Collection Guidelines

Gujarat
Biodiversity
Gene Bank
Field Collection Guidelines For Biomaterial Submission [V.1.1.2014-15]
A GSBTM, DST, Government of Gujarat initiative

Field Collection Guidelines For Biomaterial Submission
V.1.1.2014-15
BioGene Umbrella Programme
Table of Contents
Foreword .................................................................................................4
1. Introduction to BioGene.........................................................................5
1.1 Aim and Objectives ..........................................................................5

1.2 Infrastructure ..................................................................................6
1.2.1. Key Equipments ...............................................................................6
1.3. Microbial Repository ........................................................................6
1.4 Plant Gene Bank ..............................................................................7
1.5 Animal gene bank ............................................................................7
1.6 BioGene Umbrella Program on Barcoding Biodiversity of Gujarat ............8
2. Scope and objectives .......................................................................... 10
3. General Guidelines .............................................................................. 11
4. Basic Requirements ............................................................................ 12
5. Plant specimen collection Guidelines ..................................................... 13
5.1 Pressing the plants ......................................................................... 15

5.2 Mounting voucher specimens ........................................................... 16
5.3 Graphical representation of herbarium preparation ............................. 16
6. Seed collection Guidelines.................................................................... 18
6.1 Containers for collecting samples ..................................................... 18

6.2 Processing of seeds in the field ........................................................ 19
6.3 Transporting the collected material to the laboratory .......................... 19
6.4 Minimum required information with plant / seed sample ..................... 19
7. Mushroom Collection Guidelines ........................................................... 21
7.1 Collection and drying of fungal specimens ......................................... 21

7.2 Data to be recorded in the field ........................................................ 23
7.2.1 Site information ....................................................................... 23
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7.2.2 Specimen information (field) ...................................................... 23

7.2.3 Macroscopic description aid ........................................................ 25
8. Soil collection guidelines for fungus isolation .......................................... 27
9. Bryophyte, Lichen and Algae Collection Guidelines .................................. 28
9.1 Bryophyte specimens (mosses, liverworts) ........................................ 28

9.2 Lichen specimens ........................................................................... 28
9.3 Algae specimens ............................................................................ 28
9.4 Liquid preservation of mushroom, algae, lichen and bryophyte ............ 29
10. Invertebrate collection guidelines ........................................................ 30
10.1 Collecting Invertebrates for preservation and DNA barcoding ............. 30

10.2 Methods for Dry Mounting ............................................................. 32
10.3 Pining specimen on mount strips .................................................... 32
10.4 Gluing Specimens onto paper cards ................................................ 33
10.5 Preservation and transport of arthropods which are to be dry mounted
......................................................................................................... 33
11. Fish Collection Guidelines ................................................................... 35
11.1 Fixatives ..................................................................................... 36

11.2 Fixation Procedures ...................................................................... 36
12. Mammalian Tissue and Aves Feathers Collection Guidelines .................... 38
13. Amphibian and Reptile Samples Collection Guidelines ............................ 40
14. Key references ................................................................................. 41
Annexure I: Proforma for Deposition of specimen ....................................... 42
Annexure II: Essential information required for submission of specimen to
Barcode of Life Database (BOLD).............................................................. 43
Annexure III: Optional information for submission of specimen to Barcode of
Life Database (BOLD) ............................................................................. 44

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Annexure IV: Parameters for sampling, voucher preparation, transportation and
preservation of biomaterial from various sources ........................................ 45

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Akshay Saxena, IFS

Mission Director
Gujarat State Biotechnology Mission
Department of Science &Technology
Government of Gujarat
11th Block, 9th Floor, Udyog Bhavan
Gandhinagar - 382 011, Gujarat, INDIA
Phone: +917923252197, Fax: +917923252195
Foreword
Gujarat Biodiversity Gene Bank (BioGene) is an initiative of Gujarat State Biotechnology Mission,
Department of Science & Technology and Government of Gujarat for research in ex-situ conservation
of state floral, faunal and microbial biodiversity using tools of modern biotechnology.
Biomaterial collection is a key task for various activities in biodiversity and conservation
biotechnology research. This requires definite strategies and methods with detailed documentation.
Any collection, without primary information, labeling and proper collection methodology, is of little
importance and results in waste of time, effort and opportunity. As we set upon to explore,
document and undertake research on this rich biodiversity of Gujarat, it is important to comply and
follow the internationally acceptable norms and guidelines.
This document is a compilation which captures the procedures and process required to be observed
while undertaking survey and sample collection of various biological taxa. This document provides
guidelines for research students, academicians and scientists working in the areas of conservation
biotechnology of plants, microbes and animals. I am confident that “Field Collection Guidelines”
document will serve to improve documentation and research in areas of bio-banking and DNA
barcoding.
Akshay Kumar Saxena

Mission Director

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1. Introduction to BioGene
Gujarat Biodiversity Gene Bank [BioGene], is an initiative of Gujarat State Biotechnology Mission,
Department of Science and Technology, Government of Gujarat. One of the prime mandates of
BioGene is conservation of biodiversity of Gujarat in association with Department of Forest,
Government of Gujarat. Gujarat holds a very unique position in biodiversity spread, having wide
variations in eco-climatic and geographical conditions, which includes hot saline desert, humid hilly
forests and 1600 km long coastline. An estimated 70 mammalian species and 2000 plant species are
known to exist. However, Gujarat’s flora and fauna has been inadequately reported specially at the
molecular level. BioGene was thus established with the vision of securing the biodiversity, by storing
DNA, tissue of endangered species as well as socio-economically important species of Gujarat.
1.1 Aim and Objectives
The aim of Gujarat Biodiversity Gene Bank [BioGene] is long term ex-situ conservation of plant,
animal and microbial origin. BioGene actively involved in developing high end infrastructure facility,
research, training and awareness program for use of molecular sciences for conservation
biotechnology. For this purpose microbial repository, plant, seed and animal gene bank are
established.

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1.2 Infrastructure
BioGene is having a state of art research infrastructure with dedicated facilities for 1. DNA Banking,
2. Cryo preservation, 3. Culture facility for handling plant, animal and microbial biomaterial, 4.
Molecular biology facilities and 5. Genomics facilities (capillary and next generation sequencing)
1.2.1. Key Equipments
Ion Proton Ion Torrent TissueLyser II 2D-IEF

-40 Deg C Freezer(s) – 80 Deg C Freezer(s) Cryo storage system Anaerobic Chamber
24 Capillary Sequencing Biosafety Cabinet Lyophilizer Protein Purification
Platform System
4 Capillary Sequencing CO2 Incubator High Speed Automated
Platform Refrigerated Electrophoresis
Centrifuge(s) System
PCRs -20 Deg C Freezer(s) Geldoc System(s) RT-PCRs
1.3. Microbial Repository
Microbial Repository (BAB-BioGene) is microbial culture-collection of microorganisms in Gujarat. It

is currently having 3107 bacteria, 942 fungi and 68 archaea. The repository is the third largest in
India, comprising of more than 2000 NCBI accessions. Long term storage of microbes is done as
lyophilized or glycerol stock.
BioGene microbial repository is currently registered with World Data Center of Microbes (Reg. No.
1058) with the registered name “Bank A Bug”. It also maintains a web based catalogue for holding
strains which is accessible from the following link: [biogene.in/Biogene/BAB_catalogue.pdf].
Present status of microbial repository
Culture Type Total microbes NCBI Submission Accession number

Bacteria 3107 1585 1376
Fungus 942 613 569
Actinomycetes 39 24 20
Algae 1 0 0
Yeast 3 2 2
Archaea 68 57 44
Total 4160 2281 2011

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1.4 Plant Gene Bank
Plant Gene Bank stores herbarium, silica dried plant tissue, seeds and DNA. The herbarium serves
as reference for identifying plant and also fulfils the aim for dry plant tissue preservation and can
also be utilized for DNA isolation. BioGene has more than 3000 herbarium specimens of 120 plant
families. These herbariums are photographed to include them in BioGene e-herbarium collection.
Plant tissues are stored in dry condition. The material is preserved in airtight container/pouch after
freeze drying process. BioGene has tissue bank of more than 700 plant accessions.
BioGene DNA bank is established to cater the need of long-term preservation of genetic material
utilized for research purpose for the scientific community. At Present, DNA banking of 127 plant
species is completed.
BioGene activities also include Seed banking, varietal identification, Morphological, physiological
and molecular characterization of seed accessions. BioGene seed repository has more than 750
accessions comprising of 640 samples covering 100 species of forest plants and 143 accessions of
agriculturally important species which includes 74 cash crops accession covering 25 species. Seed
Bank has 37 accessions covering 14 vegetable species and 3 horticultural species.
Present Status of Plant Gene Bank

Number of vouchers/ herbarium 3100
Family 108
Number of Taxa barcoded Genus 381
Species 572
Number of Forest seeds accessions 750
Number of Agriculture seeds accessions 143
1.5 Animal gene bank
Animal gene bank is established with a purpose to store DNA and its amplified products which may
be used for the characterization of genotypes, assessment of genetic diversity, estimation of genetic
relationships within, identification of duplicates, establishment of co-relation as well as monitoring
genetic stability and integrity. DNA banks also help in obtaining knowledge to improve the efficiency
of some conservation activities or to scientifically inform decisions related to the conservation of
species.
BioGene stores animal tissue samples in the form of blood, swab and other forms such as feathers.
Currently 444 tissue samples are preserved in animal gene bank.

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Animal Gene Bank Status

Whole
Blood Tissue Feather
specimen
Mammals and Reptiles 266 90
Aves 50 8 33
Insect 73
Fish 43
Mollusc 43
1.6 BioGene Umbrella Program on Barcoding Biodiversity of

Gujarat
The shortage of trained taxonomists and access to the essential information resources (especially
museum and herbarium collections, taxonomic publications, databases on the Web) are most acute
in developing countries having rich biodiversity. DNA barcoding is process for determining sequence
variation within short and standardized regions of genome. It is a tool for species identification
providing additional information along with identification based on traditional taxonomy.
In view of above BioGene launched an umbrella program from August 2013 in collaboration with
various academic professionals of state on barcoding biodiversity of Gujarat. It provides opportunity
to post graduates students to pursue higher studies through M. Phil and Ph. D. In the short span of
one year the number of barcodes submitted to BOLD reached 1130 and are publicly accessible in the
GENG project on BOLD. A summary table is given below:
Barcodes submitted to Barcode of Life Database [BOLD]
Fungi Plant Animal Total

BOLD Project MGEN GENG ANGEN GENG
Marker
rbcL 673 673
matK 15 15
trnH-psbA 13 13
ITS 319 7 326
COI 124 124
cytB 1 1
atp6 1 1
Total submissions 1153*
*BioGene is leading in the country with highest number of DNA barcode submissions

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Bio materials deposited in Gujarat Biodiversity Gene Bank (BioGene)
Eranthemum roseum Lantana rugosa Senna alata
Ganoderma multipileum Flavodon flavus Phellorinia herculeana
Trimerotropis sp. Aplysia fasciata Eulalia viridis
Moringa olifera Oroxylum indicum Peltophorum ferrugineum

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2. Scope and objectives
The “Field collection guidelines” intend to provide students/ researchers/ academicians specific
guidelines for plant/ fungal and animal collection. We encourage adoption of these guidelines to
improve the quality, efficacy and accuracy of biomaterial collection for voucher preparation and for
the purpose of DNA barcoding.
The main objective of these guidelines is to provide the user appropriate methods for collection and
documentation. It offers general protocols for the collection of plants, fungus, insects, fishes,
amphibian, reptile and mammalian tissue. It also provides explanation for sample collection for
voucher preparation and highlights the difference in sample collection for DNA barcoding, DNA
banking and voucher preparation. The manual also is designed to provide a detailed description of
data entry rules and data entry procedures in order to standardize data entry. This standardized
form is a prime requirement for submission to Barcode of Life Database (BOLD). This is an ongoing
document, which will be updated and modified periodically.
The document is structured in a way that any user can easily get a step by step guide to collection
strategies for preparing both vouchers and DNA barcoding. A summary table is also appended to
give a snapshot of the entire guidelines. Moreover tables are also provided for documentation and
description of the data before submitting the specimen.
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3. General Guidelines
1. Always carry the collection kit.

2. Wherever required, collector(s) should take prior written permission from the authorized
agency for collection.
3. Collections must be done in two sets:
a. First for Voucher preparation in triplicates (find details in the respective guidelines).
b. Second for DNA barcoding (NO FIXATIVE BE ADDED).
4. A review of the collection site including its habitat, area and diversity will help giving an insight
into its usefulness to the collector.
5. Collection should not harm the environment.
6. When submitting the specimen to BioGene it is mandatory to submit the proforma attached
in this guideline as Annexure I and Annexure II.
7. Any submission without these proforma will not be considered.
8. Avoid duplication of the samples. Check BioGene specimen list.
9. While collecting the samples, avoid cross-contamination.
10. Surface sterilize the specimen, if required and avoid diseased/ pathogenic specimens.
11. Collected specimens should be transported immediately to the lab avoiding direct sunlight.
Refer to details mentioned in the respective guidelines for plants, animals and microbes.
12. Take high resolution photographs (preferably 300 dpi) with appropriate ISO and background
settings.

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4. Basic Requirements
Zip seal bags/

sterile Cellotape
containers
Pen
Label Sticker
Field record GPS with spare

book batteries
Camera Rucksack Bag
Blotting /
Newspapers/ First aid box
Butter Paper
Scale Forceps
Gloves Field shoes

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5. Plant specimen collection Guidelines
 Before setting out to collect plants, be sure to familiarize yourself with potentially injurious
plants. Both native/naturalized species and cultivated plants of known provenance can be
collected. Determine whether or not the plant(s) you wish to collect are planted or
spontaneous.
 Always collect from an area where there are numerous examples or sufficient plant material of
the species or cultivated variety you wish to collect. Always collect enough plant material for
preparation of at least 3 voucher specimens (herbarium). Along with the material collected
for voucher specimen 3-5 young leaves (minimum 1 g, preferably in between 10-100g, if
available) must be collected in silica gel filled in a zip seal bag for DNA barcoding.
 If plant material is collected in rainy season then ensure that plant material is free of moisture
(except in the case of aquatic species) before you collect and proper care should be taken to
avoid fungal infection.
 Herbarium specimens should have the plant features or characteristics required for positive
identification. For most plants, this means flowers or fruit/seeds should be present. Make sure
your specimen is representative of the plant’s stem and leaf patterns. Therefore, the timing of
plant collection should be an important consideration. In addition to flowers and fruit, other
identifying features of the plant should be sampled at the time of collection.
 If collecting herbaceous plants (i.e. wild flowers, graminoids or ferns) roots or underground
plant parts (rhizomes, bulbs or tubers) should be collected where possible, along with the above
ground plant parts (leaves, stems, flowers, fruit, thorns etc).
 Cyperaceae, Liliaceae, Zingiberaceae, Costaceae and plants with underground roots should be
preferably collected with roots, rhizome, corm or underground part.
 If you are collecting trees, shrubs and other woody plant material, take a cutting of a branch
with several representative leaves and flowers and/or fruit. It is often useful to include a small
sample of a tree’s bark. Do so by carefully removing a piece of bark, not larger than 2 x 2 cm,
with a knife.
 Sterilize secateurs or other cutting tools with a little rubbing alcohol before taking a sample
from a different plant to avoid the spread of pathogens and disease.
 Herbarium specimens should be pressed between newspaper or blotting paper using
herbarium press.
 Large or long-trailing herbaceous plants can be cut into sections and pressed and mounted
separately (see pressing the plants).
 For each plant specimen collected, brief information for taxonomic identification should be
noted in the field at the time of sampling as given below:
1. Plant name - Scientific name (if not known, some kind of identifier should be used).
2. Collection number, optional.
3. Date of collection.
4. Habit (Herb, Shrub, Tree, Climber, Epiphyte, Parasite, Saprophyte, Insectivore, Symbiont)

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5. Habitat (Grassland, wasteland, weed of cultivated field, stream, water bodies)

6. Root (Tap root, Adventitious roots)
7. Stem (Aerial, climbing, underground, specialized stem)
8. Leaf (Bearing of leaves)
a. Phyllotaxy (Arrangement of leaf on stem)
i. a. Alternate b. opposite c. whorled
b. Type of leaves (simple, compound)
9. Inflorescence (Arrangement of flower on floral axis)
1. Racemose (an inflorescence where the main axis does not
terminate in a flower)
2. Cymose (an inflorescence where the main axis terminate in a
flower)
3. Special type (Cyathium, Thyrsus, Verticillaster, Hypanthodium)
10. Flower (prefer to not floral formula in field)
1. Colour
2. No. of petals
3. No. of stamen
4. No. of carpels
11. Fruit
a. Simple fruit
1. Dry fruit (Capsule, legume, siliqua)
2. Fleshy fruit (Drupe, Pome, Berry, Pepo, Hesperidium)
b. Multiple or composite fruit (syconus, sorosis)
1. color
2. shape
3. seeds
12. Special notes
a. Aroma,
b. Association with other plant sp.,
c. Morphological features i.e. height,
d. vernacular name,
e. number/quantity of plants growing in the area;
f. health of the plants;
g. record photo number(s)
h. Parts of economic importance and for cultivated plants, provide information on
provenance, breeders, garden location, accession number, etc.
i. Digital photos of the plants in their habitat or growing medium may be taken. Take
close-up photograph of plant with details of reproductive organs.
13. Digital photos are complimentary to voucher specimens and can be stored in a database
and/or printed and linked to the Herbarium specimens for additional reference.

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14. A preformatted collection data sheet to register all the information that is important to
annotate during sampling may be useful.
5.1 Pressing the plants
 Plants should be pressed as soon as possible after collection. Plant material can be stored in
large polyurethane bags while in the field, and some plants will survive overnight in bags in
a refrigerator if they cannot be pressed immediately.
 Press plants using a standard plant press. If no plant press is available, smaller plants can be
pressed between several sheets of newspaper and placed under a stack of heavy books or
blocks.
 Label each sheet of newspaper according to the species name and date or collection number
that corresponds to your notes. This is important so that there is no mix up when it comes
to mounting and labelling the plants at a later date.
 When pressing plants, blot any moisture away. Lay your specimens out on one side of an
opened piece of newspaper.
 Plant parts such as leaves and petals should be laid out flat.
 The flowers should be placed so that the flower parts are distinguishable.
 Flowers and fruit may be cross-sectioned prior to drying.
 It is helpful to turn some leaves over so that examples of the top and underside
surfaces are visible.
 Close the newspaper and sandwich it between two pieces of blotting paper and
cardboard.
 If the specimen is particularly thick then a piece of foam can be folded inside the
newspaper. Alternatively, thick or bulky plant parts such as roots, seeds, bark could
be pressed in separate sheets.
 Larger plants can be cut into sections and components can be pressed individually
(i.e. base, middle & top of plant). If plants are sectioned into two or more, label
newspapers accordingly (i.e. for a plant cut into three parts, specimen 1 of 3, 2 of 3,
and 3 of 3 should be used for the top, middle and lower parts of the plant).
 If the specimens are succulent or wet (e.g. aquatic submerge or floating plants) then
they can be pressed between pieces of parchment paper.
 Layer all of your specimens into a plant press and tighten the press firmly. Store press in a
dry place with adequate air circulation.
 After a day you can check on your plants while they are still not completely dry and rearrange
them if any leaves were folded over while pressing. Change newspapers and blotting paper
as necessary to prevent molding until plants are dry.
 The length of time it takes for a species to dry depends on the plant’s water content and on
the drying conditions. The quicker a specimen is dried, the better its colour will be preserved.
After several days plants will be dry and ready to mount.

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5.2 Mounting voucher specimens
 Specimens should be mounted on acid-free standard Herbarium mounting paper.

 Poison the plant material with 0.01% HgCl2 in 60% Ethanol and 40% distilled water solution
or in spirit.
 Stamp the herbarium with label POISON
 For epiphytic orchids possibly dry the plant, inflorescence and flowers immediately in
Microwave oven to avoid abscission. Possibly stitch the plants to the herbarium.
 Place your dried plants onto a piece of 42 cm x 28 cm standard handmade paper. Arrange
them in such a way that the main features used to identify the plant are evident. Ensure
there is room for a label in the lower right hand corner.
 Use thick paper strips to place plant specimen on sheet. Arrange plant specimen and flower
/ fruit parts on handmade paper sheet. Apply glue on both the ends of strip and stick the
plant. Use large strips for bigger plant parts such as twig and stem.
 While fixing the plant on sheet additional flowers could opened or dissected to display
flower parts.
 Alternatively low glue tape can be used to reinforce areas that do not stick as well (ex. nodes,
root balls, flower heads)
 Cover the specimen with waxed paper, place it under a heavy block or between hard boards
and leave it to dry overnight.
 Store specimens in a cool, dry place and minimize exposure to light.
5.3 Graphical representation of herbarium preparation
1. Collecting: Select a typical plant and if possible two or three extra

flowers to supplement the specimen and for dissection. Ensure the
plant is healthy and collect average-sized leaves and flowers typical of
the plant, not the biggest. Photograph the plant habit and a close-up.
Attach a label with the name and date and location. Avoid collecting
material in wet weather.
2. Describing: When collecting, record the following: name of plant,

date of collection, collector, site of collection, original source of plant.
Note other details that may be lost by pressing: overall size, habit and
form, leaf or flower scent. Record color of the fresh plant using
standard color chart.

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3. Pressing: Remove soil from around the material. Use a press made
with a pair of boards of hardboard or plywood cut to the same size as
the drying paper. Dip the specimen in mercury chloride preservative
solution. Place some corrugated card on one board of press, and then
place two sheets of blotting paper on top of this. Arrange plant
material on blotting paper retaining the character of the plant. Put
flowers and dissected parts in flimsy paper. Remove leaves and
flowers of congested specimens to reduce the bulk without losing the
character of the plant.
4. Pressing: Cover the sample with two further sheets of blotting

paper and corrugated card. With both bulky and fleshy specimens,
add a sheet of foam between the blotting paper and corrugated card.
Any absorbent fabric may be useful in drawing out moisture; place it
on top of the plant material, with a thin sheet of paper between the
plant material and the fabric to prevent sticking.
5. Pressing: Once all samples have been included, cover with top
board and place bricks or heavy object, applying pressure evenly
throughout or use straps to keep the press tight. Place in a warm
place, such as a drying cabinet, airing cupboard.
6. Pressing: Inspect the material 24 hours later, replacing the

corrugated card and top layer of blotting paper with dry card. This is
your last chance to re-arrange any material while the plant material is
still moist and pliable. Inspect regularly - at least once a week.
Depending on the plants being pressed and the drying conditions a
dry specimen will be ready in anything from two to three days to two
to three weeks.

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7. Mounting: The BioGene Herbarium uses handmade acid-free

paper, measuring 419 x 276mm. Good quality A4 paper, preferably
acid-free, is sufficient for the needs of a domestic herbarium in
absence of handmade paper sheet.
8. Mounting: Attach the specimen to the paper using a combination

of neutral pH PVA adhesive and gummed linen hanging tape. The label
should the bottom right-hand corner. Alternatively paper strips can
be used to simply hold the specimen on herbarium sheet. Further
stitching and/or non-stick low friction transparent tape can be used to
place specimen on paper sheet.
9. Storage and conservation: Place the prepared specimen in a sealed

plastic bag and freeze for 72 hours. Ideally the temperature should be
-32 ºC, although most domestic freezers have a minimum of -18 ºC.
Freezing is the only method open to combat pests. The most common
pest of the herbarium specimen is the biscuit beetle, Stegobium
paniceum. Regular freezing (every six months) is recommended, as is
regular inspection to check for infestation and damage.
6. Seed collection Guidelines
Seeds should be collected at optimum maturity when seed vigor, desiccation tolerance and
longevity are expected to be highest.
6.1 Containers for collecting samples
 Use paper bags for collecting seeds.

 Use cloth bags that allow circulation of air (such as muslin bags) for collecting panicles or dry
fruits.
 Use open containers, such as baskets made of wire or bamboo or tubs to collect fleshy fruits.
 Ensure that fruits are not squashed.
 During transport, do not let fruits become too hot and ferment.
 Use nylon-net bags for collecting samples as they allow air to circulate freely. Besides their use
for collecting seeds, pods and fruits, they can be used for seed extraction and for drying extracted
seeds. They are available in a range of mesh sizes.

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6.2 Processing of seeds in the field
 Seeds collected with surface moisture should be dried in shade or a well-ventilated room. Use
newspaper or blotting paper for drying. After drying transferring them to cloth or paper bags.
 Seeds from dry dehiscent fruits (such as okra, sesamum) can be extracted by spreading the fruits
on a tarpaulin under shade.
 Mature fruits split open and release their seeds as they dry. Sometimes, additional impact such
as raking or shaking is needed.
 Remove empty fruits and debris, and transfer the seeds into cotton, nylon-net or paper bags.
 Pulpy fruits (such as tomato and cucumber), extract the seeds carefully by hand, wash them under
running water to remove pulp and mucilage, spread them in a thin layer to maximize aeration
and allow them to dry in the shade.
 Always maintain seeds in moisture-permeable containers such as cotton or paper bags, and
ensure that air circulates freely between and through them.
 Under hot and humid condition and during long collection mission dry seeds using desiccants such
as silica gel (3:1).
 Keep alternate layers of packed silica gel and packed seeds in a large airtight container to
reduce seed moisture.
 Hold seeds inside fruits when seed material is small and inside fleshy fruits.
 Avoid seed extraction if fruits require after-ripening or if seeds are delicate or recalcitrant.
 Do not collect seeds naturally fallen on ground.
6.3 Transporting the collected material to the laboratory
 Maintain proper temperature and safe moisture content during transport.

 Use cushioned seed container to avoid damage during transportation.
 Take extra care while acquiring unique / rare seeds.
6.4 Minimum required information with plant / seed sample
 Samples should be accompanied by adequate information, especially plant/cultivar name,

details of variety etc. with collection code.
 The minimum required information for plant collection is as below;
 Common name and/or genus and species.
 Collecting number (in prescribed format).
 Location of collection site (Habitat description with GPS coordinates and elevation).
 Collecting date (dd-mm-yyyy).
 Morphological features of plant with habit.
 Field photographs (preferably with habitat and close-up of reproductive organs).
 Numbers of vouchers prepared (Minimum 3 with reproductive organs).

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 Approximate quantity of seed supplied.

 Name and contact details of specimen collector ((Address, phone no. and e-mail).
 Name and contact details of specimen identifier (Address, phone no. and e-mail).

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7. Mushroom Collection Guidelines
7.1 Collection and drying of fungal specimens
 The common proforma given in annexure-1 should be filled in for all specimens to be
submitted.
 Obtain all necessary permits before setting off for any collection.
 Basic requirements for collecting macrofungi:
 Suitable collecting boxes of varying size. Small collecting boxes may be required for
smaller fungi, such as the Ascomycetes.
 Roll of aluminium foil (preferred), greaseproof paper or greaseproof bags can be very
useful for wrapping larger macrofungi. Do not use plastic bags, fungi deteriorate rapidly
when wrapped in plastic.
 Pocket knife, or trowel, to remove entire specimens from their substrate without
damaging tissues.
 GPS for recording accurate latitude, longitude and altitude readings. Alternatively, mark
the position on a topographic map and include the map date field notebook. This can be
a pocket-sized notebook, or a book of pre-printed specimen labels or the QMS field
recording sheet (See Table-1).
 Camera for photographing the form, colour and natural habitat of the fungi.
Photographs should be linked to each specimen by a unique reference number.
 Set of gloves, for protection when collecting in litter.
 Tags for labelling the collection.
 Choose specimens which are in good condition and representative of the population’s
variability. Ideally, the fruiting bodies should be
produced by an individual mycelium (some of these
can cover an extensive area) and collection from an
isolated patch is therefore a preferred practice.
 A good specimen includes all parts of the fungus -
cap, hymenium (spore-bearing tissues) and stipe;
and ring and volva where present. For some species
the colour of the attached mycelium is an important
diagnostic character and should be noted. The
fungal material should be fertile i.e. it should have
mature spores, as these are vital for identification.
 Note carefully the substrate (what the specimen was growing on/in) and the associated
organisms (the species or genera of plants it was growing with).
 High Resolution Photographs should include the specimen growing in its natural habitat, a
display of the specimen which shows the gills or pores, any unusual, distinctive or interesting
features, a tag to provide scale and its unique reference number.

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 Specimens should be wrapped in waxed paper or foil (Never use plastic) and placed in a
suitable container for transport back to the laboratory.
 Good collection of medium–large fungi normally consists of at least five fruiting bodies in
which representatives of each stage of development are present. For medium sized fruiting
bodies, at least 10 would be required, for small-medium, at least 20, and for very small about
30 or so. In the case of extremely large fungi, a single sporocarp may suffice. Ensure that
sufficient photographs are taken, however, to capture any variations that may occur in the
taxon.
 Use a pocket-knife or a trowel to extract specimens from the substrate, taking care to collect
the whole specimen including the base. Don’t collect by cutting the stalk of the fungus.
 Never press the specimen as done for plants.
 The transport to the laboratory should be done as quickly as possible in not more than two
days so that mushrooms are as fresh as possible. If it is not possible to transfer them quickly
to the lab morphological characters including size, color, and spore print should be taken
and then the mushroom should be dried in a cool oven with a temperature not more than
40-42°C.
 Two to three mushrooms should be stored as museum specimen in glass jars containing
70:20:10 (ethanol: water: formaldehyde) as described in the liquid preservation section.
 Never freeze fresh samples.
 Clean all tools and implements that you have used on your foray/survey with 70%
methylated spirits (or ethanol, if you have access). This is necessary in order to prevent the
possibility of any transfer of contaminants, e.g. pathogens, between sites.
 For taking spore print follow the below given steps:
 Spore prints are generally made on acid free white paper. In case the spore color is
very pale or pink black paper can be used.
Steps to create a spore print:
Fold a square piece of white paper.
Cut in the middle.

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Place the stipe through the slot in the paper and suspend on
the mouth of a plastic beaker/cup.
Once a spore print has been obtained, label it and place this on
the dryer with the collection and other labels.
7.2 Data to be recorded in the field
Fungi collections unaccompanied by field notes are of very little use.
7.2.1 Site information
1. Site name: If the name is not known, then describe how to get to the site from the nearest
known locality.
2. GPS location: This can be recorded as lat/long. Most GPS devices will also record and store an
altitude reading.
3. Habitat data: Should include landforms, slope, dominant plant species, and vegetation
structure, for example ‘open forest’, ‘open woodland’ or ‘grassland’.
4. Record any evident management system or any disturbance, for example grazed paddock,
recently burnt, or timber extraction.
7.2.2 Specimen information (field)
The following information about fungal specimens should be recorded in the field:
1. Reference/voucher number: to uniquely denote each collection.

2. Genus and/or species name: if the name is not known, then a description of the type of
fungus, e.g. agaric.
3. Substrate: what the fungus was growing on. e.g. leaf, insect, soil, log, twig, sawdust pile.
4. Associated organism: which can be either the species name for the vegetative material on
which the fungus was growing, or in the case of mycorrhizal fungi, the name of the nearest
likely mycorrhizal associate.
5. Color of the fruiting body, hymenium, and smell should be noted down.

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6. Collector: the name and initials of the collector.

7. Determiner: Name and initials of the person who identified the fungus in the field.
8. Photographer: Name and initials of the photographer, if different from the collector.
9. Morphological characters: Record the morphological characters as given in Table 1.
10. Do not forget to fill in the Annexure II before submitting the specimen.
Table 1: Morphological characters required of identification of mushrooms (refer section 7.2.3)
Locality:
Associated species:
Substrate:
Cap:
Shape:
Diameter:
Texture:
Surface:
Color:
Cap margin:
Flesh:
Smell:
Color:
Gills (Gills/ Pores/ Teeth)
Color:
Attachment:
Stipe
Color:
Size: Height ____mm Width ____mm
Texture:
Shape: Ring or Volva
Spore
Color:
Size: Height ____mm Width ____mm

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7.2.3 Macroscopic description aid

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8. Soil collection guidelines for fungus isolation
 Ten to 30 g of soil should be collected using soil coring tube or

similar device sterilized in 70% ethanol and dried just prior to
collection.
 Before sampling the overlying litter or humus should be removed.
 The sample should be placed in sterile container or bag, labeled and
stored in an ice box or cooler.
 The soil sample must be plated out within 4 days or frozen if plating cannot be done.
 The soil and sediment sample may be collected from terrestrial or shallow water
environment using sterile containers. Presence of filamentous fungi is common in these
environments.
 Standard plating techniques using serial dilution, four flame
streaking or pour plate method on standard fungal media
generally results in isolated colonies.
 Two to three sub-cultures generally result in pure colonies of
the fungus.
 Once a pure culture is obtained the spore morphology must
be recorded.
 A high resolution photograph must be taking showing the
spores as well as mycelia.
 For DNA isolation freshly grown mycelia work best.
 For long-term preservation the fungal spores must be cryopreserved in glycerol or
lyophilized.
 Permanent slide of spore should also be prepared.

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9. Bryophyte, Lichen and Algae Collection Guidelines
9.1 Bryophyte specimens (mosses, liverworts)
 Collect fertile material with capsules, if possible.

 If the bryophyte is growing on a tree collect some bark.
 Collect at least a 4 × 4 cm patch.
 Place collection in a paper envelope (never plastic) and air dry as rapidly as possible in a well-
ventilated area. Do not press the specimens.
 Bryophytes can often be found growing intertwined. It is important to check carefully that
capsules belong to the plant that they seem to be growing from.
 Bryophytes should be kept cool and moist or air dried immediately.
 Liquid preservation is done in commercial formalin as described in liquid preservation
section.
9.2 Lichen specimens
 Simply place the lichen specimen in a paper bag or envelope. Do not place in plastic bags.
Dry in a well-ventilated area, as rapidly as possible.
 Collect some of the substrate, such as rock or wood, but trim excess substrate.
9.3 Algae specimens
 Aquatic algae can be temporarily stored in water from the collecting site for a couple days
but must be transported on ice at the earliest and no later than two days to the lab
 Marine algae must be covered by waxed paper or muslin before pressing between
newspaper (which otherwise adheres to the specimen).
 Preparation of vouchers must be done in field itself.
 For preparing the voucher specimen use the floating out technique followed by pressing.
With the specimen in a shallow dish of water, place a piece of stiff white paper under the
specimens and then carefully lift out the paper with the specimen on top. Press between
newspapers.
 If possible one should go with a field microscope to do a general microscopy
 Specimens collected should be in three sets (i) Specimens for microscopic analyses should
be fixed in 4%formaldehyde solution, (ii) unfixed subsamples dried on herbarium paper as
voucher and (iii) another in a vial with silica gel for DNA extraction and barcoding.
 Liquid preservation is done in commercial formalin as described in Liquid preservation
section.

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9.4 Liquid preservation of mushroom, algae, lichen and

bryophyte
Algae/Mushroom/Bryophyte can be stored initially in a bucket, jar, bottle or plastic bag, with some
water from the collecting site. The container should be left open or only half filled with liquid and
wide shallow containers are better than narrow deep jars. Note that glass is reportedly not
satisfactory for some due to its inherent alkalinity damaging cells. However, glass vessels are
commonly used to collect specimens. Samples can also be refrigerated or kept on ice soon after
collection from field and can be kept alive for short periods (a day or two). Place collected sample at
cool place with reduced light. For long-term storage, specimens can be preserved in liquid (see
below), dried, or made into a permanent microscope mount (preferably all three). Even with ideal
preservation, examination of fresh material is sometimes essential for an accurate determination.
For liquid preservation the following preservatives/ fixative can be used.
1. Specimen can be directly preserved in formalin (40% formaldehyde) at 1/10 to use as

fixative. (Note: Formaldehyde is thought to be carcinogenic hence avoid contact with skin, eyes
and air passages).
2. Specimen can be preserved in FAA (by volume, 40% formaldehyde 1: glacial acetic acid 1: 95%
alcohol 8: water 10) or 6-3-1 (by volume, water 6: 90% alcohol 3: 40% formaldehyde 1) solutions
3. Instead of FAA solution, standard alcohol and water mix (e.g. 70% ethyl alcohol or industrial
methylated spirit) can also be used to preserve specimen.
4. Algae can be kept in diluted formalin for a number of years, but the solution is usually replaced
by 70% ethyl alcohol with 5% glycerin (the latter to prevent accidental drying out).
5. Lugol's solution is commonly used for short-term (e.g. a few months, but possibly a year or
more) storage. Dissolve one gram of iodine crystals and two grams of potassium iodide in 300
ml of water. Use three drops of this solution in a 100 ml sample.

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10. Invertebrate collection guidelines
10.1 Collecting Invertebrates for preservation and DNA

barcoding
 All collections must be processed in duplicate sampling: (i) for voucher preparation and (ii) for
DNA barcoding.
o For voucher preparation: At least three specimens must be collected, fixed and stored as
per the table given in Annexure-IV.
o For DNA barcoding: The sample size depends on the size of the specimen collected. For
small insects 3-5 specimens are required corresponding to a minimum of 100 mg material
for DNA extraction. The sample which is to be used for DNA extraction should not be
stored in formalin.
 The invertebrates should be collected using proper techniques to prevent damage. Damaged
or incomplete specimens should be avoided.
 The collections can be made using passive and active technique. Passive technique uses Pitfall
trapping, Leaf Litter extraction, Yellow pan trap, Flight intercept traps, glue trap, pheromone
trap.
Yellow pan trap
The collection fluid attracts insects in search of water but more

specifically many wasps and certain flies are attracted to the
color yellow, which is essential for the method.
Leaf litter extraction
Flight Intercept trap
A variety of intercept traps have been designed to catch flying

insects but all work on the same principle, that is an insects
flight is impeded and in the process the insect is channeled into
a container with preservative.

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 Active technique involves hand picking, chemical knockdown, beat sampling, light
sampling, sweep sampling.
 Beating tray, sweeping net, butterfly net and pond net may also be used
Beating Tray Sweeping Net Butterfly Net Pond Net
 Several kind of traps may also be set such as pitfall trap, Aspirator, Malaise trap, Mercury
Vapor Lamp, Tullgren funnel
Aspirator Pitfall Trap Mercury Vapor Tullgren funnel

Lamp
 After collection the invertebrate must be immobilized in a relaxed state.

 Insects must never be transported in zip seal bags. Always use sterile containers of suitable
size for the transfer of insets.
 Invertebrates destined for DNA isolation should ideally be collected in 70-95% ethanol
while still alive. Container must be leak-proof. Container should be kept in ice-box with
icepack immediately after collection and while transport.

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 Invertebrates must be labeled with collection place, unique collection number and
collection date.
 High resolution photograph of the insect in its habitat, dorsal and ventral photographs
should accompany the invertebrate submission.
 For voucher preparation and long term storage of invertebrates group specific fixatives
and preservatives are given in Annexure IV:
 The preservation procedure generally follows three steps:
Step I: Formalin - 1 week (fix soft tissue)
Step II: One day (leach out the formalin)
Step III: Alcohol - long term storage
 All specimen stored in liquid medium must be transport in cool and dark condition.
10.2 Methods for Dry Mounting
Dry-mounting and pinning is recommended or even necessary for the Lepidoptera, Coleoptera,
Hymenoptera, Diptera, Heteroptera, Orthoptera, Odonata and Neuropterida. All other taxonomic
insect groups as well as insect larvae, Arachnida, Myriapoda, and Crustacea are best killed and
preserved in 70 -80% ethanol.
Two methods for dry mounting are well known; best suited alternative according to the insect type
should be used.
10.3 Pining specimen on mount strips
 Direct pinning of specimens should be done with an insect pin that fits to the specimen’s size.
The standard size for insect pins is 1 or 2 which fits for most Lepidoptera, large Hymenoptera
and many Coleoptera. Larger specimens should be pinned with size 3, 4 or 5, while for smaller
specimens, pins with size 0, 00, or even 000 are available. However, it should be noted that pins
with size 0 or smaller are difficult to handle. Pinning through the labels or through the paper
layer of insect boxes should be done with great care as the thin pins are easily twisted.
 Direct pinning of insects should be done in a way that the pin is in
a right angle to the body. The insect specimens should rest about
1/3 of the pin length away from the top. This gives enough space
to handle the specimens, i.e. to grip the top of the needle by the
thumb and the index finger without damaging the specimens with
the fingertips or fingernails the specimens should not rest further
away from the top of the needle as the bottom space is needed
for collection and determination labels. The pin is usually inserted
through the mesothorax but the exact insertion point depends on Inserted needle is in a
the insect group. Bugs (Heteroptera) are pinned submedially right angle to the body
through the scutellum. In Hymenoptera and Diptera the insertion of the insect
point is slightly removed laterally from the median axis. This allows median sculpture or bristle

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patterns to remain intact and visible medially and also on one side. Beetles (Coleoptera) are
pinned through the right elytron. In butterflies and moths (Lepidoptera) the insertion point is in
the middle of the mesothorax.
10.4 Gluing Specimens onto paper cards
Very small insects (body length below 3 mm) should never be directly pinned as specimens will
always be damaged or lost over time. Card mounting is the method of choice for small beetles, bugs
and Micro-Hymenoptera.
 Beetles and bugs should be glued on rectangular cards.
 Small Hymenoptera can either be glued on rectangular cards or on the tip of card points, which
are small triangles of stiff paper.
 The paper cards with the mounted insects should be pinned with common insect pins of larger
size (sizes 3 to 5). Pins of that size can easily be inserted through the paper cards. The glue should
be water-soluble or ethanol soluble so that specimens can easily be removed from the card in
case they need to be re-mounted without being damaged. Seccotine (fish glue) is a water soluble
glue and also recommended for rectangular cards and shellac (a resin produced by lac bugs,
Coccoidea) is recommended point cards.
10.5 Preservation and transport of arthropods which are to

be dry mounted
 Use cardboard tubes of different diameter for transport. Both sides of a tube are to be closed
with a cotton plug. The freshly killed sample should be placed directly inside the tube. It can be
transferred into a soften chamber afterwards for preparation of setting and mounting. This
method is feasible for strongly sclerotized specimens (e.g., beetles) which need to be stored
during fieldwork before they can be dry mounted in the laboratory. However, scaled, pilose, and
coated specimens could be rubbed off during transport. Cardboard tubes are preferred over
glass or plastic ones as they are lightweight, fracture-proof and absorb moisture. The tubes are
to be stored inside of feasible sealed transport boxes containing crumbs of thymol which
prevents moulding.
 Use butterfly envelopes of different size, made of vellum. This method should be used to
transport or even store dry unset Macro-Lepidoptera and winged insect orders like Odonata and
Neuroptera. It is important to “close” the specimens inside of the envelope with the wings folded
upwards. This protects the more important upper sides of the wings (as identification characters)
against rubbing and facilitates later setting and spreading. If this is not possible in case of rigor
mortis, the specimens have to be injected by syringe with ammonium chloride to soften rigor.
Placing more than one specimen into one envelope should be avoided as they may damage each
other during transport.

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Card Mounting. A label can be Card points are small triangles of Cardboard tubes are ideal for
sticked to the other card and stiff paper that allow specimens to hard bodied insects, such as
inserted in parallel to the be observed from all sites if beetles.
specimen card. specimens are glued laterally to the
tip of the triangle.

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11. Fish Collection Guidelines
 An important part of any field program is to obtain and carry all relevant permits for
sampling activities in the study area.
 It is advised to take help of a local person as a guide.
 Always carry a field identification key/ guide.
 It is important to acquaint with the general knowledge of the different body parts of the fish.
Typical fish labeled with the body parts is given below;
 As soon as the fish is captured the length of the fish should be measured. Fork length, total
length and standard length should be measured as shown below

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 If possible the weight of the fish should also be taken after draining the excess liquid.
 Where-ever possible all collections of fish should be made in two sets, one for (i) Voucher
preparation and the other for (ii) DNA barcoding.
For voucher preparation the fish must be anaesthetized to kill so that it remains in relaxed condition.
Thereafter it must be fixed and stored in isopropyl alcohol.
11.1 Fixatives
 Formalin is commonly used to preserve collected specimens and it is available in liquid or

powder forms (Full strength liquid formalin is actually 37% formaldehyde dissolved in water).
It is recommended that a solution of 10% formalin be used for the preservation of fish
specimens. To make a 10% solution of buffered formalin, combine 1 part full strength
formalin with 9 parts distilled water and add approximately 3 ml of borax (buffering agent)
per liter of solution. Formalin is slightly acidic and will de-calcify and soften bony structures.
The addition of a buffering agent helps to slow down this process.
 Alcohols, such as ethanol and iso-propanol, are also commonly used to fix and preserve fish
specimens, especially if skeletal structures such as otoliths are to be examined. Alcohol is a
poor fixative and is not recommended for fixation.
 An alternative for preserving specimens is to quickly freeze them in dry ice or liquid nitrogen.
This is one of the best methods to preserve the colors and tissues of the specimen. Samples
must remain frozen until they arrive at the laboratory and can be permanently preserved.
11.2 Fixation Procedures
 Specimens should be fixed soon after collection to limit deterioration of the tissues. All
specimen must be killed prior to fixation. To fix the specimen, place it in a wide-mouthed
glass, and fill the jar with the fixative solution.
 Specimens should be inserted into jars head first to make them easier to remove from the
jars in the laboratory. Different species captured in the same set can be fixed and stored
together.
 The fish must be preserved in as natural a state as possible. Where possible, the specimen
should float freely in the jar to avoid curling or bending. Before immersing large specimens,
fixative should be injected directly into the body cavity to facilitate penetration and
preservation of the internal organs. The stomach should also be incised for internal fixation
in order to prevent rotting due to digestive juices.
 Two labels are needed for the specimen: a waterproof specimen attached to the jaw or
inserted into the mouth or opercular area of each specimen and a waterproof data label on
the outside of each jar. All labels must be written in pencil. The specimen label contains.
 the fish identification number
 species name of the fish

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 collection method code

 collection date
 A brief description of the habitat and catch may also be included.
For DNA barcoding Tissue samples for DNA extraction should be frozen or preserved in fresh 95%
EtOH and stored in a cool place, preferably in a freezer.
 Large pieces of tissue should be cut into small pieces (5-7 mm) to permit adequate fluid
penetration.
 Two archival quality tissue samples will be immediately collected from each specimen;
 one frozen to preserve the broadest array of molecular characters possible,
 one placed into a preservation fluid, such as EtOH, to serve as a back-up in case of a
meltdown or loss of the frozen specimen.
 Several tissues are suitable for DNA extraction from fishes. These include the following:
 Musculature: remove one or more cubes (5 – 7 mm) of lateral muscle from the right
side of the specimen.
 Gill tissue: remove one or more gill arches with attached filaments from the right
side.
 Eye: remove the right eye from extremely small specimens such as larvae.
 For species with small body size, entire specimens can be placed in preservative in lieu
of subsampling. This should be avoided unless a series of conspecifics are available for
fixation in formalin for standard morphological analysis.

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12. Mammalian Tissue and Aves Feathers Collection

Guidelines
 Because of their important economical, conservation, and biomedical status, animals are
among the most highly regulated living organisms when it comes to collecting, deposition,
and international transfer. It is imperative that any prospective contributor to the animal
barcoding campaign is well aware of the national, international, and institutional
regulations pertaining to the collection, storage and distribution of specimens and any
derivative biomaterials and conducts his/her operations in a compliant manner. All
necessary permits must be obtained prior to any collection.
 Any infection to the animal should be notified while submitting the tissue.
 Vital field for DNA barcoding involve detailed locality with coordinates and elevation,
collection date, name(s) of collector(s), sex and age of the vertebrate.
 When collecting tissue for DNA barcoding.
 Avoid tissue from kidney or liver as they are enzymatically rich which adversely
effects DNA. If kidney or liver cannot be avoided then they should be submitted
within 24h to the lab for processing.
 Preferred source of tissue for barcoding studies is muscle or gonads stored in 95-
100% ethanol. Ethanol preserved tissue should be stored at -20 ˚C or lower.
 The tissue can also be stored in tissue protectant which comprise of 70% PBS (NaCl
8g, KCl 0.2 g, Na2HPO4 1.44g, KH2PO4 0.24 g in 1L) and 30% glycerol.
 The tissue must be minced thoroughly before fixation and the fixative should be
changed after the initial first week.
 The volume of tissue to fixative should be less than 1:10.
 If the tissue is dry i.e. obtained from a carcass, museum can be stored as it is and
fixative should not be added.
 When collecting feather (in case of birds)
 Make sure that the superior umbilicus of the feather is present.
 A minimum of 3-4 feathers should be collected.
 In case of large feathers (rectrices) only the section of the feather shaft containing
the superior umbilicus is used as sample for DNA isolation and barcoding.
 If possible freshly plucked feathers (from breast or retrices) should be preferred.
 Feather characteristics include macroscopic attributes like plumage pattern, texture
and size should be noted followed by microscopic attributes of the plumulaceous
barbs.
 High resolution photograph of the animal in its habitat should accompany the tissue/
feather submission.

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Figure: Feather specimen with umbilicus

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13. Amphibian and Reptile Samples Collection Guidelines
 All necessary permits must be obtained prior to any collection.

 Any infection to the animal should be notified while submitting the tissue.
 For amphibians and reptiles, a broad spectrum of tissue samples can be used for the DNA
extraction, amplification, and sequencing of the DNA barcoding process.
 Wherever possible the DNA barcoding study on amphibians and reptiles should be
supplemented with adequate collection of representative voucher specimens for subsequent
taxonomic study.
 In some cases, however, the killing of the animals and their deposition in public collections is
undesirable or impossible; for instance, in critically endangered species blood samples (most
reptiles with middle to large body size), tail tips (e.g., lizards and snakes), toe clips (frogs and
salamanders), fin clips (aquatic salamanders and tadpoles), and scale clips (snakes) can be
taken. In moderate- to large-sized specimens, buccal saliva swabs give good results. Swabs can
be air-dried and then stored, but in humid environment, storage in pure ethanol is preferable.
A further alternative source of DNA are the exuviae (shed skin, especially of snakes) but if
encountered in the wild, the identity of the specimen will not always be ascertainable.
 For dead animals including museum specimens that have not been fixed using formalin, liver,
heart, or muscle tissues represent optimal sources of genomic, and especially mitochondrial
genomic DNA. In general, ethanol-fixed and ethanol-preserved museum specimen are ideal
for DNA barcoding.
 All tissues must be stored in 95% ethanol or tissue protectant 70% PBS (For preparing 1 L of
PBS: NaCl 8 g, KCl 0.2 g, Na2HPO4 1.44 g, KH2PO4 0.24 g) and 30% glycerol.
 Whenever samples are collected, documentation of color in life should be a crucial component
of each study. Digital high resolution photos should be made both of dorsal and ventral sides.
If voucher specimens are collected, the photos will later serve to know their life color since
many amphibians and lizards (like colorful geckos or chameleons) will lose their color within
hours in preservative (and sometimes in life while being handled).
 If voucher specimens are not collected, photographic documentation becomes even more
important and should include lateral, dorsal, and ventral photos as well as close-ups (e.g., of
head and ventral sides of hands and feet), to make sure diagnostic characters will be
recognizable and allow to subsequently verify species identification of barcoded specimens.
 If possible in case of frogs vocalizations should be noted i.e. observation of the movement of
the vocal sac must be noted and recorded as “call voucher”. If this can be achieved, this “call
voucher” specimen should be collected separately, documented photographically, and its call
recording and tissue sample marked unambiguously.
 Preferably capture amphibians by hand but wash hand frequently and in between two
specimens. It is recommended that gloves be used and changed between two specimens.

V.1.1.2014-15
14. Key references
 CCAC species-specific recommendations on: amphibians and reptiles (Canadian Council of

Animal Care),
www.ccac.ca/Documents/Standards/Guidelines/Add_PDFs/Wildlife_Amphibians_Reptiles.p
df
 Fish sampling techniques, http://nptel.ac.in/courses/120108002/module5/lecture11.pdf
 How To Preserve Fish Specimens for Long-Term Storage or Shipment,
http://research.amnh.org/vz/ichthyology/congo/other05.html
 http://www.ento.csiro.au/education/preserving.html#spiders
 http://www.lib.ncsu.edu/agnic/sys_entomology/preserve.html
 Iliffe R, 2006, British Mycological Society Recording Network: Guidance Notes In: Collecting
and recording fungi, British Mycological Society.
 Invertebrate Collection Manual: A guide to traditional invertebrate collection methods,
http://australianmuseum.net.au/Uploads/Documents/9382/The%20Invertebrate%20Collec
tion%20Manual.pdf
 Iwanycki N, 2009, Guidelines for Collecting Herbarium Specimens of Vascular Plants, Royal
Botanical Garden, https://www.rbg.ca/Document.Doc?id=125
 Lijtmaer DA, Kerr KCR, Stoeckle MY, Tubaro PL, 2012, DNA Barcoding Birds: From Field
Collection to Data Analysis, Methods in Molecular Biology, Vol. 858, pp 127-152.
 Patrick Leonard (2010) A Guide to Collecting and Preserving Fungal Specimens for the
Queensland Herbarium (Version 3.2).
 Rudnick JA, Katzner TE, Bragin EA, Woody JA, 2007, Species identification of birds through
genetic analysis of naturally shed feathers, Molecular Ecology Notes, 7(5), 757-762.
 Steinke D, Hanner R, 2011, The FISH-BOL collaborators’ protocol. Mitochondrial DNA,
22(S1): 1014.
 Vences M, Nagy ZT, Sonet G, Verheyen E, 2012, DNA barcoding amphibians and reptiles,
Methods in Molecular Biology, Vol. 858, pp 79-107.
 Wilson R, 2005, Marine invertebrate sample processing procedures,
http://researchdata.museum.vic.gov.au/amit/Marine_sample_processing_8Sep2005.pdf

V.1.1.2014-15
Annexure I: Proforma for Deposition of specimen
1. Name of the depositor:

2. Designation:
3. Address of the organization:
4. Phone (with STD Code):
5. Mobile:
6. E-mail address:
7. Number of samples to be submitted:
8. Collection site with GPS details:
Terms and conditions for submission:
1. The material/ specimen should be collected as per the guidelines.

2. All the details of the specimen should be provided in prescribed format (Annexure II).
3. The sole responsibility of sample deposition will be of the depositor. It is understood that
samples/ specimens submitted to Gujarat Biodiversity Gene Bank (BioGene) are collected in
compliance of national/ international regulatory requirements.
4. All specimen(s) once deposited to BioGene will be sole belongings of BioGene, GSBTM,
DST,GoG and will not be returned back.
5. I/we own the copyright and have the consent and permission of each author to transfer
copyright of the said contribution. I hereby assign to the Gujarat Biodiversity Gene Bank
(BioGene) full copyright and all rights under it, including, but not limited to all rights for
publication in paper, electronic, and facsimile, formats, and for electronic capture,
reproduction, and licensing in all formats, in whole or in part, now and in perpetuity, in the
original and all derivative works.
6. BioGene will make best efforts for the preservation and maintenance of the specimen; but
it will not take any responsibility of loss of specimen because of any accidental hazards.
7. The detailed information asked in proforma reflects the academic authenticity. This will help
to develop the information base which is required and desirable for match making, or culture
exchange and culture transfer for potential organisms. Hence the depositor is requested to
provide the detailed scientific information of the organism.
I/ We have read the above terms and conditions and hereby agree to above terms and conditions
for depositing specimen.
Signature with official stamp
Date:
Place:

V.1.1.2014-15
Annexure II: Essential information required for

submission of specimen to Barcode of Life Database
(BOLD)
Sr. No. Collection data

1 Habitat including details of sample collection like place, ecological description,
season
2 Sample ID
3 Field ID
4 Collection code
5 Collection Date
6 Collector
7 Phylum
8 Class
9 Order
10 Family
11 Genus
12 Species
13 Identifier
14 Identifier Email
15 Identifier Institution
16 Identification Method
17 Voucher Status
18 Country
19 State
20 Region
21 Sector
22 Exact Site
23 Latitude (in degree decimal format)
24 Longitude (in degree decimal format)
25 Elevation (meter)
26 Photographer
27 Digital photos of specimen in habitat (preferably in 300 dpi)
28 Close-up photograph of specimen with taxonomic / identification features
(preferably with scale in 300 dpi)
Note: Refer BOLD Handbook for submission parameter

(http://www.boldsystems.org/libhtml_v3/static/BOLD_Handbook_Oct2013.pdf)

V.1.1.2014-15
Annexure III: Optional information for submission of

specimen to Barcode of Life Database (BOLD)
Sr. No. Collection data

1 Vernacular name
2 Identification method
3 Taxonomy notes
4 Voucher status
5 Tissue descriptor
6 Associated taxa
7 Associated specimen
8 Sex
9 Reproduction
10 Life stage
11 Specimen collection depth / elevation (meter)
12 Habit
13 Sampling protocol
14 Measurement
15 GPS source
16 Coordinate Accuracy
17 Event Time
18 Collection Date accuracy
19 Collection notes
20 Site Code
Note: Refer BOLD Handbook for submission parameter

(http://www.boldsystems.org/libhtml_v3/static/BOLD_Handbook_Oct2013.pdf)

Field Collection Guidelines
Annexure IV: Parameters for sampling, voucher preparation, transportation and

preservation of biomaterial from various sources
Required Sample and Information

Specimen
Sr. Sample Required Voucher Storage/ Specimen
DNA Barcoding (5 Remarks
No. Type Biomaterial Preservation Transportation
Specimen Photograph replicates) Medium
(Preferably 3-5) (Pref. 300 dpi)
1 Soil Pure Fungus 3 lyophilized copy Plate photograph 20-100 mg Glycerol stock Plates containing full
cultured on Petri and 3 live copy on having mycelia mycellia for each for mycellial growth
Plate (preferably solid media; 3 and spores; replicate cryopreservati under ice conditions;
in mycellial form) permanent slide microscopic on; Generally glycerol stock in dry
of spores photograph of PDA (may be ice/ liquid nitrogen
spore specific for
some species)
2 Mushroo Complete 3 dried Dorsal and 20-100 mg of Mycellial form Fresh mushroom
m Fruiting body mushrooms and 3 ventral tissue from the on PDA, Dried/ wrapped in butter
with fungus - preserved in FAA photographs internal part of FAA preserved paper in cold dark
cap, hymenium or Lugol solution showing color/ fruiting body / lyophilized condition; dried /
(spore-bearing (see detailed shape of collected under fruiting body lyophilized
tissues) and guidelines); Spore hymenium sterile conditions mushroom at RT
stipe; and ring print and proper
and volva where sterilization
present
3 Lichen Thallus with 100-200 mg tissue Photographs in 100-200 mg of Dried in paper In a paper bag under
some amount of in paper bag habitat, close-up tissue bag preserving dry conditions
substratum of thallus the structure
Gujarat Biodiversity Gene Bank | GSBTM | DST | GoG Page | 45

4 Algae Thallus Specimens for Photographs in 100-200 mg of Voucher Dried material in dry
microscopic habitat, close-up tissue specimen in dark condition/ if
analyses should of thallus dark/ dry material fresh then
be fixed in condition; ship in ice box
4%formaldehyde sample in silica
solution, and containing zip
unfixed seal bags for
subsamples dried DNA isolation
on herbarium
paper as voucher
5 Bryophyt Fertile material Specimens for Photographs in 100-200 mg of Voucher Dried material in dry
e with capsule microscopic habitat, close-up tissue specimen in dark condition/ if
analyses should of the dark/ dry material fresh then
be fixed in sporophytic condition; ship in ice box
4%formaldehyde stage sample in silica
solution, and containing zip
unfixed seal bags for
subsamples, air DNA isolation
dried as voucher
6 Higher Plant specimen Plant vouchers on Plant photograph 5-7 young leaves Herbarium Voucher sample from
Plants collected during herbarium sheet in habitat/ close- in zip seal bag under dark/ field with herbarium
reproductive up photograph containing dried dehumidified press and DNA
stage (with covering details silica (minimum conditions; sample with silica gel
flower and/or of reproductive 20 mg for each Silica gel or in icebox with ice
fruiting organ) organs replicate) sample under pack
dark condition;
Lyophilized
sample at RT

7 Porifera Whole animal 70% ethanol Photo in habitat 100-200 mg of 70% ethanol Preferably snap Formalin will
(colony) as tissue tissue in ethanol chilled samples in render most
and voucher liquid nitrogen; sponges
source Alternatively it can unidentifiabl
be preserved and e
shipped on ice or
with cool packs
8 Cnidaria( Whole animal 70% ethanol Photo in habitat 100-200 mg of 70% ethanol Preferably snap Formalin will
Octocora tissue in ethanol chilled samples in dissolve
llia) liquid nitrogen; spicules and
Alternatively it can render many
be preserved and octocorals
shipped on ice or unidentifiabl
with cool packs e.
9 Cnidaria Whole animal Fixed in 4% Photo in habitat 100-200 mg of 70% ethanol Preferably snap
(Scyphoz formalin and tissue in ethanol chilled samples in
oa) preserved in 70% liquid nitrogen;
ethanol Alternatively it can
be preserved and
shipped on ice or
with cool packs
10 Cnidaria Whole animal Fixed in 4% Photo in habitat 100-200 mg of 70% ethanol Preferably snap
(others) formalin and tissue in ethanol chilled samples in
preserved in 70% liquid nitrogen;
ethanol Alternatively it can
be preserved and
shipped on ice or
with cool packs

11 Platyhel Mature stage of Fixed in 4% Dorsal and 100-200 mg of 70% ethanol Preferably snap Fix living
minthes animal formalin and ventral side of tissue in ethanol chilled samples in specimens on
preserved in 70% animal. Prefer to liquid nitrogen; frozen 4%
ethanol take photo in Alternatively it can formalin or
habitat be preserved and narcotise
shipped on ice or (freezing or
with cool packs propylene
phenoxytol
or MgCl2).
Otherwise
probably
unidentifiabl
e.
12 Annelida Whole animal as Specimen fixed in Dorsal and 100-200 mg of 70% ethanol Preferably snap Leeches and
tissue source 4% formalin ventral side of tissue in ethanol chilled samples in some
followed by 70% animal. Prefer to liquid nitrogen; polychaete
ethanol (see detail take photo in Alternatively it can families are
guideline) habitat. be preserved and easier to
shipped on ice or identify if
with cool packs anaesthetize
d
13 Arthropo Whole animal / Specimen fixed in Dorsal and 100-200 mg of 70% ethanol Preferably snap
da muscle tissue 4% formalin ventral side of tissue in ethanol chilled samples in
(Crustace followed by 70% animal. Prefer to liquid nitrogen;
a) ethanol (see detail take photo in Alternatively it can
guideline) habitat. be preserved and
shipped on ice or
with cool packs

14 Arthropo Leg with muscle / Insect must be Dorsal and 100-200 mg of Dry mounted Room Temperature See detail
da whole animal stored in dry ventral side of tissue in ethanol specimen guidelines
(Insects) condition using animal. Prefer to
mounting and take photo in
pinning habitat.
15 Mollusca Whole animal / Fixed in 4% Photo in habitat 100-200 mg of 70% ethanol Preferably snap Narcotise
(Opistho muscle tissue formalin, 70% tissue in ethanol chilled samples in (freezing or
branchia) (Use knife or ethanol or in 1% liquid nitrogen; propylene
sharp blade to Chloral hydrate, Alternatively it can phenoxytol
remove animal Store at -20˚C be preserved and or MgCl2) if
from shell) shipped on ice or at all
with cool packs possible;
recording
colour in life
are also very
useful
16 Mollusca Whole animal / Fixed in 4% Photo in habitat 100-200 mg of 70% ethanol, Preferably snap
(Other) muscle tissue (If formalin, 70% tissue in ethanol Outer shell chilled samples in
required use ethanol or in 1% liquid nitrogen;
hand digging Chloral hydrate, Alternatively it can
tools) Store at -20˚C be preserved and
shipped on ice or
with cool packs
17 Echinode Soft tissue 70% ethanol Dorsal and 100-200 mg of 75-85% Preferably snap Formalin will
rmata ventral side of tissue in ethanol ethanol chilled samples in render many
animal. Prefere liquid nitrogen; echinoderms
to take photo in Alternatively it can unidentifiabl
habitat. be preserved and e, especially
shipped on ice or holothurians
with cool packs

18 Tunicata Soft tissue Fixed in 4% Dorsal and 100-200 mg of 70% ethanol Preferably snap
formalin and ventral side of tissue in ethanol chilled samples in
preserved in 70% animal. Prefer to liquid nitrogen;
ethanol take photo in Alternatively it can
habitat. be preserved and
shipped on ice or
with cool packs
19 Fish Entire fish if 3-5 fishes for Lateral Tissue of Storage in FAA Preferably snap
smaller than 12- smaller specimens photographs Musculature (5 – 7 chilled samples in
15 inch (<12 inches) snap with fin details; mm) from right liquid nitrogen;
chilled or dorsal and lateral Alternatively it can
preserved in FAA; ventral muscle; Gill be preserved and
for longer fishes photographs for tissue; Eye tissue shipped on ice or
(>12 inch) take flat fishes preserved in with cool packs
field photograph Ethanol
20 Amphibia Preferably Fixed and Dorsal and Prefer 1-2 ml Storage in FAA Preferably snap
and mature animal, if preserved in FAA ventral side of blood samples chilled samples in
Reptiles collecting animal. Prefer to with EDTA; For liquid nitrogen;
developmental take photo in large animals take Alternatively it can
stages prefer to habitat. 3-5 buccal saliva be preserved and
collect it with a swabs;Minimum shipped on ice or
mature organism 500 mg tissue with cool packs
for identification sample from tail
tips (e.g., lizards
and snakes), toe
clips (frogs and
salamanders), fin
clips (aquatic
salamanders and
tadpoles), and

scale clips
(snakes)
21 Aves and Blood, tissue, Stuffed bird, Front and lateral Prefer 1-2 ml Stuffed Bird, Preferably snap
Mammal feather, hair or Feather having photograph. blood samples FAA preserved chilled samples in
s nail with Follicle superior Prefer to take with EDTA; material liquid nitrogen;
tissue umbilicus, Muscle photo in habitat. Minimum 500 mg Alternatively it can
tissue muscle tissue; 3-5 be preserved and
feathers / hairs / shipped on ice or
nail with follicle with cool packs
tissue

Gujarat Biodiversity Gene Bank
Gujarat State Biotechnology Mission
Department of Science & Technology, Government of Gujarat
9/11, Udyog Bhavan, Gandhinagar – 382011
Ph. 91-79-23252197, Fax: 91-79-23252195, url: btm.Gujarat.gov.in; biogene.in

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Gujarat

A GSBTM, DST, Government of Gujarat initiative

1.1 Aim and Objectives ..........................................................................5

3. General Guidelines .............................................................................. 11

4. Basic Requirements ............................................................................ 12

5. Plant specimen collection Guidelines ..................................................... 13

5.1 Pressing the plants ......................................................................... 15

6.1 Containers for collecting samples ..................................................... 18

7.1 Collection and drying of fungal specimens ......................................... 21

Gujarat Biodiversity Gene Bank | GSBTM | DST | GoG P a g e |1

7.2.2 Specimen information (field) ...................................................... 23

9. Bryophyte, Lichen and Algae Collection Guidelines .................................. 28

9.1 Bryophyte specimens (mosses, liverworts) ........................................ 28

10.1 Collecting Invertebrates for preservation and DNA barcoding ............. 30

11.1 Fixatives ..................................................................................... 36

13. Amphibian and Reptile Samples Collection Guidelines ............................ 40

14. Key references ................................................................................. 41

Annexure I: Proforma for Deposition of specimen ....................................... 42

Annexure II: Essential information required for submission of specimen to

Barcode of Life Database (BOLD).............................................................. 43

Annexure III: Optional information for submission of specimen to Barcode of

Life Database (BOLD) ............................................................................. 44

Gujarat Biodiversity Gene Bank | GSBTM | DST | GoG P a g e |2

Annexure IV: Parameters for sampling, voucher preparation, transportation and

preservation of biomaterial from various sources ........................................ 45

Gujarat Biodiversity Gene Bank | GSBTM | DST | GoG P a g e |3

Akshay Saxena, IFS

Akshay Kumar Saxena

Gujarat Biodiversity Gene Bank | GSBTM | DST | GoG P a g e |4

1.1 Aim and Objectives

Gujarat Biodiversity Gene Bank | GSBTM | DST | GoG P a g e |5

1.2.1. Key Equipments

Ion Proton Ion Torrent TissueLyser II 2D-IEF

1.3. Microbial Repository

Microbial Repository (BAB-BioGene) is microbial culture-collection of microorganisms in Gujarat. It

Present status of microbial repository

Culture Type Total microbes NCBI Submission Accession number

Gujarat Biodiversity Gene Bank | GSBTM | DST | GoG P a g e |6

1.4 Plant Gene Bank

Present Status of Plant Gene Bank

1.5 Animal gene bank

Gujarat Biodiversity Gene Bank | GSBTM | DST | GoG P a g e |7

Animal Gene Bank Status

1.6 BioGene Umbrella Program on Barcoding Biodiversity of

Barcodes submitted to Barcode of Life Database [BOLD]

Fungi Plant Animal Total

Gujarat Biodiversity Gene Bank | GSBTM | DST | GoG P a g e |8

Bio materials deposited in Gujarat Biodiversity Gene Bank (BioGene)

Eranthemum roseum Lantana rugosa Senna alata

Ganoderma multipileum Flavodon flavus Phellorinia herculeana

Trimerotropis sp. Aplysia fasciata Eulalia viridis

Moringa olifera Oroxylum indicum Peltophorum ferrugineum

Gujarat Biodiversity Gene Bank | GSBTM | DST | GoG P a g e |9

2. Scope and objectives

Gujarat Biodiversity Gene Bank | GSBTM | DST | GoG P a g e | 10

1. Always carry the collection kit.

Gujarat Biodiversity Gene Bank | GSBTM | DST | GoG P a g e | 11

Zip seal bags/

Field record GPS with spare

Camera Rucksack Bag

Gloves Field shoes

Gujarat Biodiversity Gene Bank | GSBTM | DST | GoG P a g e | 12

5. Plant specimen collection Guidelines