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

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Publication Date
14-Jul-2020
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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/TS 21310:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO/TS 21310:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
---------------------- Page: 2 ----------------------
ISO/TS 21310:2020(E)
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

© ISO 2020 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO/TS 21310:2020(E)
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
---------------------- Page: 4 ----------------------
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]
© ISO 2020 – All rights reserved 1
---------------------- Page: 5 ----------------------
ISO/TS 21310:2020(E)
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
---------------------- Page: 6 ----------------------
ISO/TS 21310:2020(E)
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.
© ISO 2020 – All rights reserved 3
---------------------- Page: 7 ----------------------
ISO/TS 21310:2020(E)
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 %
...

TECHNICAL ISO/TS
SPECIFICATION 21310
First edition
Traditional Chinese medicine —
Microscopic examination of
medicinal herbs
PROOF/ÉPREUVE
Reference number
ISO/TS 21310:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO/TS 21310:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii PROOF/ÉPREUVE © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 21310:2020(E)
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 ...................................................................................................................................................................... 2

8 Test report ................................................................................................................................................................................................................... 3

Annex A (informative) Preparation methods for microscopy .................................................................................................... 4

Bibliography .............................................................................................................................................................................................................................10

© ISO 2020 – All rights reserved PROOF/ÉPREUVE iii
---------------------- Page: 3 ----------------------
ISO/TS 21310:2020(E)
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 PROOF/ÉPREUVE © ISO 2020 – All rights reserved
---------------------- Page: 4 ----------------------
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]
© ISO 2020 – All rights reserved PROOF/ÉPREUVE 1
---------------------- Page: 5 ----------------------
ISO/TS 21310:2020(E)
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.
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

2 PROOF/ÉPREUVE © ISO 2020 – All rights reserved
---------------------- Page: 6 ----------------------
ISO/TS 21310:2020(E)

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:—;
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.
© ISO 2020 – All rights reserved PROOF/ÉPREUVE 3
---------------------- Page: 7 ----------------------
ISO/TS 21310:2020(E)
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: appro
...

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