Instrumentations Manual in

BIOLOGY

Editors
J B Bhandari
C Gurung

Narosa Publishing House

New Delhi Chennai  Mumbai   Kolkata
Instrumentations Manual in Biology
114 pages

Editors
J B Bhandari
C Gurung
Department of
T
T

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 FOREWORD

It gives me immense pleasure to know that the Department of Botany of my

college is publishing the workshop papers presented by the Resource Persons of
the first UGC sponsored Instrumentation Workshop entitled “Recent Trends In
Applications of Modern Scientific Instruments for Biological and Bio-medical
Researches”organized by the Department during December 20-22, 2013 in the
form of a book “Instrumentaion Manual in Biology”.
Instrumentation is central to the study of Biological Sciences, especially at the
molecular level. The development of numerous techniques and highly sophisticated
instruments in Biology today have created an urgent need to understand the
physical principles involved in the operation of research instruments and the
parameters required in using them. Keeping this need in mind, the editors have
tried their best to present the compilation of some of the papers in the form
of “Instrumentaion Manual in Biology”. I hope this book will truely fulfill this
need by addressing the different aspects of instrumentation from basic operation
to advanced instrumentation which hold the keys to cutting-edge research and
innovative applications.
Malay Kanti Karanjai
 HERBARIUM TECHNIQUES
A. P. Das
Department of Botany, University of North Bengal, Siliguri 734013,
West Bengal, India
E-mail IDs: apdas.nbu@gmail.com

ABSTRACT
Preservation of plant materials is a huge subject. Techniques differ for different
groups or habits of plants and is also related to the purpose of preservation.
Herbariums are the natural history museums storing and displaying specially
mounted dried and poisoned plant specimens meant for numerous scientific and
social benefits. The article discussed the methods of herbarium-sheet preparation
starting from plant collection leading to the final storage, maintenance and
utilization.
Some special techniques like colour preservation in herbarium-specimens and the
digitization of herbariums are also discussed with details of some easily applicable
methods.
Herbaria round the world are recording the planet’s plant diversity and the people
of future generations will be enriched about the lost plants of the world as the rate
of extinction of species is increasing very fast.

RECOGNITION IS IMPORTANT
Recognition of everything is a major aspect of our daily life and a recognized
article or matter needs a name to refer it to another person. In fact, recognition and
naming are basic instincts in human life, which are now scientifically expressed in
the subject of Taxonomy. As it understood now, ‘Identification’ and ‘Nomenclature’
are two main elements of working Taxonomy. Lawrence (1951) defined taxonomy
as “It is a science that includes identification, nomenclature and classification of
objects ….”

IDENTIFICATION AND NAMING

When we face any object, living or non-living, we try to recognize it and refer it
with a name. This part of the total effort is identification. In this process if we find
 Herbarium Techniques 79

there is no existing name for an object then we coined it with a freshly prepared
name following the social cultures and customs or rules and this part of the effort
is naming and this entire methodology is referred to as nomenclature.

NEED OF PERMANENT VOUCHER

After recognition of a plant as ‘new to science’ if we provide it a new scientific
binomial and if that name is referred to another person then, again, it will not
be possible for the second person to recognize the plant as he has seen the plant
never before. This problem led to the formation of another method of tagging a
voucher with each name and the maintenance of those vouchers. This is also a
huge subject and the methodology differs according to the nature of the object.
For the nomenclature of plants ICN (2012) is the only recognized guideline which
indicated the importance of keeping vouchers for each and every published name
of lower rank, with no exception.

HOW MANY SPECIES?

The total number of plants on earth is estimated to be around 4 million (https://
www.bgci.org/policy/1521/) and the total number of biological species i.e. plants,
animals and microbes (lichens, fungi, bacteria) is well over 7.2 million (Chapman
2009; https://www.currentresults.com/Environment-Facts/Plants. Animals estimate
-of-worlds- total-number-of-species.php). And, being a student of plant science it
is essential to recognize 4 million species by their names. This is one impossible
task for a person to identify all species of plants. To resolve this great problem the
idea of keeping vouchers for each minor taxon was perceived. A ‘voucher’ will be
a representative specimen for a taxon, generally of species and infra-specific ranks
(ICN, 2012).

PRESERVATION METHOD DIFFERS

Structurally all plants are not of similar nature and certainly method of their
preservation also differs, especially when numerous species are either microscopic
or extremely slender thread-like, uni- or few-celled and devoid of vascular strands
(DeWolf, 1968; Humphries & Huxley, 1999). So, the preservation technique for
species of Spirogyra and of Mangifera can’t be same. For Spirogyra we keep a
large number of its extremely slender filaments as specimens in a bottle containing
liquid preservative (e.g. FAA, diluted formalin) or in a prepared permanent slide.
But, for the species of Mangifera one twig of the plant is dried within blotters,
poisoned and paste the dry specimen on a thick rag-paper mount-board. Each of
these mounted vouchers are referred as a ‘Herbarium specimen’.
80 Instrumentations Manual in Biology

HERBARIUM SPECIMENS
This is a method of preservation of voucher specimens for vascular plants, i.e.
for pteridophytes, gymnosperms and angiosperms. However, sometimes, for some
non-vascular plants also specimens are preserved in this manner (Thiers, 2017).
For this a twig or one entire small plant is collected and dried in a plant-press
keeping within the blotters, poisoned and then mounted on a strong paper board,
commonly referred as ‘herbarium sheet’ using adhesive glue. The standard size
of herbarium sheet is 29 × 43 cm (Woodland, 1997). One ‘Herbarium Label’ is
attached generally to its lower right corner to record different data related to that
particular specimen. Such mounted and labelled herbarium sheets are stored in a
museum that is generally referred as ‘Herbarium’.

HERBARIUM
The name ‘Herbarium’ [Herbaria in plural] is referred to one or more rooms or a
building where the mounted and labelled herbarium-sheets are stored systematically.
In fact, it is a museum but referred as ‘Herbarium’ as it stores such specific type of
specimens only. Generally an accepted system of plant classification is followed
for the arrangement of specimens in a series of ‘Herbarium cabinets’.
History: In around the year 1700 for the first time Turnfort used the term
‘Herbarium’ for a collection of dry specimens of medicinal plants and later on Carl
Linné (Carolus Linnaeus) started using the term regularly. However, originally, the
word Herbarium referred to a book of such mounted specimens of medicinal plants
(Stearn, 1957; Bridson & Forman, 1998). As far the records are available, Luca
Ghini (1490 – 1556), a professor of botany in Italy, first dried plants under pressure
and pasted those on papers as long-term records (Arber, 1938). At the beginning
herbarium-sheets were bound as book-like volumes, later on with the increase
of collections and for easy handling sheets were kept free like reference cards.
Gradually, earlier alphabetical arrangements were replaced by accepted taxonomic
classifications to trace a specimen easily and to understand their relationships. This
also helps in easy maintenance of the Herbarium. Depending on the regional extent
and purpose of different Herbaria those can be termed variously but the important
ones may be recognized as Regional, National or International.
Uses: The use of Herbarium is linked to a large number of fields of study including
morphology, taxonomy, ethnobotany, pharmacology, biochemistry, cytology,
genetics, molecular biology, material science, ecology, conservation, plant
pathology, plant breeding and physiology (Metsger & Byers, 1999). Bridson and
Forman (1998) summed up the services of a herbarium as:
i. How plants can be recognised – i.e. Identification
ii. How they can be named in order to transfer information about those plants –
i.e. Nomenclature
 Herbarium Techniques 81

iii. To trace the close relatives of a useful plant as those might have similar
properties – i.e. Classification
iv. Locating the areas of their occurrence – i.e. Distribution
v. Understanding their habitat preference – i.e. Ecology
vi. To know their useful properties – i.e. Uses

BASIC TECHNIQUES FOR PREPARING HERBARIUM SHEETS

Schepanek & Waller (1999) delineate the overall management guidelines for
running a Herbarium. However, the purpose of the present article does not warrant
the details of the usefulness of Herbaria but the methodology for the preparation
of herbarium-sheets need to be discussed with possible details. The book entitled
‘A Handbook of Field and Herbarium Methods’ (Jain & Rao 1977) is one such
important publication that can be used by any collector/ curator.
For the preparation of mounted herbarium-specimens different important steps
include: (i) collection of specimens; (ii) making the specimens ready for drying;
(iii) drying of specimens; (iv) poisoning of dry specimens; (v) mounting; (vi)
labelling; (vii) storing; and (viii) housing.
1. Collection: Collection of specimens for herbarium in general is expected
to be a good representative part of the plant or it may be one entire plant.
Good representative specimen indicate that it should be in reproductive stage
bearing flowers and/or fruits along with leaves. But, that is a difficult task
because in good proportion of species flowering and vegetative growth takes
place in different seasons. Again, some characters are available only during
active vegetative stage or even during winter or draught perennation. Majority
of the species remain in vegetative stage for major part of the year and it is not
possible to visit all remote habitats in different seasons to collect their flowers
and fruits or may be the vegetative parts. Some species also produce flowers
for a very brief period and the total life span of many ephemeral herbs are
just 1 – 2 weeks only. For deciduous species it is not always possible to get
leaves and flowers at a time as many species flowers before the appearance of
leaves. Palms create different type of problem as their leaves, inflorescence
and in many cases fruits too are so big to accommodate in a herbarium sheet.
Leaves and inflorescence of many aroids are also extremely large. Families
like Palmae, Araceae, Typhaceae, Pandanaceae, Agavaceae, etc. pose such
problems. For small herbs one entire plant including root is to be collected.
Plants with underground modified stem (rhizome, tuber, bulb, etc.) are to be
preserved along with those specialized organs. So, for all such cases one need
to take some logical decisions and the preserved specimen should have proper
representation of different parts of plant-body. For further details regarding
82 Instrumentations Manual in Biology

the collection of difficult plant materials one can go through the publications
like Bailey (1946), Marchant et al. (2001), Stone (1983), Fish (1999) and
Harris et al. (2008).
After plucking the sample it is to be kept inside a ‘vasculum’ or in its absence
in good polythene bags with mouth kept close, preferably tied with thread or
rubber-band. During dry season little water needs to be sprinkled inside the
bag and before tying one should make it inflated by blowing in with mouth.
This avoid the chances of damage due to pressure from outside.
Use knife or scissors or secateurs for cutting the branches and for underground
parts use a suitable digger.
Use a GPS to record the geographical location and an altimeter to record the
elevation of the precise habitat. Also take close photographs of different plant
parts including bark.
Record each plant in the ‘Field-Note-Book’ along with different other
perishable characters and relevant field-data. It is better not to rely on your
memory and the field-records must be noted in the field only. Also, tie the tag
with the specimen that will carry the field-number against which the plant has
been recorded in the Field-Note-Book.
Process the specimens at the earliest in the field-camp itself.
2. Pre-treatment: Before putting the specimens in the Plant-Press few more
steps are to be taken: (i) tagging with field numbers to all the duplicates;
(ii) trimming to a suitable size; (iii) fixing the entire specimen or to the
fragile points like leaf-bases, flowers, etc. using some chemicals like diluted
formalin; (iv) for colour retention dipping in special solution, etc.
When, it is scheduled to return to the laboratory after a long time then a
different method can be followed. Keep the trimmed and shaped specimens
within the blotters, add diluted formalin (2:3, formalin : water) (Fosberg &
Sachet, 1965; Smith, 1971) on the specimens using droppers or a brush so
that some amount is also soaked into the blotters. These are then made into
thick bundles, insert in a polythene bag, seal the bag tightly and then put it
in another similar bag and seal it again. Put one identity/ reference mark on
the packet. Or, instead of putting formalin solution on each blotter, soak the
entire packet taking the solution in a bucket or in such a similar big container
for about two hours; keep it outside to drain off the excess solution and then
seal it twice in polythene bags. Such packed specimens can be stored for a
longer period and after drying, these specimens may not be poisoned again.
One important caution, while using formalin switch-off the overhead fan. If
available use a pedestal or table fan from your back side otherwise evaporated
formalin will affect the eyes. Or, if you are working in open area then check
the direction of air-flow so that evaporated formalin moves away from you.
 Herbarium Techniques 83

And, the use of gloves during such works is a must!

3. Drying: This is a crucial stage for herbarium-sheet preparation. If the field
or the study area is a remote place and the collectors are not returning to the
main laboratory on the same day then one small portable plant-press is to be
used for initial drying in the field and specimens are to be shifted to a larger
and heavy plant-press on return to the laboratory. Folded blotting papers or,
in its absence, folded newsprints can be used as blotters. Good quality glossy
newspapers are not suitable for this purpose as such papers will absorb water
very slowly.
While placing the specimens on the blotters it is essential to keep at least one
leaf showing the lower (dorsal) surface. It is important as the lower surface
of lamina bears many important characters. Avoid to keep many specimens
in one sheet and as far as possible there will be no overlapping. At this stage
check carefully that all the specimens, including duplicates, are tagged with
field-number or not.
Timing for changing blotters is very much related to the nature of the plant
specimen. If it is a soft aquatic plant, like the species of Ottelia or Vallisneria,
then for the first day change the blotters in every hour to avoid any damage to
the specimen. Also, do not exert heavy pressure from the beginning. Increase
the pressure slowly and that may be from the second day. However, for normal
mesophytes give changes every day for the first 3 – 4 days at the beginning
and then gradually increase the duration until complete drying.
However, for drying of huge bulk of specimens some special arrangement is
essential. Also, almost similar arrangement is essential during rainy season
and especially in moist and cold areas. Generally for large amount of materials
horizontal plant-press is used. In such press use of corrugated aluminium
sheets is very important for proper aeration that helps in the removal of water-
vapour and carrying heat into the blotters. In cold areas and in moist seasons
the entire set is covered with tarpaulin and one heater is placed inside. In the
field camp a kerosene stove can replace the electrical heater.
4. Poisoning: Poisoning of dried specimens is very important as those are prone
to become food for many insects and microorganisms. So, to save the plant-
specimens from these organisms one or more chemicals are used. The most
popular is the mercuric-chloride. Generally 4 – 6 % solution of HgCl2 in
rectified spirit (90 – 95 % ethanol) is taken in a PVC or enamel coated tray as
HgCl2 is one highly corrosive chemical. Trays must be 40 – 60 cm long. The
side of the tray which is away from the worker’s body is generally kept little
up using some sort of padding. A stainless-steel wire-net is kept across part
of the tray or on a separate tray for draining off the excess solution from the
specimens. Well-dried specimens are dipped into the solution, kept for 1 – 2
minutes then taken out from the solution using broad-tipped forceps and then
kept on the wire-net for dripping off the excess liquid for some time. Moist
84 Instrumentations Manual in Biology

specimens are to be transferred in to the blotters and need to be dried under

pressure. If those are allowed to dry outside then there will be severe curling
of the specimens that may lead to the fragmentation and loss of the material.
But, now, the use of HgCl2 is no more recommended.
Many herbaria now successfully use Lauryl pentachlorophenate (LPCP), also
known as dodecanoic acid or mystox or phenyl pentachlorophenate. A 3.75 %
solution of LPCP in white spirit (a highly volatile petroleum distillate) is used.
Bundles of specimens are dipped in it for few minutes and then again dried
under pressure (Fosberg & Sachel, 1965). This is a much safer alternative.
Few herbaria also use Sodium pentachlorophenet (Eusebio & Stern, 1964).
5. Mounting: Mounting of dry and poisoned specimens on a herbarium-mount
need some additional artistic sense also. Placement of the main part of the
specimen, additional parts like extra flowers, fruits, seeds and any other
desirable part needs basic morphological knowledge and at the same time it is
also essential to make it beautiful.
Old herbaria, even today, use temperature sensitive glue as adhesive for
mounting. It is having the benefit of opening the specimen easily by heating
below the herbarium sheet and that could be re-fixed by cooling.
However, now-a-days, most of the Herbaria use PVA (Polyvinyl Acetate) glue
(Down et al., 1996). If purchased in powdered condition then it could be
dissolved in water and made into a desirable consistency. After drying such
paste will be almost permanent and no longer remain water-soluble. In Indian
market most easily available brand of PVA solution is Fevicol. Starch based
adhesives are not suitable for mounting herbarium specimens as those have
several draw-backs.
The glue can be added in different manner. Keep the specimen’s lower-
surface up on a clean blotter or on a glass-plate. Smear the glue on it using a
moderately soft brash. It should be smeared uniformly on all parts. Then, one
blank herbarium mount is to be kept on it, putting little pressure and turn the
lower side up keeping one hand below the blotter. Now, remove the blotter
slowly and carefully so that all plant parts remain attached to the herbarium
mount only. Also, fix the tag to the mount-sheet using adhesive. Now, transfer
the freshly mounted sheets inside a folded blotting paper and keep it under
little pressure till drying.
In traditional practice, the glue is smeared on a clean glass plate. Keep the
specimen on it and allow the glue to smear on the lower surface of the
specimen. Slowly take out the specimen from the glass plate and place it on
a blank herbarium mount. Transfer the sheet into a folded blotting paper and
keep it under pressure till drying.
In some Herbaria instead of glue specimens are attached to the mount using
3M archival mounting tape. This tape is available in different width. Narrow
 Herbarium Techniques 85

tapes are used for delicate plant parts and the wider ones for the stems of
different thickness. The main benefit of using these sticky tapes is opening of
specimens from the mount-sheet can be done very easily and can be re-fixed.
However, commonly used fixing tapes like sellotape (or cellotape) is strictly
prohibited (Lawrence, 1951) as it loses its grip very soon. Herbarium
specimens are made for storing and using for hundreds of years so the most
stable sticking method is to be followed.
Sometimes, some parts of specimens need to fix with the herbarium mount by
stitching using needle and strong-durable threads. Thick stem, fruits, seeds,
cymba of palms, etc. are to be fixed in such manner otherwise specimens may
be separated from the mount-sheet and the entire specimen may be damaged.
Paste a Herbarium label on the mounted Herbarium sheet, preferably near the
lower-right corner.
6. Labelling: This is also one important part of the entire process. A ‘Herbarium
Label’ is generally formatted as per need of the collection (Voss, 1999).
However, some basic data should be there in any such label. These include:
(i) Field number; (ii) Date of collection; (iii) Place of collection; (iv) Name of
the plant; (v) Family; (vi) Habit; (vii) Habitat; (viii) Availability; (ix) Local
name; (x) some characters those will not be available from a dry specimen,
etc. In fact, all these data were recorded in the field-note-book at the time of
collection and those field-data are now transferred to labels.
While writing the date of collection it is suggested to write the year in full
form. So, one should write “July 22, 2016” and not “July 22, ’16”
So, now the herbarium-sheets are ready to work and for inserting in Herbarium
Cabinets.

TEMPORARY STORING
Generally herbarium-sheets are not inserted in the main Herbarium immediately
after preparation. Those are commonly stored in a separate wooden or steel cabinet
for their immediate use in the running project. It is better to keep the specimens in
such temporary store following alphabetical sequence of family or genus names.
However, these are to be transferred to the main Herbarium at an earliest possible
time so that specimens can be accessed by all the users. Sometimes, duplicates are
distributed in other herbaria.

HOUSING OF MOUNTED SPECIMENS

Prepared herbarium specimens are finally deposited and inserted in one organized
Herbarium. There are a few steps in inserting such specimens those include:
(i) Fumigation of specimens; (ii) Recording in the Herbarium Accession Book/
Register; (iii) Putting the sheets in folders for species or infra-specific categories;
86 Instrumentations Manual in Biology

and (iv) Finally, inserting those folders at proper place in the pigeon-holes of
Herbarium-cabinets.
Special arrangement for storing Type specimens is very important as these are most
important irreplaceable materials. These are generally kept in specially designed
room and cabinet to protect the specimens from any incidence of fire.
Fumigation is one very important step especially for the specimens entering the
Herbarium from outside. Through fumigation any possible entry of insects and
microbes into the Herbarium is avoided. Previously the highly poisonous carbon-
tetrachloride was used to fumigate the specimens. For this one highly controlled
Fumigation Chamber was essential. But, the use of this chemical in Herbaria
is now highly restricted. To replace such poisonous chemicals low temperature
refrigerators are commonly used. Bundles of specimens are kept in the refrigerator
at below –20° C for 7 – 14 days [www.floridamuseum.ufl.edu/herbarium/policy/
pestcontrol.htm]. It is a safer technique but takes quite a long time for each
treatment.
The Accession Book/ Register maintains the stock of the sheets stored in a
Herbarium. The serial Accession number is then written or printed on the accessed
herbarium-sheet. The Accession number of a sheet is its recorded identity.
Recently use of barcode numbers for accession is in use where most of the
herbarium data is stored in computers. Use of a barcode reader help the reader
to access the data related to a specimen almost immediately. While Accession
numbers are printed to the lower left corner of the sheet, the barcode-label is pasted
towards the upper-right corner. However, this left/ right placement of numbers
and/or stickers is not mandatory.

FUMIGATION OF HERBARIUM
Regular fumigation of Herbarium is very important. Some herbaria are air-
conditioned and some are not! The Herbaria facing the natural environmental
conditions are more prone to regular entry of different insects and microbes.
Though less, unwanted organisms also inter the air-conditioned Herbaria. So,
regular fumigation of Herbarium is very important and the entire Herbarium is
fumigated generally using some chemicals.
The chemicals used for fumigation include methyl bromide, phosphine, ethyl
oxide, permethrin, deltamethrin, pestigas and pyrethrum. Some other chemicals
are also used less commonly like carbon tetrachloride, carbon disulphide, hydrogen
cyanide, ethylene dichloride and sulfuryl fluoride (Hall, 1988). Some Herbaria
uses a standard insect bomb or some surface spray like Cislin. The use of methyl
bromide is controversial but it is still in use in many Herbaria. [http://www.chah.
gov.au/insect-control/index.html].
 Herbarium Techniques 87

Herbarium fumigation also can be done using some common gasses like nitrogen
and carbon-di-oxide. It can also be done by keeping the specimens in 45°C for 2 –
3 hours. In freezing method, specimen bundles are kept at –18°C to –30°C at least
for 48 hours. Other methods like gamma radiation and microwaves were also used
in different herbaria.
For regular maintenance use of common insect-repellents like naphthalene and
para-dichlorobenzene (PDB) is common. But, these two materials should not be
used together.

HERBARIUM ACRONYMS
The name of a Herbarium may be quite long and made of a number of long and short
words. That creates problem in the reference system. Probably, due to this reason
each Herbarium is now referred with a short ‘acronym’. The Central National
Herbarium or the Calcutta Herbarium, located inside The Acharya Jagadish
Chandra Bose Indian Botanic Garden near Kolkata is known by the acronym
‘CAL’. It is ‘K’ for the Kew Herbarium and ‘NBU’ for the North Bengal University
Herbarium. Acronyms of different herbaria are recorded in Index Herbariorum.
This was originally published by the International Association of Plant Taxonomy
(IAPT) and its 8th printed edition was edited by Patricia Holmgren from St. Louis
Botanical Garden [https://www.nybg.org/science-project/index-herbariorum]

FIELD AND LABORATORY EQUIPMENT

For the proper collection of vascular plant material and processing of specimens
leading to the final insertion to the Herbarium-cabinet a number of tools/ kits/
equipment/ facilities are required (Victor et al., 2004; ANBG, 2017). These may
be listed as follows:
I. For the Field:
i. Field Press: light-weight portable one, preferably made of wooden plates and
a broad strap for binding
ii. Driers: Blotting papers, old newsprints
iii. Field-Note-Book [FNB]: to record different field data regarding the collected
specimens and also the habitat
iv. Tags: generally FNBs are provided with tags (including thread for tying); if
such a FNB is not used then separate string-tags may be used; the collection
number as recorded in the FNB that should be written on (preferably with a
pencil or with permanent ink) the tag
v. Diggers & Clippers: garden trowel – preferably with a steel shank or any
other type of locally available light-weight metallic digger; also knife or
scissors or secateurs for cutting the branches.
88 Instrumentations Manual in Biology

vi. Hand lens: a 10X hand-lens for quick observation

vii. Vasculum or collection bags: now-a-days vasculum (a light-weight metallic
chamber with a strap for hanging from the shoulder) is rarely used; polythene
bags are more popular, easy to use and can carry more specimens but any
pressure on specimen-filled such bags can damage the specimens
viii. Small bottles, envelopes, etc.: to collect loose materials; same field-numbers
need to be written on those
ix. Camera: with provision for taking close-up and zoomed pictures is a must
x. GPS: to record the precise location of the study area and/or of the plant
xi. Altimeter: to record the altitude of the collection site
xii. Maps & colour-charts: very much essential in the field.
Apart from these some other materials also may be required during field works
and those include (a) liquid preservatives [diluted formalin, FAA, 70% ethanol],
(b) waxed paper, (c) trays, (d) first-aid-box, etc. If need to stay in field camps
then all necessary camping materials need to be taken (including tent, insecticides,
repellents, etc.)
II. In the Laboratory:
i. Heavy Plant-Press: this may be wooden press for limited amount of
specimens; for large bulk of specimens large parallel press with tarpaulin
cover and heating arrangement (oil or electric based) may be used
ii. Blotters: blotting papers and/or old newsprints in good amount
iii. Corrugated Aluminium sheets: these are inserted in regular intervals within
the blotters; these helps in aeration that take out the water-vapour easily from
the blotters and specimens and carry the heat inside
iv. Hot-Air-Oven: sometimes used to achieve desired level of drying
v. Formaldehyde and/or other fixing chemicals: to avoid decomposition of
soft plant parts. However, to save formalin sensitive materials those may
be kept in boiling water for 12 – 15 seconds, or the plants may be taken in
polythene bags and sprayed with enough alcohol for 12 – 24 hours to check
the development of abscission layer
vi. Trays, Wire-nets & broad-tipped forceps: basic arrangement for poisoning
the dried specimens
vii. Rectified spirit & Mercuric chloride or White spirit & Lauryl
pentachlorophenate (LPCP): for poisoning the dried specimens
viii. Blank Herbarium-mount, Herbarium labels, small paper packets, needle
& thread, scissors, wax-paper, etc. – all these are required for proper
mounting.
 Herbarium Techniques 89

xiii. Suitable papers for species and genus covers; family covers are also
sometimes essential especially in small Herbaria.

COLOUR PRESERVATION
While drying the plant specimens under normal condition in a Plant-Press those
lose their natural colour mainly due to the oxidation of phenolic compounds. Not
only morpho-taxonomists, for pathological and virological works retention of the
natural colour of the specimen is very important. There are many methods and/or
chemical formulations available for colour preservation in plant specimens. Only
two simpler methods are presented below:
i. Calcium-chloride method: Drying over a layer of granular Calcium-chloride
(CaCl2) is probably the most convenient method (Kyriakopoulou, 1982). On
a flat table a bed of CaCl2 is made inside a polythene bag and a plastic net is
kept over it to work as a separator. Now, the specimen is first taken in a folded
blotting paper and keep under heavy pressure for a few hours to lose some
amount of water. Then it is to be taken in a fresh and clean blotting paper and
insert into the polythene bag and placed on the separator (plastic net). Seal
the mouth of the polythene bag to make it air tight. Put some pressure on it.
The material will be dried. The entire set may be kept in room-temperature or
in a refrigerator for 3 – 10 days, depending on the nature of the material, for
proper drying.
ii. Liquid solution method: Prepare a solution with following chemicals (Keefe,
1926; Blaydes, 1937):
90 ml of 50 % ethanol
5 ml of formalin
2.5 ml of glycerine
20 gm of cupric chloride, and
2.5 gm of uranium nitrate.
Properly cleaned and trimmed materials are kept in a good quality polythene
bag or in a PVC tray. Pour sufficient amount of the solution. Keep it closed
for few days and then transfer to the plant-press for drying. This preserve the
greenness of the plant material nicely.

DIGITAL HERBARIUM
Three main factors are working behind the digitization of Herbarium. The space is
a big factor as to store and conserve millions of specimens along with processing
and maintenance facilities. Then, the regular handling of specimens is liable to
damage and thereby reducing the life-span. But, we need to use these materials for
an indefinite period and specially the Types that are irreplaceable materials. And,
90 Instrumentations Manual in Biology

the third factor is the accessibility of specimens to the larger section of scientists
particularly those who live in faraway places. Digitization of herbarium-specimens
can avoid all these three situations.
For digitization, herbarium specimens are either (i) scanned using an inverted
scanner, as mounted herbarium sheets are not permitted to keep up-side-down,
and the images are stored in the computer in digital form; or (ii) the specimens are
photographed using a good digital camera and then the images are transferred to a
computer (preferably in external Hard Disk) for storing.
Use of an inverted scanner is supposed to consume longer time and scanning and
scanned-data processing. It is suitable for small herbaria. However, use of a digital
camera is preferable and with little effort a workstation can be prepared. Following
are the minimum requirements (modified after Harris & Marsico, 2017):
i. A digital SLR camera, minimum 18 megapixel, with remote control hand-set
ii. A smooth flat bed for placing the herbarium-sheets below the camera
iii. A monopodial camera-stand with an adjustable mount to which the camera
can be fixed
iv. Two or three sources of fluorescent (CFL or LED) light, need to be placed
properly to avoid the formation of shade
v. A small colour-chart
vi. A scale
vii. One computer
viii. One high capacity external hard-disk
ix. Accession Register [this can also be done in a computer]
In larger herbaria, specimens are kept on trays, which, in turn are placed on conveyer
belts so that the cameraman can work non-stop. Through this much larger number
of sheets can be imaged in a day.
But, each herbarium-sheet should have one accession number that can be
recognized by the computer. For this instead of using a conventional Accession
Register, barcode labels are used for naming files and linking images with the
database (Nelson et al., 2015). At the same time, nomenclature need to be verified
before imaging a specimen.
Now a work-flow can be designed as follows:
Step-1. Bundle of specimens be despatched to the Digitization Room keeping
proper record
Step-2. Check the nomenclature from www.theplantlist.org and the up-to-date
name may be written on a determinativit-slip.
 Herbarium Techniques 91

Step-3. Fixing Barcode labels on the sheet and record the details of the specimen
on a computer against the provided code
Step-4. Put the herbarium-sheet on the tray and transfer the tray on the conveyor
belt
Step-5. The belt will carry the sheet to the flat-bed for imaging
Step-6. Imaging the specimen with camera
Step-7. Taking out of the belt for re-packing and returning to the herbarium-cabinet
Step-8. Connecting the image with the data-sheet as prepared at Step-3
Step-9. Upload to the website of the Herbarium.
However, photographs of freshly collected plants, different parts plant, especially
the floral parts are also very much useful. That will provide much more useful data
and will be of much use for the identification of plants. But, this is not possible for
stored dried specimens, in international herbaria, collected or deposited during its
life-time from widely distributed geographical areas.

CONCLUDING REMARKS
Preservation of plant specimens is a huge subject as the diversity in plant features is
endless. Numerous people has given much thoughts over it, made experiments and
devised innumerable group or habit or purpose specific methods of preservation.
All such works and publications, gradually, enriched the subject and improved the
status of preservation, utilization and accession of millions of specimens deposited
in thousands of herbaria round the world. So far, Index Herbariorum has recorded
approximately 3000 well established Herbarium round the world that are supported
by at least 12,000 associated curators and biodiversity specialists [www. nybg.org/
science/ih/]. Now, one can easily access important specimens like Types and other
authenticated specimens sitting in any remote area. Without having such preserved
and documented specimens it was almost impossible to reach the taxonomist’s
goal of giving effect to ‘one taxon one name’ hypothesis. There are innumerable
publications and Hicks and Hicks (1978) published a bibliography of such works
and that formed a basis for further investigation and improvement in Herbarium
Techniques. However, after 1978, many more such articles were published and the
bibliography needs to be updated.
At the same time, new ideas like cryopreservation, recorded molecular information,
etc. are also important aspects and are helpful in documentation practices. So, such
techniques are to be scanned and recorded properly for better utilization.
With the rapid change of climate and overall environmental conditions the rate of
extinction of species is increasing very fast. And, in near future, when only a small
human population will survive in artificial environment, then they will not find any
92 Instrumentations Manual in Biology

living plants or will find only very few plants on the earth and the Herbaria, round
the world, will carry the evidence of the rich plant diversity once occupying the
most hospitable habitats of this green planet.

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