35

Tables Table 1 — Settling times for Lugol preserved seawater samples [12].......................................................11 Table F.F1 — Maximum allowable variance for Poisson approximation (µ = mean, σσσσ 2 = variance).......35 Table F.F2 — Multinomial homogeneity test................................................................................................36

EN 15204:2006 (E) 3 Foreword This document (EN 15204:2006) has been prepared by Technical Committee CEN/TC 230 “Water analysis”, the secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by February 2007, and conflicting national standards shall be withdrawn at the latest by February 2007. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard : Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.

EN 15204:2006 (E) 4 Introduction The European Water Framework Directive (2000/60/EC) has created a need for a uniform procedure to assess ecological quality of surface waters using phytoplankton abundance and composition. This European Standard will meet this need and will help laboratories improve the quality of their analytical results. A single standard procedure for the assessment of phytoplankton composition and abundance cannot be given as the questions which drive monitoring programmes are diverse in character and therefore require specific protocols. This European Standard, therefore, aims to provide guidance on basic aspects of microscopic algal analyses and to provide statistical procedures for the design, optimization and validation of methods and protocols. Though mentioned in Annex C, a method for the estimation of biovolume is not included. WARNING — Persons using this European Standard should be familiar with normal laboratory practice. Long periods of microscopic phytoplankton analysis can cause physical fatigue and affect eyesight. Attention should be given to the ergonomics of the microscope and advice from a health and safety practitioner should be sought to ensure that risks are minimized. The use of chemical products mentioned in this European Standard can be hazardous and users should follow guidelines provided by the manufacturers and take necessary specialist advice. This European Standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate health and safety practices and to ensure compliance with any national regulatory guidelines.

EN 15204:2006 (E) 5 1 Scope The procedure described in this European Standard is based on the standard settling technique as defined by Utermöhl in 1958 [31]. It describes a general procedure for the estimation of abundance and taxonomic composition of marine and freshwater phytoplankton by using inverted light microscopy and sedimentation chambers, including the preceding steps of preservation and storage. Emphasis is placed on optimizing the procedure for the preparation of the microscopic sample. Many of the general principles of the approach described may also be applied to other techniques of enumerating algae (or other entities) using a (conventional) microscope, some of which are described in Annex E. This guidance standard does not cover field collection of samples or the analysis of picoplankton, quantitative analysis of free-floating mats of Cyanobacteria or specific preparation techniques for diatoms. 2 Normative references Not applicable. 3 Terms and definitions For the purpose of this document, the following terms and definitions apply. 3.1 accuracy closeness of agreement between a test result or measurement result and the true value 3.2 algal object unit/cluster of one or more algal cells encountered during the phytoplankton analysis that is discrete from (liable to settle independently of) other particles in the sample 3.3 detection limit minimum number and/or size of a specific taxon or group of organisms in a sample at which its presence can be detected with a specified probability NOTE This definition is analogous to the definition used in chemistry (smallest true value of the measurand which is detectable by the measuring method). 3.4 error difference between an individual result and the true value 3.5 fixation protection from disintegration of the morphological structure of organisms 3.6 microscope counting field delimited area (e.g. a square or grid) in the microscope field of view, used for enumeration 3.7 nanoplankton small algae between 2 µm and 20 µm in size

EN 15204:2006 (E) 6 3.8 numeric aperture (NA) difference in refraction index of the medium between objective and object multiplied by the sine of half the angle of incident light 3.9 performance characteristic characteristics of a specific analysis protocol which encompass qualitative and quantitative aspects for data precision, bias, method sensitivity and range of conditions over which a method yields satisfactory data 3.10 phytoplankton community of free-living, suspended, mainly photosynthetic organisms in aquatic systems comprising Cyanobacteria and algae 3.11 picoplankton very small algae between 0,2 µm and 2 µm in size 3.12 precision closeness of agreement between independent test/measurement results obtained under stipulated conditions

3.13 preservation process that protects organic substances from decay 3.14 (analysis) protocol specific analytical procedure concerning (sub)sample volume, magnification, number of cells to count, taxonomic level of identification etc. 3.15 repeatability precision under repeatability conditions 3.16 repeatability conditions conditions where independent test/measurement results are obtained with the same method on identical test/measurement items in the same test or measuring facility by the same operator using the same equipment within short intervals of time NOTE This definition should be interpreted as the error occurring between replicate sub-samples from the same sample, counted using the same counting chamber, performed by one analyst using one microscope in a continuous run on one day. 3.17 reproducibility precision under reproducibility conditions

3.18 reproducibility conditions conditions where independent test/measurement results are obtained with the same method on identical test/measurement items in different test or measurement facilities with different operators using different equipment

EN 15204:2006 (E) 7 3.19 uncertainty parameter associated with the result of a counting that characterizes the dispersion of values that could reasonably be attributed to the measurand 3.20 validation confirmation by examination and the provision of effective evidence that the particular requirements for a specific intended use are fulfilled 4 Principle After preservation and storage, if applicable, the sample is homogenized and a sub sample is placed in a sedimentation chamber. When the algae have settled to the bottom of the chamber, they are identified and counted using an inverted microscope. 5 Equipment and preservatives 5.1 Sampling bottles A sampling bottle should meet the following requirements (the relevance of some of these may depend on the duration of storage of the sample):  the bottle should be clean and easily be cleaned. It should not be permeable to, or react with, the preservative used;  the bottle should be transparent (so that the state of preservation and the presence of aggregates can be examined easily), but stored in the dark.;  the combination of bottle and screw cap should ensure a closure that is watertight (to facilitate homogenisation) and almost gastight (to minimize evaporation) to allow long periods of storage;  the neck of the bottle should be wide enough for filling the counting chamber. The bottle should not be too large for easy handling and filling of the counting chamber: generally, a volume of some 100 ml to 200 ml is satisfactory;  to facilitate homogenisation, bottles should not be filled completely with sampling water (preferably fill to around 80 %). 5.2 Sedimentation chamber Sedimentation chambers consist of a vertical column, with a base through which the contents can be observed with an inverted microscope. The column is filled with a sample and the particles in the sample are allowed to settle on the bottom of the chamber. By using a relatively small cross-sectional area in comparison with column height, the sample can be concentrated effectively. A common type of chamber has 2 pieces: a top-piece column that is placed above a well in a base-piece, the top-piece being slid aside and replaced with a cover glass once the algae have settled on the bottom. Sedimentation chambers may be square or circular. The thickness of the base plate should not exceed 0,17 mm as this directly affects image quality. Counting chambers should be calibrated so that the volume of sub-sample contained can be determined. Counting chambers should be cleaned and dried between uses. For best results, cleaning should include washing with detergent using a soft paintbrush or small scrubbing brush; afterwards, the chamber should be rinsed in distilled water. Other agents that can be used, depending on the chamber material, are methanol, ethanol (90 %), commercial ‘denatured’ alcohol or isopropanol.

EN 15204:2006 (E) 8 5.3 Inverted microscope The use of an inverted microscope allows the algae, settled on the bottom of the chamber, to be brought into clear focus (see Annex A). The optical properties of the microscope determine the discriminating potential and hence the identification possibilities. For phytoplankton counting, an inverted microscope should be equipped with a condenser with a NA of at least 0,5 and plan objectives with a NA of 0,9 or more (see Annex A). Phase-contrast and/or Normarski interference-contrast is usually used in marine phytoplankton analysis. It can assist greatly in the identification of certain taxa, including flagellates, diatoms and delicate forms such as chrysophytes. Ideally, the microscope should be equipped with a (digital) camera. The microscope should have binocular, wide-field × 10 or × 12,5 eyepieces. One eyepiece should be equipped with a calibrated ocular micrometer. The other eyepiece should be equipped for counting by use of an appropriate calibrated counting-graticule: a) for counting of randomly-selected microscope fields, the graticule should have a square field or grid (available commercially, e.g. a Whipple disc), or the equivalent using 4 crossing threads, or b) for counting transects or the whole chamber, 2 parallel threads within the eyepiece forming a transect, preferably with a third vertical thread crossing the other two in the centre. The ocular micrometer and counting-graticule shall be calibrated for each magnification being used, and for each microscope. To do this, a stage micrometer slide composed of 100 µm × 10 µm divisions is viewed and focused through the ocular micrometer/counting-graticule and used to measure the scale of the ocular micrometer and the dimensions (to permit calculation of area) of the counting-field. Though inverted microscopy is the recommended method for enumeration of phytoplankton, conventional (non-inverted) compound light microscopes may also be used for enumerating phytoplankton under some conditions (see Annex E). 5.4 Preservatives 5.4.1 Acid Lugol’s iodine [35] Dissolve 100 g of KI (potassium iodide) in 1 l of distilled or demineralised water; then add 50 g of iodine (crystalline), shake until it is dissolved and add 100 g of glacial acetic acid. As the solution is near saturation, any possible precipitate should be removed by decanting the solution before use. Lugol’s solution can be stored in a dark bottle at room temperature for at least 1 year. 5.4.2 Alkaline Lugol’s iodine (modified after [37]) Dissolve 100 g of KI (potassium iodide) in 1 l of distilled or demineralised water; then add 50 g of iodine (crystalline), shake until it is dissolved and add 100 g of sodium acetate (CH3COO-Na). As the solution is near saturation, any possible precipitate should be removed by decanting the solution before use. The use of 5 ml of Lugol’s solution per litre of sample is standard. However, this is dependent on the algal density: for meso- and especially oligotrophic waters more than 2 ml might already cause over-saturation rendering the algae difficult to identify, in which case a lower volume of Lugol’s should be used. In general enough Lugol should be added to turn the sample to a cognac or straw colour. 5.4.3 Formaldehyde 37 % volume fraction For long term storage, formaldehyde should be added to give a final concentration of 4 %. This should only be done if no reanalysis of the sample is planned, since naked small flagellates will be destroyed. Another risk is a quick decoloration of the sample. Most preservatives are commercially available. The reader is referred to Annex B for more details on the use of different preservatives.

EN 15204:2006 (E) 9 6 Sample processing 6.1 General Samples should be divided into two with one part being preserved and stored at low temperature for later analyses whilst the other sub-sample is kept unpreserved to allow examination of live material (6.1 and 6.2). Before a sub-sample is taken for analysis acclimatize the sample to the appropriate temperature and homogenise (6.3 to 6.5). Thereafter transfer a sub-sample directly to a calibrated counting chamber (6.5 to 6.6). Then count the number of algal objects in a known area of the chamber with the aid of eyepiece graticules, and from this determine the concentration of algal units (Clause 7). The precise counting protocol used will vary depending on, for example, the purpose and objectives of the study, the nature of the samples being analysed, and the resources/equipment available. The error associated with each protocol will differ and so it is important to validate each protocol before it is used (Clause 8 and Annex F). The detailed analyses of certain groups of organisms may require special treatment. For (benthic) diatoms, guidance can be found in EN 13946 and EN 14407. 6.2 Preservation of samples Samples should be preserved as soon as possible after they have been taken, with one of the specified preservatives. Living samples should also be retained for preliminary analysis of the algal flora (6.3.1). 6.3 Storage 6.3.1 Living samples Living samples for preliminary analysis (7.2) should be kept in the dark at a temperature between 4 °C and 10 °C. Samples taken from ambient water at a higher temperature may need to be cooled gradually in order to avoid damage to phytoplankton cells. A maximum storage time of 36 h should not be exceeded, prior to analysis. NOTE In samples with a very high density of organisms, blooms or surface scums, depletion of oxygen (and, hence, degradation) should be prevented by diluting the sample with filtered (0,45 µm) water from its origin. 6.3.2 Preserved samples Samples preserved with Lugol’s solution (or formaldehyde) should be stored in the dark and cooled to 1 °C to 5 °C, unless they are analysed within three weeks, in which case they can be stored in the dark at room temperature. The level of the sample in the bottle should be marked on the bottle prior to storage. Storage at low temperature will slow down the rate of physical and chemical processes thus leading to a reduction in sample quality. Storage in the dark is always necessary to prevent photo-oxidation. The maximum storage time for Lugol preserved samples in the dark and between 1 °C and 5 °C is 12 months. Preservation and storage for longer periods is possible only after addition of formaldehyde (Annex B). New samples should be checked after a couple of days for oxidation of the Lugol’s iodine. The sample should have a Cognac or straw colour. If not, Lugol’s solution should be added until the sample has regained this colour. When properly sealed sample bottles are stored at a temperature between 1 °C to 5 °C no significant evaporation should occur. 6.4 Acclimatization In order to promote a random distribution of plankton in the sedimentation chamber, the sample and all equipment used should be of a similar temperature. Usually, an acclimatization period to room temperature of some 12 h is adequate but this depends upon actual ambient temperatures and the sample volume.

EN 15204:2006 (E) 10 NOTE Temperature differences between sedimentation chamber and medium may produce convection currents that have different effects on the settling of phytoplankton species, depending on their physical properties. Furthermore, bubbles may develop in relatively cold samples as the solubility of gases declines with the gradual rise of the temperature of the sample. Therefore, acclimatization is an important step in a controlled procedure for phytoplankton analyses. 6.5 Sample homogenisation The first critical step in preparing a sample for microscopic analysis is homogenisation of the sample. During sample storage, suspended particles settle out and (small) algae become indiscernible by incorporation in detritus aggregates or by adhesion to other large algal cells. Re-suspension and separation of particles can be achieved by shaking the sample as gently as possible. This may be performed manually or preferably by the use of an appropriate shaking device. Devices (tumbler mixers) based on a three-dimensional motion (figure-of-eight movement with rotation: Paul-Schatz principle) are preferred over standard orbital shakers, which induce a vortex movement of the water sample leading to incomplete mixing. The method used for manual shaking should be described clearly in order to minimise differences between operators. A combination of alternating horizontally rolling and vertical turning upside down of the sample bottle for a specific number of times provides better mixing than straightforward shaking. NOTE 1 Vigorous shaking may lead to the disintegration of fragile colonies, which is a problem if colony size has to be determined. When a lot of small bubbles are produced (which will affect sedimentation adversely), then allow one hour before taking the sub-sample, gently re-shaking the bottle before doing so. NOTE 2 Disintegration of colonies to facilitate cell counts can be promoted by exposure to ultrasonic vibration taking care not to damage cells by over exposure [25], [3] or by hydrolysis of the colony mucus [2], after which the sample may need to be homogenised by gentle shaking. Some colonies (e.g. Phaeocystis, certain chlorophytes and colonial chrysophytes as Synura and Uroglena will partly or fully disintegrate shortly after preservation in acid Lugol´s solution. 6.6 Sub-sample preparation After homogenisation, a known volume of sample should be used to fill the counting chamber. The filling of the counting chamber is crucial, as it affects the final distribution of settled particles. Random distribution allows for simple and uniform counting strategies and statistical procedures to assess measurement uncertainty. The chamber should be filled directly from the sample bottle. The exact volume depends on the phytoplankton density, the volume of the counting chamber and its surface to volume ratio. Larger sub-sample volumes (up to 100 ml) will be required from oligotrophic waters (see also B.3). At high phytoplankton biomass a dilution step may be necessary to ensure that the concentration of particles is sufficiently low to prevent clogging of particles by adhesion and to optimise the counting process (6.6). Dilution should be performed with relatively large volumes (e.g. using a graduated measuring cylinder). For marine samples filtered (0,45 µm) sea water should be used for dilution. NOTE The height of sedimentation tubes should not be higher than 5 times of chamber diameter [19]. This means for instance that 100 ml-tubes with a diameter of 26 mm are not suitable for counting of nanoplankton and picoplankton. If more than 50 ml sedimentation volume is required a pre-sedimentation is indicated (see Annex B.3). When using small volume pipettes (1 ml to 5 ml) with removable tips, the end of the tip should be cut off to widen the opening to a diameter between 3 mm and 4 mm, to ensure that large taxa such as Ceratium are not excluded. Since cutting the tip will affect the accuracy of the pipette, calibrate each individually. The following points should be noted for optimal filling of sedimentation chambers:  ensure that all equipment (including filling tips etc.) is allowed to equilibrate to the ambient temperature of the room where the analyses are to be performed. The ambient temperature should be as constant as possible;  place the chamber on a horizontal flat surface that is a poor heat conductor (e.g. a thin acrylic plate). Wait until all materials have reached an equal temperature;

EN 15204:2006 (E) 11  take enough sample (diluted if necessary), to completely fill the chamber in a single addition (with no air spaces at the top);  if it becomes obvious that many algal cells are obscured by adhesion to detritus, the quality of the sub-sample can be improved by prolonged and/or more intensive shaking of the sample and appropriate dilution;  close the chamber with a cover glass; avoid trapping air bubbles in the process;  the sedimentation should take place in the dark at a constant ambient temperature that is similar to the temperature of the sub-sample; avoid vibrations;  for freshwater samples preserved with Lugol’s iodine a settling time of at least 4 h per cm is recommended [19], [20] and for seawater samples preserved with formaldehyde a settling time of at least 16 h per cm [9]. For Lugol preserved seawater samples the following settling times are advised [12];

Table 1 — Settling times for Lugol preserved seawater samples [12] Volume of chamber Height of chamber Settling time ml cm h 2 1 3 10 2 8 25 5 12 50 10 24 100 20 48

 after sedimentation, slide the chamber column aside and place a cover glass on the counting chamber to close it; avoid enclosing air bubbles; these can be eliminated by topping up with water using a small dropper pipette whilst sliding the coverslip back;  gentle moving the counting chamber to the microscope will not affect the settled particles when the chamber is filled completely and a cover glass is used. However, settled algae in an open sedimentation chamber are easily disturbed even when moved carefully. Open chambers should not be used for this reason;  an intense light source (including that from the microscope) might cause settled algae to float again even in a closed counting chamber. It should be checked for each microscopic configuration and procedure whether this phenomenon will indeed affect the analysis. When incomplete sedimentation has been observed or when there are specific sampling or historical indications that buoyant algae like many Cyanobacteria or for instance the lipid containing green alga Botryococcus may be present, the fluid in the upper column of the chamber should be centrifuged at an appropriate speed to estimate whether the amount of buoyant algae is significant. Furthermore, some small Cyanobacteria remain in suspension just above the bottom glass under some circumstances. This can be checked by focusing above the bottom prior to counting. If necessary, gas vesicles of Cyanobacteria can be collapsed, after which a new sub-sample may be prepared. Gas vesicles can be collapsed by putting a sample in a large plastic syringe from which the needle has been removed, leaving an opening of 1 mm or 2 mm in diameter. If the needle end is hit firmly against a wall whilst holding the piston, the sudden increase of pressure inside the syringe will collapse the gas vesicles. Another option is to put a rubber stopper in the opening of a sample bottle and then hit it carefully with a hammer. Collapsing of gas vesicles may sometimes be achieved using acidified Lugol with a slightly higher concentration of glacial acetic acid.

EN 15204:2006 (E) 12 When all particles are settled, the overall distribution pattern should be judged using a stereo-zoom microscope to confirm a random distribution. At a low mag

...

Questions, Comments and Discussion

Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.