EN 17477:2021
(Main)Algae and algae products - Identification of the biomass of microalgae, macroalgae, cyanobacteria and Labyrinthulomycetes - Detection and identification with morphological and/or molecular methods
Algae and algae products - Identification of the biomass of microalgae, macroalgae, cyanobacteria and Labyrinthulomycetes - Detection and identification with morphological and/or molecular methods
This document specifies a method for the detection and identification of microalgae, macroalgae, cyanobacteria and Labyrinthulomycetes by using morphological methods and/or molecular methods.
The morphological methods in this document are applicable to harvested wet biomass and to harvested dried unground biomass from microalgae, macroalgae, cyanobacteria and Labyrinthulomycetes that have been grown and/or harvested for further processing and/or use.
The molecular methods in this document are applicable to harvested wet biomass and to harvested dried and/or ground biomass from microalgae, macroalgae, cyanobacteria and Labyrinthulomycetes that have been grown and/or harvested for further processing and/or use.
This document describes a toolbox, consisting of several identification methods that can be chosen according to the applicability and purpose of the identification:
- morphological methods based on observation and referring to scientific literature on taxonomy:
- macroscopic identification;
- light microscopic identification.
- molecular methods for sequencing and blasting of sequences:
- 16S rDNA sequencing;
- 18S rDNA sequencing;
- rbcL DNA sequencing;
- ITS sequencing;
- COX1 gene sequencing;
- tufA gene sequencing.
This document does not deal with genetic purity of the biomass or quantification of the identified taxa.
This document is not suitable for the analysis of highly processed biomass with highly degraded DNA where the fragments’ length are not sufficient for amplification of the targets and the morphological characteristics cannot be assessed.
Algen und Algenprodukte - Identifizierung der Biomasse von Mikroalgen, Makroalgen, Cyanobakterien und Labyrinthulomycetes - Erkennung und Identifizierung mit morphologischen und/oder molekularen Verfahren
Dieses Dokument legt ein Verfahren für den Nachweis und die Identifizierung von Mikroalgen, Makroalgen, Cyanobakterien und Labyrinthulomycetes (Netzschleimpilze) mittels morphologischer und/oder moleku-larer Verfahren fest.
Die in diesem Dokument dargelegten morphologischen Verfahren sind anzuwenden für geerntete feuchte Biomasse und geerntete trockene, ungemahlene Biomasse von Mikroalgen, Makroalgen, Cyanobakterien und Labyrinthulo¬mycetes, die für die Weiterverarbeitung und/oder Verwendung kultiviert und/oder geerntet wurden.
Die in diesem Dokument dargelegten molekularen Verfahren sind anzuwenden für geerntete feuchte Biomasse und geerntete trockene und/oder gemahlene Biomasse von Mikroalgen, Makroalgen, Cyanobakterien und Labyrinthulomycetes, die für die Weiterverarbeitung und/oder Verwendung kultiviert und/oder geerntet wurden.
In diesem Dokument wird eine Sammlung von Verfahren beschrieben, die aus mehreren Identifizierungs-verfahren besteht, welche je nach Anwendbarkeit und Zweck der Identifizierung gewählt werden können:
- morphologische Verfahren, die auf Beobachtung unter Bezug auf die wissenschaftliche Literatur zur Taxonomie basieren:
- makroskopische Identifizierung;
- lichtmikroskopische Identifizierung;
- molekulare Verfahren zur Sequenzierung und zum Sequenzvergleich („Blasting“):
- 16S rDNS Sequenzierung;
- 18S rDNS Sequenzierung;
- rbcL DNS Sequenzierung;
- ITS Sequenzierung;
- COX1 Gensequenzierung;
- tufA Gensequenzierung.
Dieses Dokument behandelt weder die genetische Reinheit der untersuchten Biomasse noch die Quantifizierung der identifizierten Taxa.
Dieses Dokument ist nicht geeignet für die Analyse von stark verarbeiteter Biomasse mit hochgradig abgebauter DNS, bei der die Länge der Fragmente für die Amplifikation der Targets nicht ausreichend ist, und bei der die morphologischen Merkmale nicht beurteilt werden können.
Algues et produits d'algues - Identification de la biomasse de microalgues, macroalgues, cyanobactéries et Labyrinthulomycètes - Détection et identification à l'aide de méthodes morphologiques et/ou moléculaires
Le présent document spécifie une méthode de détection et d’identification des microalgues, macroalgues, cyanobactéries et Labyrinthulomycètes à l’aide de méthodes morphologiques et/ou moléculaires.
Les méthodes morphologiques utilisées dans le présent document sont applicables à la biomasse humide récoltée et à la biomasse sèche non broyée récoltée provenant des microalgues, macroalgues, cyanobactéries et Labyrinthulomycètes qui ont été cultivées et/ou récoltées en vue d’un traitement ultérieur et/ou d’une utilisation ultérieure.
Les méthodes moléculaires utilisées dans le présent document sont applicables à la biomasse humide récoltée et à la biomasse sèche et/ou broyée récoltée provenant des microalgues, macroalgues, cyanobactéries et Labyrinthulomycètes qui ont été cultivées et/ou récoltées en vue d’un traitement ultérieur et/ou d’une utilisation ultérieure.
Le présent document décrit une boîte à outils, comprenant plusieurs méthodes d’identification qui peuvent être choisies en fonction de l’applicabilité et de l’objectif de l’identification :
- méthodes morphologiques reposant sur l’observation et faisant référence aux ouvrages scientifiques sur la taxonomie :
- identification macroscopique ;
- identification au microscope optique ;
- méthodes moléculaires de séquençage et d’alignement de séquences :
- séquençage de l’ADNr 16S ;
- séquençage de l’ADNr 18S ;
- séquençage de l’ADN rbcL ;
- séquençage de l’ITS ;
- séquençage du gène COX1 ;
- séquençage du gène tufA.
Le présent document ne traite ni de la pureté génétique de la biomasse ni de la quantification des taxons identifiés.
Le présent document ne convient pas à l’analyse de la biomasse hautement traitée contenant de l’ADN fortement dégradé dans lequel la longueur des fragments n’est pas suffisante pour amplifier les cibles et dans lequel les caractéristiques morphologiques ne peuvent pas être évaluées.
Alge in izdelki iz alg - Ugotavljanje biomase pri mikroalgah, makroalgah, cianobakterijah in labirintulomicetah - Odkrivanje in prepoznavanje z morfološkimi in/ali molekularnimi metodami
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-oktober-2021
Alge in izdelki iz alg - Ugotavljanje biomase pri mikroalgah, makroalgah,
cianobakterijah in labirintulomicetah - Odkrivanje in prepoznavanje z morfološkimi
in/ali molekularnimi metodami
Algae and algae products - Identification of the biomass of microalgae, macroalgae,
cyanobacteria and Labyrithulomycetes - Detection and identification with morphological
and/or molecular methods
Algen und Algenprodukte - Identifizierung der Biomasse von Mikroalgen, Makroalgen,
Cyanobakterien und/oder Labyrinthulomycetes - Erkennung und Identifizierung mit
morphologischen und/oder molekularen Methoden
Algues et produits d'algues - Identification de la biomasse de microalgues, macroalgues,
cyanobactéries et/ou Labyrinthulomycètes - Détection et identification à l'aide de
méthodes morphologiques et/ou moléculaires
Ta slovenski standard je istoveten z: EN 17477:2021
ICS:
13.020.55 Biološki izdelki Biobased products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 17477
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2021
EUROPÄISCHE NORM
ICS 13.020.55
English Version
Algae and algae products - Identification of the biomass of
microalgae, macroalgae, cyanobacteria and
Labyrinthulomycetes - Detection and identification with
morphological and/or molecular methods
Algues et produits d'algues - Identification de la Algen und Algenprodukte - Identifizierung der
biomasse de microalgues, macroalgues, cyanobactéries Biomasse von Mikroalgen, Makroalgen,
et Labyrinthulomycètes - Détection et identification à Cyanobakterien und Labyrinthulomycetes - Erkennung
l'aide de méthodes morphologiques et/ou moléculaires und Identifizierung mit morphologischen und/oder
molekularen Verfahren
This European Standard was approved by CEN on 7 June 2021.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17477:2021 E
worldwide for CEN national Members.
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 7
3 Terms and definitions . 7
4 Abbreviations . 11
5 Reagents . 12
5.1 Reagents for morphological methods . 12
5.1.1 Isotonic solution . 12
5.2 Reagents for molecular methods . 12
5.2.1 Primer . 12
5.2.2 Deoxynucleotide triphosphate mix (dNTPs) . 12
5.2.3 Thermostable DNA polymerase . 12
5.2.4 PCR reaction buffer . 12
5.2.5 Agarose gel . 12
5.2.6 Gel electrophoresis buffer . 13
5.2.7 Loading buffer . 13
5.2.8 DNA Ladder . 13
6 Apparatus . 13
6.1 General . 13
6.2 Apparatus for morphological identification methods . 13
6.2.1 Low-magnifying optical system . 13
6.2.2 Light microscope . 13
6.2.3 Scientific literature on taxonomy . 13
6.2.4 Microscope slide . 14
6.2.5 Microscope cover glass . 14
6.3 Apparatus for molecular identification methods . 14
6.3.1 Thermocycler . 14
6.3.2 Gel electrophoresis device . 14
6.3.3 DNA sequencer . 14
6.3.4 Plastic consumables, DNA free, disposable . 14
7 Principle . 14
7.1 General . 14
7.2 Morphological methods . 15
7.3 Molecular methods . 15
8 Procedure. 15
8.1 General laboratory requirements . 15
8.2 Choice of methods . 15
9 Morphological identification methods . 17
9.1 General . 17
9.2 Macroscopic identification with the naked eye or a magnifying glass . 17
9.3 Light microscopy . 17
9.3.1 General . 17
9.3.2 Staining . 17
9.3.3 Preparation of microscope slides . 17
9.3.4 Microscopic identification . 18
9.3.5 Use of identification keys . 18
10 Molecular identification methods . 18
10.1 General . 18
10.2 DNA extraction and purification . 19
10.3 DNA Amplification . 19
10.3.1 Principle of DNA Amplification . 19
10.3.2 Method . 19
10.4 Selection of primers . 20
10.5 Control reactions . 20
10.6 Evaluation of PCR products . 21
10.7 PCR product cloning . 21
10.8 PCR product sequencing . 21
10.9 Evaluation of sequence data . 21
10.10 Sequence analysis/comparison with reference sequences in public databases . 22
11 Test report . 23
Annex A (informative) Examples of applicable primers . 24
Annex B (informative) Scientific literature that may be used for identification . 26
Bibliography . 28
European foreword
This document (EN 17477:2021) has been prepared by Technical Committee CEN/TC 454 “Algae and
algae products”, the secretariat of which is held by NEN.
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 2022 and conflicting national standards
shall be withdrawn at the latest by February 2022.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association, and supports essential requirements of EU
Directive(s) / Regulation(s).
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United
Kingdom.
Introduction
This document has been prepared by the experts of CEN/TC 454 'Algae and algae products'.
The European Committee for Standardization (CEN) was requested by the European Commission (EC) to
draft European standards or European standardization deliverables to support the implementation of
Article 3 of Directive 2009/28/EC for algae and algae-based products or intermediates.
This request, presented as Mandate M/547, also contributes to the Communication on “Innovating for
Sustainable Growth: A Bio economy for Europe”.
The former working group CEN Technical Board Working Group 218 “Algae”, was created in 2016 to
develop a work programme as part of this Mandate. The technical committee CEN/TC 454 'Algae and
algae products' was established to carry out the work programme that will prepare a series of standards.
The interest in algae and algae-based products or intermediates has increased significantly in Europe as
a valuable source including but not limited to, carbohydrates, proteins, lipids, and several pigments.
These materials are suitable for use in a wide range of applications from food and feed purposes to other
sectors, such as textile, cosmetics, biopolymers, biofuel and fertilizer/biostimulants. Standardization was
identified as having an important role in order to promote the use of algae and algae products.
The work of CEN/TC 454 should improve the reliability of the supply chain, thereby improving the
confidence of industry and consumers in algae, which include macroalgae, microalgae, cyanobacteria,
Labyrinthulomycetes, algae-based products or intermediates and will promote and support
commercialisation of the European algae industry.
This document has been developed with the aim to cover the horizontal definitions for algae and algae
based products or intermediates. Hence, other terms and definitions are given in the other standards
developed by CEN/TC 454 “Algae and algae products”.
For food, feed and non-food, non-feed applications additional definitions may exist in other product
specific standards.
1 Scope
This document specifies a method for the detection and identification of microalgae, macroalgae,
cyanobacteria and Labyrinthulomycetes by using morphological methods and/or molecular methods.
The morphological methods in this document are applicable to harvested wet biomass and to harvested
dried unground biomass from microalgae, macroalgae, cyanobacteria and Labyrinthulomycetes that have
been grown and/or harvested for further processing and/or use.
The molecular methods in this document are applicable to harvested wet biomass and to harvested dried
and/or ground biomass from microalgae, macroalgae, cyanobacteria and Labyrinthulomycetes that have
been grown and/or harvested for further processing and/or use.
This document describes a toolbox, consisting of several identification methods that can be chosen
according to the applicability and purpose of the identification:
— morphological methods based on observation and referring to scientific literature on taxonomy:
— macroscopic identification;
— light microscopic identification.
— molecular methods for sequencing and blasting of sequences:
— 16S rDNA sequencing;
— 18S rDNA sequencing;
— rbcL DNA sequencing;
— ITS sequencing;
— COX1 gene sequencing;
— tufA gene sequencing.
This document does not deal with genetic purity of the biomass or quantification of the identified taxa.
This document is not suitable for the analysis of highly processed biomass with highly degraded DNA
where the fragments’ length are not sufficient for amplification of the targets and the morphological
characteristics cannot be assessed.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 24276:2006, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and
derived products — General requirements and definitions
EN 17399:2020, Algae and algae products — Terms and definitions
3 Terms and definitions
For the purposes of this document, the terms and definitions in EN 17399:2020, ISO 24276:2006 and the
following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at https://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp
3.1
16S rDNA sequencing
process of determining the sequence of nucleotides in a complete or partial gene coding for the 16S
ribosomal ribonucleic acid
Note 1 to entry: The largest amount of 16S rDNA gene sequencing work concerns prokaryotes.
3.2
18S rDNA sequencing
process of determining the sequence of nucleotides in a complete or partial gene coding for the 18S
ribosomal ribonucleic acid
Note 1 to entry: The 18S rDNA gene sequencing work concerns eukaryotes.
3.3
alignment
process or result of matching up the nucleotide residues of two or more biological sequences to achieve
maximal levels of identity
3.4
basic local alignment search tool
BLAST
sequence comparison algorithm optimized for speed that is used to search sequence databases for
optimal local alignments to a query
Note 1 to entry: This algorithm directly approximates alignments that optimize a measure of local similarity, the
maximum signal pair (MST) score or high-scoring segment pair (HSP) score.
[SOURCE: ISO 20813:2019, 3.1, modified – Note 2 to entry and Note 3 to entry have been deleted]
3.5
blasting of sequences
sequence comparison against commonly used gene sequence databases using the BLAST algorithm
3.6
COX1 gene sequencing
process of determining the sequence of the COX1 gene that codes for the cytochrome C oxidase subunit 1
Note 1 to entry: In literature, COX1 gene can also be called CO1 or COI.
3.7
detection
discovery of the target organism using a suitable method
3.8
DNA extraction
sample treatment for the release and separation of DNA from other cellular components
[SOURCE: ISO 16577:2016, 3.44, modified – the word 'liberation' has been replaced by 'release']
3.9
DNA purification
method resulting in a more purified DNA
Note 1 to entry: In this context, purity refers to the reduction of observable and measurable effects of PCR
inhibitors.
[SOURCE: ISO 24276:2006, 3.2.2]
3.10
DNA sequence
order of nucleotides within a deoxyribonucleic acid molecule
3.11
external amplification control
spiked amplification control
DNA added to an aliquot of the extracted nucleic acid in a defined amount or copy number serving as a
control for amplification in nucleic acid-based reactions
[SOURCE: ISO 16577:2016, 3.60]
3.12
FASTA
GIR (genomic information representation) that includes a name and a nucleotide sequence for each
sequencing read
[SOURCE: ISO/IEC 23092-2:2020, 3.7, modified – Note 1 to entry has been left out]
3.13
GenBank
comprehensive public database of genetic reference sequences
Note 1 to entry: GenBank at National Center for Biotechnology Information (NCBI) is part of the International
Nucleotide Sequence Database Collaboration, which comprises the DNA DataBank of Japan (DDBJ), the European
Nucleotide Archive (ENA). These three organizations exchange data on a daily basis.
3.14
internal amplification control
gene sequence naturally present in template DNA that is amplified to serve as a control for amplification
in nucleic acid-based reactions
Note 1 to entry: A housekeeping gene with known copy numbers/genome can be used as an internal
amplification control.
3.15
Internal Transcribed Spacer
ITS
piece of non-coding DNA located between structural ribosomal rDNA subunits
3.16
ITS sequencing
process of determining the partial or complete sequence of the internal transcribed spacer (ITS)
Note 1 to entry: The largest amount of ITS sequencing work concerns eukaryotes and prokaryotes.
3.17
macroscopic identification
identification with the naked eye, based on taxonomic identification keys
3.18
microscopic identification
identification with magnification by using magnifying glasses, binoculars or microscopes, based on
taxonomic identification keys
3.19
molecular identification method
set of tools that rely on the comparison of the nucleic acid sequences of DNA obtained from an organism
using a PCR-based method with public/documented data of known organisms
Note 1 to entry: The data obtained using the respective follow-up tools like gene sequencing, can be compared
with sequences of known species accessible via public databases (see 3.4).
Note 2 to entry: These methods allow detection of low concentrations of DNA in non-viable organisms.
3.20
negative DNA target control
well-characterized DNA preparation material that does not contain target nucleic acid sequences
[SOURCE: ISO 16577:2016, 3.118]
3.21
negative extraction control
negative control reaction generated by performing all required steps in an extraction procedure except
for the addition of the test portion
Note 1 to entry: For example, by substitution of water for the test portion.
Note 2 to entry: This control is used to demonstrate the absence of contamination during extraction steps.
3.22
negative process control
well-characterized reference sample lacking target analyte and that should be put through the exact same
process steps as the test samples
[SOURCE: ISO 16577:2016, 3.119, modified – the word 'recognized' has been replaced by 'well-
characterized']
3.23
morphological identification method
identification method based on morphological characteristics
3.24
polymerase chain reaction
PCR
in vitro enzymatic technique to increase the number of copies of a specific DNA fragment by several
orders of magnitude
Note 1 to entry: PCR is used to selectively amplify DNA target.
3.25
PCR product
DNA molecule / fragment amplified by PCR
Note 1 to entry: If necessary the PCR product can be purified by using commercial kits.
[ISO 16577:2016, 3.138 – modified – note added]
3.26
positive DNA target control
well-characterized DNA preparation material containing intact target nucleic acid sequences for PCR
Note 1 to entry: Reference DNA or DNA extracted from a certified reference material is generally used to
demonstrate that PCR reagents are working as intended.
[SOURCE: ISO 16577:2016, 3.150, modified – partly rewritten]
3.27
positive PCR control
known positive (identified) sample representing the DNA-sequence of the organism under study
Note 1 to entry: This control is used to demonstrate that the PCR reagents are working as intended.
3.28
positive process control
well-characterized reference sample containing a detectable amount of a target analyte that should be
put through the exact same process steps as the test samples
Note 1 to entry: The positive process control goes through exactly the same process steps as the test samples.
[SOURCE: ISO 16577:2016, 3.151, modified – partly rewritten and added 'that should be put through the
exact same process steps as the test samples']
3.29
rbcL DNA sequencing
process of determining the sequence of the rbcL in a complete or partial gene coding for the large subunit
of the protein ribulose-1,5-bisphosphate carboxylase/oxygenase
Note 1 to entry: The largest amount of rbcL DNA gene sequencing work concerns eukaryotes.
3.30
sanger sequencing
cycle sequencing method using fluorescent-labelled dideoxynucleotides
3.31
sample taxon identification
determination of the genus and preferably also of the species name of an isolate
3.32
tufA gene sequencing
process of determining the sequence of the tufA in a complete or partial gene coding for the elongation
factor Tu
Note 1 to entry: Some tufA gene sequencing work concerns prokaryotes and eukaryotes.
Note 2 to entry: The plastid tufA gene is encoding the transcription factor Tu and is evaluated as a suitable
barcode marker gene for green algae.
4 Abbreviations
A Adenine
BLAST Basic Local Alignment Search Tool
C Cytosine
COX1 Cytochrome c oxidase subunit 1
DDBJ DNA Data Bank of Japan
DIC Differential Interference Contrast
DNA Deoxyribonucleic acid
dATP Deoxyadenosine 5'-triphosphate
dCTP Deoxycytidine 5'-triphosphate
dGTP Deoxyguanine 5'-triphosphate
dNTPs Deoxyribonucleotide triphosphate mix
dTTP Deoxythymidine 5'-triphosphate
ENA European Nucleotide Archive
G Guanine
GenBank GenBank at National Centre for Biotechnology Information (NCBI)
ITS Internal Transcribed Spacer
NCBI National Centre for Biotechnology Information
PBS Phosphate-Buffered Saline
PCR Polymerase Chain Reaction
rbcL Ribulose-1,5-bisphosphate carboxylase/oxygenase
rDNA Ribosomal DNA
T Thymine
tufA Elongation factor Tu
5 Reagents
5.1 Reagents for morphological methods
5.1.1 Isotonic solution
An isotonic solution should be used when examining biomass for morphological identification. The
isotonic solution should contain a sufficient amount of salts, so that when examining the sample, bursting
or shrinking of cells is prevented. It can be used to prepare a sample for microscopic identification.
Distilled water shall not be used for this purpose.
NOTE Examples of suitable isotonic solutions could be tap water, culture medium, phosphate-buffered saline
(PBS) or water from the local environment.
5.2 Reagents for molecular methods
5.2.1 Primer
A primer for sequencing is a synthetic DNA oligonucleotide of approximately 15–30 bases. They are
designed to bind (via sequence complementarity) to sequences that flank the region of interest in the
template DNA. Primers are used for PCR and sequencing reactions. For example, during PCR reaction, the
DNA polymerase extends the primers from their 3’-ends. Thus, the primers’ binding sites shall be unique
to the vicinity of the target gene with minimal homology to other sequences of the input DNA to ensure
specific amplification of the intended target gene.
See Annex A for PCR and sequencing primers recommendations.
5.2.2 Deoxynucleotide triphosphate mix (dNTPs)
A deoxynucleotide triphosphate mix (dNTPs) consists of four basic nucleotides — dATP, dCTP, dGTP, and
dTTP. They are the building blocks for new DNA strands. These four nucleotides are typically added to
the PCR reaction in equimolar amounts for optimal base incorporation. dNTPs are commercially
available.
5.2.3 Thermostable DNA polymerase
Thermostable DNA polymerase is an enzyme that is derived from a thermophilic bacterium, functions at
high temperature and is used in PCR. Thermostable DNA polymerase should be used as directed by the
manufacturer’s protocol.
5.2.4 PCR reaction buffer
PCR reaction buffers are generally sold with the thermostable DNA polymerase (5.2.3). PCR reaction
buffers can include MgCl or they can come with a separate MgCl solution.
2 2
5.2.5 Agarose gel
Use an agarose gel of suitable concentration (e.g. 1 % to 2 % (w/v)).
5.2.6 Gel electrophoresis buffer
Gel electrophoresis buffers in gel electrophoreses are used to enable an electrical current to flow through
the gel matrix and allow the migration and separation of nucleic acids samples. Common gel
electrophoresis buffers used to evaluate PCR products are Tris/Acetate/EDTA (TAE) buffer and
Tris/Borate/EDTA (TBE) buffer.
5.2.7 Loading buffer
A loading buffer (or sample buffer) is combined with the PCR product to be loaded into an agarose gel
(5.2.5) well to perform a gel electrophoresis.
5.2.8 DNA Ladder
The DNA ladder is a molecular weight size marker. It is a set of standards that are used to identify the
approximate size of a molecule such as a PCR product run on a gel during electrophoresis (see 6.3.2). DNA
ladders are commercially available.
6 Apparatus
6.1 General
The laboratory shall use properly maintained equipment according to the manufacturer's instructions.
If available, calibration should be routinely performed on equipment as performance could impact the
data produced.
6.2 Apparatus for morphological identification methods
6.2.1 Low-magnifying optical system
For the morphological investigation of details, which are necessary for the identification of macroalgae
or tissues, a commercially available magnifying glass or a dissecting microscope might be sufficient. These
have lenses with magnifying factors between 0,65x and 5x. In the case of a dissecting microscope this is
multiplied by the magnifying factor of the eyepiece (usually 10x) or lens in the light path of the camera
(often 10x as well). This results in a total magnification between 6,5x and 50x. Use as instructed in the
apparatus’ manual.
6.2.2 Light microscope
For the morphological investigation of details of microscopic organisms, a commercially available light
microscope, optionally equipped with different contrasting and/or fluorescence units can be used. These
usually have front lenses with magnifying factors between 4x and 100x complemented by the magnifying
factor of the eyepiece (usually 10x) or lens in the light path of the camera (often 10x as well). This results
in a total magnification between 40x and 1000x. Optional contrasting equipment such as phase contrast
or differential interference contrast (DIC, also called Nomarski contrast) or also dark field mode might be
useful depending on the taxonomic group to be studied. Use as instructed in the apparatus’ manual.
6.2.3 Scientific literature on taxonomy
Scientific literature should be chosen according to the organism to be identified. A selection is given in
Annex B. This literature might carry a dichotomous or polytomous identification key to guide you through
the identification process. Additional drawings or photos could help in identifying the correct taxon.
6.2.4 Microscope slide
Microscope slides are composed of glass and are commercially available in the typical size of 76 × 26 mm
(length x width) and a thickness of 1 mm to accommodate the x/y-table of a microscope. Glass quality is
optimized not to interfere with the optical light path and the front lenses. Before use, the microscope slide
should be cleaned so that no particles can interfere with the microscope details of the organism to be
investigated.
6.2.5 Microscope cover glass
Microscope cover glasses are composed of glass and are commercially available in various sizes and
forms (round, square, rectangular). For broad applications, square microscope cover glasses with a size
of 18 mm × 18 mm and a thickness of 0,13 mm-0,16 mm are recommendable. The thickness of the
microscope cover glass is important if higher magnifications are applied, such as when using lenses > 50x
magnifying factor. These often are immersion objectives usually used with immersion oil. The thickness
of the microscope cover glass is optimized for the light path providing an optimal vision of the organism
studied.
6.3 Apparatus for molecular identification methods
6.3.1 Thermocycler
A thermocycler is a device used to amplify DNA samples by PCR. The thermocycler is programmed to
raise and lower the temperature of the samples in a thermal block allowing for denaturation, primer
annealing and elongation, and thus exponential amplification of DNA in a sample with various reagents.
Amplified genetic products can be used in many downstream applications such as cloning, sequencing,
expression analysis and genotyping.
6.3.2 Gel electrophoresis device
A gel electrophoresis device is an instrument that is used for separating the components of samples that
contain nucleic acid. The procedure is performed by placing the samples with a loading buffer (5.2.7) in
small wells at one end of an electrophoresis gel. A gel electrophoresis buffer (5.2.6) is used to provide
ions to help sample migration. An electrical source is attached and runs for a certain time. Use as
instructed in the apparatus’ manual.
6.3.3 DNA sequencer
A DNA sequencer is a device used to determine the nucleotide order. This is reported as a text called read
(sequence). Some DNA sequencers can be considered optical instruments as they analyse light signals
originating from fluorochromes attached to nucleotides. Use as directed in the apparatus’ manual.
6.3.4 Plastic consumables, DNA free, disposable
NOTE Negative PCR control can be used for confirmation that the consumables are DNA free.
EXAMPLE PCR tubes in single strips or plates, pipette tips
7 Principle
7.1 General
The selection of the identification method depends on the type of material and the target organism to be
detected and/or identified (Figure 1).
7.2 Morphological methods
Morphological and anatomic features of the whole thallus (macroalgae), portions or sections of the
specimens, filaments and/or single cells (microalgae, cyanobacteria and Labyrinthulomycetes) are
determined using the naked eye or magnifying devices. All data obtained from the characterization of
morphological features are compared with data in scientific literature (6.2.3) and often, using single-
access or multi-access identification keys (dichotomous or polytomous), the correct identity of a taxon
can be identified.
7.3 Molecular methods
DNA is extracted from the biomass by applying an appropriate method. Some algae like encrusting and
free-living coralline algae, might need a pre-processing step before the DNA extraction by removing the
surface of the thallus.
NOTE This can be done chemically or mechanically, e.g. with a drill bit. Furthermore, secondary metabolites,
especially of brown algae (e.g. specific carbohydrates) can hamper the DNA extraction when using commercially
available extraction kits. Full or partial length of 16S rDNA, 18S rDNA, ITS, COX1, tufA or rbcL are amplified by PCR.
PCR products can be either cloned or directly used for Sanger sequencing. The former procedure involves cloning
(transformation, bacterial incubation) and DNA isolation steps to prepare template DNA for sequencing. The
nucleotide sequence of the PCR product is then determined by a suitable DNA sequencing method (e.g. Sanger
sequencing).
The sequence is evaluated by comparison to reference sequences in databases, thus allowing the
assignment to a species or genus according to the degree of identity with the sequence of reference of the
genus or species in question. For macroalgae genus level genetic identification might be sufficient.
Other sequencing methods, like next generation sequencing, could also be used.
8 Procedure
8.1 General laboratory requirements
ISO 24276 shall be used for determining the general laboratory requirements.
8.2 Choice of methods
Several methods can be used for the identification of microalgae, macroalgae, cyanobacteria and
Labyrinthulomycetes as shown in Figure 1.
Figure 1 — Choice of methods
NOTE 1 There are special cases of rare algal taxa, for which the decision tree in Figure 1 might not be best
suitable. In such cases special processing of the algal sample might be necessary as well as other methodological
approaches to reach taxonomic identification.
NOTE 2 Alternative approaches for identification of alga taxa are described by Fawley and Fawley [18].
9 Morphological identification methods
9.1 General
This method should preferably be applied to wet (fresh) biomass, but may also be applied to dried
biomass. Dried biomass shall need to be rewetted in an isotonic solution (5.1.1) to obtain a suitable
preparation. Data obtained from morphological features needed for identification vary greatly between
the different taxonomic groups of organisms.
Features to be characterized in detail can refer to overall morphology (e.g. shape) or special anatomic
characteristics, depending on the taxon. A selection of scientific literature (6.2.3) that may be used for
identification is given in Annex B.
Depending on the characteristics requested by the identification keys and the biomass available, apply
either macroscopic (9.2) or microscopic identification (9.3) or both. Use of positive controls and/or
drawings or photos from scientific literature (6.2.3) of the target organism for comparison with the
organism in question can be of help for the identification based on morphological characteristics.
9.2 Macroscopic identification with the naked eye or a magnifying glass
In case of identifying macroalgae or filamentous microalgae, observation with the naked eye or the use
of a low-magnifying optical system (6.2.1) might be sufficient for identification. Use details such as colour,
shape, size, branching and the presence of organs (for example sexual organs) for the identification.
9.3 Light microscopy
9.3.1 General
When using light microscopy, cell shape, size and any other characteristics as outlined in the specific
scientific literature (6.2.3) on taxonomy are used to identify the organisms. If characteristics of single
cells and cell organelles are needed for identification, use a light microscope (6.2.2).
9.3.2 Staining
If relevant, specific stains should be applied to the cells prior or after preparation of the microscope slide
(9.3.3). Examples are Lugol's solution (iodine potassium iodide solution) e.g. for staining of starch or
making the cell nucleus better visible or methylene blue (methylthioninium chloride) also making the
nucleus better visible. Staining can be useful to visualize specific cell organelles or metabolites in the cell
if asked for in the identification key. These stains can either be visualized in normal light microscopy
mode or, in case of fluorescent dyes, when using fluorescence microscopy. In the latter case the
microscope needs to be equipped with a special light source, suitable filter systems and light path optics.
9.3.3 Preparation of microscope slides
To prepare microscopic slides, the following steps should be followed.
Suspend an aliquot of the wet or dry cells from the sample in an isotonic solution (5.1.1).
Apply a droplet of this suspension to one side of a microscope slide (6.2.4) and cover it with a microscope
cover glass (6.2.5).
Make sure that no air bubbles are trapped between the microscope cover glass and the microscope slide
as this might make microscopic identification difficult. To avoid air bubbles, first place one side of the
microscope cover glass at an inclined angle onto the droplet with the organism, then slowly lower on the
preparation completely.
9.3.4 Microscopic identification
Place the prepared microscope slide (9.3.3) on the x-y table of a light microscope (6.2.2) and fix it
according to the microscope's manual. Switch on the light source, revolve a front lens of suitable
magnification into the light path and focus for clear view on any cell part or organelle needed for
determining the characteristics stated in the identification key. Use brightfield or any contrasting option
such as phase contrast or differential interference contrast (DIC, Nomarski contrast) from the
microscope's setup if needed. Different viewing modes can be useful to visualize the different
morphological details of the cell or cell organelles that are necessary for identification according to the
identification keys mentioned in the scientific literature (6.2.3).
9.3.5 Use of identification keys
A choice of scientific literature (6.2.3) with identification keys is given in Annex B.
Go through each step of the identification key by observing the thallus, cell or organelle feature in
question. At each branching of the identification key, a question shall be answered, which will guide you
step by step through the identification key. If unsure, first proceed along one branch of the identification
key until either clear identification, or, in case this branch proves to assign your organisms of question to
an undoubtful wrong taxon, go back to the last branch and try the other option. Drawings and pictures
for each species in the identification ke
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