ISO/TS 21310:2020
(Main)Traditional Chinese medicine — Microscopic examination of medicinal herbs
Traditional Chinese medicine — Microscopic examination of medicinal herbs
This document specifies the methods for microscopic examination of medicinal herbs. It covers the equipment, sampling, preparation and observation methods. This document is applicable to medicinal herbs used in traditional Chinese medicine, including Chinese materia medica (whole medicinal materials) and decoction pieces derived from plants. It is not applicable to medicinal materials derived from animals or minerals.
Médecine traditionnelle chinoise — Examen microscopique des herbes médicinales
General Information
Relations
Buy Standard
Standards Content (Sample)
TECHNICAL ISO/TS
SPECIFICATION 21310
First edition
2020-07
Traditional Chinese medicine —
Microscopic examination of
medicinal herbs
Médecine traditionnelle chinoise — Examen microscopique des herbes
médicinales
Reference number
©
ISO 2020
© ISO 2020
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Sampling . 2
5 Apparatus . 2
6 Preparation for microscopic examination . 2
6.1 Cross-section or longitudinal-section slides . 2
6.2 Powder slides . 2
6.3 Mounting and swelling agents . 2
7 Observation of components . 3
8 Test report . 3
Annex A (informative) Preparation methods for microscopy . 4
Bibliography .10
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 249, Traditional Chinese medicine.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2020 – All rights reserved
TECHNICAL SPECIFICATION ISO/TS 21310:2020(E)
Traditional Chinese medicine — Microscopic examination
of medicinal herbs
1 Scope
This document specifies the methods for microscopic examination of medicinal herbs. It covers the
equipment, sampling, preparation and observation methods. This document is applicable to medicinal
herbs used in traditional Chinese medicine, including Chinese materia medica (whole medicinal
materials) and decoction pieces derived from plants. It is not applicable to medicinal materials derived
from animals or minerals.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
microscopic examination
examination of a test specimen by microscope with a magnification of generally × 50 to × 500
[SOURCE: ISO 17639:2003, 3.2, modified.]
3.2
medicinal herbs
raw materials derived from various parts of plants for drugs used in herbal medicine
Note 1 to entry: Herbal medicine includes traditional Chinese medicine, Korean medicine and Kampo.
3.3
slide
flat rectangular plate of glass on which an object is mounted for microscopic examination
[SOURCE: ISO 10934-1:2002, 2.133]
3.4
cover glass
rectangular or circular piece of thin glass used to cover a microscopical preparation
[SOURCE: ISO 10934-1:2002, 2.34, modified — Note 1 to entry removed.]
3.5
micrometer
device for measuring small lengths
[SOURCE: ISO 10934-1:2002, 2.96]
4 Sampling
Small-sized, cut or powdered material (50 g to 250 g) samples shall be taken after mixing thoroughly.
Large-sized or whole material (250 g to 500 g) samples shall be taken after mixing thoroughly. After
that, select a representative sample of the material. If necessary, the samples should be preserved in
airtight containers.
5 Apparatus
Use the usual laboratory apparatus and, in particular, the following:
5.1 Optical microscope or slide scanner.
5.2 Optical or in-software micrometer.
5.3 Imaging devices such as drawing attachments, embedded camera or digital imaging sensor for
the microscope.
5.4 Slides and cover glasses.
5.5 Botanical dissecting instruments such as tweezers, surgical knife, razor blade, microtome.
6 Preparation for microscopic examination
6.1 Cross-section or longitudinal-section slides
a) According to the sample condition, moisturizing, fixation or maceration process can be added. See
A.1 to A.3 for additional information.
b) Select representative pieces of the material being examined and cut into suitable lengths.
c) After softening, cut the material with a razor blade or a microtome to a thickness of 10 μm to 20 μm.
d) Place a section on a slide glass, add two or three drops of a mounting agent or chloral hydrate
solution and place a cover glass over it, taking precautions against the inclusion of bubbles.
e) Embed the material in hard paraffin for cutting, if necessary. See A.4.
6.2 Powder slides
a) Place about 0,1 g of powdered sample in a watch glass containing two or three drops of a swelling
agent or chloral hydrate solution, stir well with a small rod to prevent the inclusion of bubbles and
allow to stand for more than 10 min to swell the sample.
b) Using a small glass rod, smear the slide glass with a small amount of the swollen sample, add one
drop of the mounting agent and place a cover glass on it so that the tissue sections spread evenly
without overlap, taking precautions against the inclusion of bubbles.
6.3 Mounting and swelling agents
Mounting and swelling agents may be made of a mixture of glycerine and water (1:1) or a mixture of
glycerine, 95 % ethanol and water (1:1:1) as mounting and swelling agents. Other agents which have
characteristics of mounting and swelling agents can be used.
2 © ISO 2020 – All rights reserved
7 Observation of components
Observation can be conducted in the order of the outer portion, inner portion and cell contents. In
case of a powdered sample, observation can be made in the order of characteristic component, matter
present in large amounts, rarely existing matter and cell contents. For histochemical detection of the
sample, see A.6.
8 Test report
The test report shall include the following information:
a) all information necessary for the complete identification of the sample;
b) the sampling method used;
c) the test method used, with reference to this document, i.e. ISO/TS 21310:2020;
d) the test result(s) obtained;
e) all operating details not specified in this document, or regarded as optional, together with details
of any incidents which may have influenced the test result(s);
f) any unusual features (anomalies) observed during the test;
g) the date of the test.
Annex A
(informative)
Preparation methods for microscopy
A.1 Moisturizing
Dried parts of a plant may require softening before preparation for microscopy, preferably by soaking
in water. Use a desiccator for larger quantities of material, placing water into the lower part instead of
the drying agent.
Bark, wood and other dense and hard materials need to be soaked in water or in equal parts of water,
ethanol and glycerol for a few hours or overnight until they are soft enough to be cut. Boiling in water
for a few minutes can sometimes be necessary.
Any water-soluble contents can be removed from the cells by soaking in water. Starch grains can
be gelatinized by heating in water. In certain cases, material can be moistened with water for a few
minutes to soften the surfaces and allow sections to be cut.
A.2 Fixation
Fixation is the process of preserving the tissue by placing the tissue in fixatives. The permeation of
fixatives into the tissue can be dependent upon the size of the sample. Before fixation, it is recommended
that samples be cut smaller than 6 mm ⨯ 6 mm ⨯ 6 mm.
The following water solutions are used as fixatives:
— ethanol: 50 % to 70 %
— formalin: under 5 %
— acetic acid: approximately 100 %
— chromic acid: approximately 1 %
— F.A.A. solution: formalin 5 ml, acetic acid 5 ml, 50 % to 70 % ethanol 90 ml
— Craf III solution: 1 % chromic acid 30 ml, 10 % acetic acid 20 ml, formalin 10 ml, water 40 ml.
A.3 Maceration
A.3.1 General
For maceration, cut or slice the sample into small pieces about 2 mm in thickness. Depending on the
feature of the material, one of the following three methods can be used. For medicinal herb samples
with only a few or scattered woody tissues or with parenchyma tissues, use the potassium hydroxide
method. For hard materials mainly composed of woody tissues or woody tissues grouped into bundles,
use the chromic-nitric acid or potassium chlorate method.
A.3.2 Potassium hydroxide method
Place the sample in a test tube, add an adequate quantity of aqueous potassium hydroxide solution
(a volume fraction of 5 %), then heat until the residue can be easily separated when pressed with a
glass rod. Decant the alkaline solution and wash the residue with water. Transfer a small amount of
4 © ISO 2020 – All rights reserved
macerated material onto a slide and tease it out with a needle. Mount in dilute glycerine and examine
under a microscope.
A.3.3 Chromic-nitric acid method
Place the sample in a test tube and add an adequate quantity of chromic-nitric acid test solution, then
leave to stand until the material can be easily separated when pressing with a glass rod. Decant the
acidic solution, wash the residue with water and prepare the slide as directed in A.3.2.
A.3.4 Potassium chlorate method
Place the sample in a test tube, add dilute nitric acid (volume fraction of 50 %) and a few crystals of
potassium chlorate. Warm gently until the effervescence subsides, then add a few crystals of potassium
chlorate to maintain a slight effervescence. When the tissue shows a tendency to disintegrate, break
the material with a glass rod. Decant the acidic solution, wash the macerated material with water and
prepare the slide as directed in A.3.2.
A.4 Section
...
TECHNICAL ISO/TS
SPECIFICATION 21310
First edition
2020-07
Traditional Chinese medicine —
Microscopic examination of
medicinal herbs
Médecine traditionnelle chinoise — Examen microscopique des herbes
médicinales
Reference number
©
ISO 2020
© ISO 2020
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Sampling . 2
5 Apparatus . 2
6 Preparation for microscopic examination . 2
6.1 Cross-section or longitudinal-section slides . 2
6.2 Powder slides . 2
6.3 Mounting and swelling agents . 2
7 Observation of components . 3
8 Test report . 3
Annex A (informative) Preparation methods for microscopy . 4
Bibliography .10
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 249, Traditional Chinese medicine.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2020 – All rights reserved
TECHNICAL SPECIFICATION ISO/TS 21310:2020(E)
Traditional Chinese medicine — Microscopic examination
of medicinal herbs
1 Scope
This document specifies the methods for microscopic examination of medicinal herbs. It covers the
equipment, sampling, preparation and observation methods. This document is applicable to medicinal
herbs used in traditional Chinese medicine, including Chinese materia medica (whole medicinal
materials) and decoction pieces derived from plants. It is not applicable to medicinal materials derived
from animals or minerals.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
microscopic examination
examination of a test specimen by microscope with a magnification of generally × 50 to × 500
[SOURCE: ISO 17639:2003, 3.2, modified.]
3.2
medicinal herbs
raw materials derived from various parts of plants for drugs used in herbal medicine
Note 1 to entry: Herbal medicine includes traditional Chinese medicine, Korean medicine and Kampo.
3.3
slide
flat rectangular plate of glass on which an object is mounted for microscopic examination
[SOURCE: ISO 10934-1:2002, 2.133]
3.4
cover glass
rectangular or circular piece of thin glass used to cover a microscopical preparation
[SOURCE: ISO 10934-1:2002, 2.34, modified — Note 1 to entry removed.]
3.5
micrometer
device for measuring small lengths
[SOURCE: ISO 10934-1:2002, 2.96]
4 Sampling
Small-sized, cut or powdered material (50 g to 250 g) samples shall be taken after mixing thoroughly.
Large-sized or whole material (250 g to 500 g) samples shall be taken after mixing thoroughly. After
that, select a representative sample of the material. If necessary, the samples should be preserved in
airtight containers.
5 Apparatus
Use the usual laboratory apparatus and, in particular, the following:
5.1 Optical microscope or slide scanner.
5.2 Optical or in-software micrometer.
5.3 Imaging devices such as drawing attachments, embedded camera or digital imaging sensor for
the microscope.
5.4 Slides and cover glasses.
5.5 Botanical dissecting instruments such as tweezers, surgical knife, razor blade, microtome.
6 Preparation for microscopic examination
6.1 Cross-section or longitudinal-section slides
a) According to the sample condition, moisturizing, fixation or maceration process can be added. See
A.1 to A.3 for additional information.
b) Select representative pieces of the material being examined and cut into suitable lengths.
c) After softening, cut the material with a razor blade or a microtome to a thickness of 10 μm to 20 μm.
d) Place a section on a slide glass, add two or three drops of a mounting agent or chloral hydrate
solution and place a cover glass over it, taking precautions against the inclusion of bubbles.
e) Embed the material in hard paraffin for cutting, if necessary. See A.4.
6.2 Powder slides
a) Place about 0,1 g of powdered sample in a watch glass containing two or three drops of a swelling
agent or chloral hydrate solution, stir well with a small rod to prevent the inclusion of bubbles and
allow to stand for more than 10 min to swell the sample.
b) Using a small glass rod, smear the slide glass with a small amount of the swollen sample, add one
drop of the mounting agent and place a cover glass on it so that the tissue sections spread evenly
without overlap, taking precautions against the inclusion of bubbles.
6.3 Mounting and swelling agents
Mounting and swelling agents may be made of a mixture of glycerine and water (1:1) or a mixture of
glycerine, 95 % ethanol and water (1:1:1) as mounting and swelling agents. Other agents which have
characteristics of mounting and swelling agents can be used.
2 © ISO 2020 – All rights reserved
7 Observation of components
Observation can be conducted in the order of the outer portion, inner portion and cell contents. In
case of a powdered sample, observation can be made in the order of characteristic component, matter
present in large amounts, rarely existing matter and cell contents. For histochemical detection of the
sample, see A.6.
8 Test report
The test report shall include the following information:
a) all information necessary for the complete identification of the sample;
b) the sampling method used;
c) the test method used, with reference to this document, i.e. ISO/TS 21310:2020;
d) the test result(s) obtained;
e) all operating details not specified in this document, or regarded as optional, together with details
of any incidents which may have influenced the test result(s);
f) any unusual features (anomalies) observed during the test;
g) the date of the test.
Annex A
(informative)
Preparation methods for microscopy
A.1 Moisturizing
Dried parts of a plant may require softening before preparation for microscopy, preferably by soaking
in water. Use a desiccator for larger quantities of material, placing water into the lower part instead of
the drying agent.
Bark, wood and other dense and hard materials need to be soaked in water or in equal parts of water,
ethanol and glycerol for a few hours or overnight until they are soft enough to be cut. Boiling in water
for a few minutes can sometimes be necessary.
Any water-soluble contents can be removed from the cells by soaking in water. Starch grains can
be gelatinized by heating in water. In certain cases, material can be moistened with water for a few
minutes to soften the surfaces and allow sections to be cut.
A.2 Fixation
Fixation is the process of preserving the tissue by placing the tissue in fixatives. The permeation of
fixatives into the tissue can be dependent upon the size of the sample. Before fixation, it is recommended
that samples be cut smaller than 6 mm ⨯ 6 mm ⨯ 6 mm.
The following water solutions are used as fixatives:
— ethanol: 50 % to 70 %
— formalin: under 5 %
— acetic acid: approximately 100 %
— chromic acid: approximately 1 %
— F.A.A. solution: formalin 5 ml, acetic acid 5 ml, 50 % to 70 % ethanol 90 ml
— Craf III solution: 1 % chromic acid 30 ml, 10 % acetic acid 20 ml, formalin 10 ml, water 40 ml.
A.3 Maceration
A.3.1 General
For maceration, cut or slice the sample into small pieces about 2 mm in thickness. Depending on the
feature of the material, one of the following three methods can be used. For medicinal herb samples
with only a few or scattered woody tissues or with parenchyma tissues, use the potassium hydroxide
method. For hard materials mainly composed of woody tissues or woody tissues grouped into bundles,
use the chromic-nitric acid or potassium chlorate method.
A.3.2 Potassium hydroxide method
Place the sample in a test tube, add an adequate quantity of aqueous potassium hydroxide solution
(a volume fraction of 5 %), then heat until the residue can be easily separated when pressed with a
glass rod. Decant the alkaline solution and wash the residue with water. Transfer a small amount of
4 © ISO 2020 – All rights reserved
macerated material onto a slide and tease it out with a needle. Mount in dilute glycerine and examine
under a microscope.
A.3.3 Chromic-nitric acid method
Place the sample in a test tube and add an adequate quantity of chromic-nitric acid test solution, then
leave to stand until the material can be easily separated when pressing with a glass rod. Decant the
acidic solution, wash the residue with water and prepare the slide as directed in A.3.2.
A.3.4 Potassium chlorate method
Place the sample in a test tube, add dilute nitric acid (volume fraction of 50 %) and a few crystals of
potassium chlorate. Warm gently until the effervescence subsides, then add a few crystals of potassium
chlorate to maintain a slight effervescence. When the tissue shows a tendency to disintegrate, break
the material with a glass rod. Decant the acidic solution, wash the macerated material with water and
prepare the slide as directed in A.3.2.
A.4 Section
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.