The document provides information on preparing, preserving, and storing plant specimens in a herbarium. It defines what a herbarium is and its functions. It describes how to collect, press, dry, poison, mount, label, and store plant specimens. It discusses important herbarium collections around the world and includes techniques for staining specimens. The overall document serves as a guide for building and maintaining a herbarium collection.

1. DURGESH SIROHI
DEPARTMENT OF BOTANY
C.C.S.UNIV. (MEERUT)
PREPARATION OF HERBARIUM SHEETS,
PRESERVATION,STORAGE &
STAINING OF MATERIALS

2. CONTENTS
 Herbarium definition
 Functions of herbaria
 Kinds of herbaria
 Important herbaria of the world
 Plant collection
 Pressing & drying of specimens
 Poisoning
 Mounting
 Labelling
 Storing
 Uses
 Staining techniques
 References

3. HERBARIUM
 A herbarium is a store house of plant specimens. In it dried, pressed, preserved
& mounted plant specimens are arranged in a sequence of an accepted system
of classification for future reference & study.
 The collected plant specimens from far & wide, mounted on appropriate sheets
& kept in pigeon holes of steel or wooden cupboards for study at the same
place & time.
 According to Fosberg & Sachet a modern herbarium is a great filling system
for information about plants both primary in the form of actual specimens &
secondary in the form of published information, pictures & recorded notes.

4. FUNCTIONS OF HERBARIUM
 Conservatory of material & data.
 To allow accurate identification of plants.
 To certify that a plant name is correct (by providing a vital
reference collection of authenticated material. This is often based
on the original material used to frame the plant’s description and
is called ‘Type material’).
 To act as a source of information about plants (e.g. on plant
distribution, ecology or plant medicinal usage).
 To allow the validation of scientific observations (e.g. on climate
change, genetics and conservation matters).
 To support the research and teaching activities.

5. KINDS OF HERBARIA
The kinds of herbaria depend upon the contents, purpose, regions/place,
plant groups-present in these:
 The herbaria of medicinal plants
The herbaria include specimens of plants of known medicinal value or
supposed to have medicinal properties.
ACECR Central Herbarium of Medicinal Plants, Halejerd region,
Karaj, Alborz province.
 Herbaria of weeds
The herbaria have weeds of cultivated fields & waste places.
 Regional herbaria
The herbaria of particular region or place e.g. Herbarium of Arid Zone
Circle, Jodhpur; Herbarium of Central Circle, Allahabad; Herbarium of
a University or Institution.

6. IMPORTANT HERBARIA OF
THE WORLD
The first herbarium of world was founded in 1545 in the
University of Padua, Italy. In the world, there are about one
thousand five hundred recognised herbaria, besides many smaller
herbaria with institutions, universities, pharmacies, etc.

7. SOME IMPORTANT
HERBARIA OF THE WORLD
HERBARIUM PLACE /
COUNTRY
YEAR OF
FOUNDING
NO. OF SHEETS
(APPROX.)
Royal Botanic Garden,
Kew
London , U.K. 1853 6,500,000
Botanical Institute Leningard,
USSR
1823 5,000,000
U.S. National Museum Washington 1868 3,000,000
Royal Botanic Garden Edinburgh 1761 2,500,000
Arnold Arboretum Boston 1872 700,000
Herbarium of the
Rangoon University,
Rangoon
Burma 1947 15,000

8. HERBARIUM PLACE /
COUNTRY
YEAR OF
FOUNDING
NO. OF SHEETS
(APPROX.)
Central National
Herbarium
Calcutta 1793 2,500,000
Indian Council for
Forest Research &
Institute
Dehradun 1874 3,00,000
Botanical Survey of
India , Southern
circle
Coimbatore 1874 2,00,000
Botanical Survey of
India , Eastern circle
Shillong 1956 100,000
Botanical Survey of
India , Western circle
Poona 1956 135,000
Botanical Survey of
India , Northern
circle
Dehradun 1956 60,000
Botanical Survey of
India , Cental circle
Allahabad 1955 40,000

9. PLANT COLLECTION
 Good collection is the corner- stone of whole edifice. To build new
herbarium or to enrich our herbaria, not to fill them with poor
scraps, the botanical collection must have a purpose. The purpose
of botanical collection can be following:
 To build new herbarium
 To enrich the herbaria
 To write a flora
 To carry out research on a particular genus/ species.

10. WHAT TO COLLECT?
 What plants shall be collected depends on
the purpose of study.
The possible main approaches are:
1. To collect as much as one possibly can. This method is desirable
in little- known areas but only when the collector is able to
cope with the tiring work involved.
2. To collect certain groups, either those in which a taxonomist is
particularly interested, or of which herbaria require better
representation.
3. At least six specimens of each plant are to be collected, to
facilitate the exchange & detailed studies.
4. In herbaceous plants rootstock should be collected.

11.  We do not collect rare or endangered species. For common
species, several representative specimens are collected, especially if
they are small or have only a few flowers, leaves or fruits. For
herbaceous plants the specimen(s) should include, if possible, all
plant parts (underground parts, stems, leaves, flowers and fruits).
Large parts, such as cones, large fruits, seeds and needle leaves,
can be stored in boxes, bags or envelopes associated with the
specimen. When collecting, one should keep in mind that the
“final” specimen, after pressing and drying has to fit on a
herbarium sheet of 42 x 28cm.

12. PROCEDURE FOR COLLECTING
THE PLANT SPECIMENS
 Planning
 One should try to get available maps & collect local information.
 One should try to have the best guide & for carrying the
equipment one should select preferably people who are more or
less acquainted with forest work.

13. EQUIPMENTS
 A collecting pick for digging up roots & rhizomes of herbaceous
plants so that the collected specimen is complete.
 A strong knife is sometimes required for cutting branches &
other plant parts.
 A pair of pruning shears or secateur for cutting woody & hard
material.
 A vasculum for accommodating collected specimens.
 Blotting papers
 Collecting bags
 Hand lens
 A field book (field diary) for noting down the details of collected
plants.

14. Herbarium Cabinets
http://talkingplants.blogspot.in/2010_10_01_archive.html
vasculum

15. Out in the field, collecting plant material to press

16. Some collected material, showing labeling

17. FIELD NOTES
 The main objective of field notes is to record data of
taxonomic value that will not be apparent form the
specimen.
1. Date
2. Number of plant
3. Name of plant
4. Province
5. Locality
6. Altitude
7. Habitat
8. Description

18. Collector on expedition,
writing notes about their
plant

19. HANDLING OF FRESH MATERIAL
 To press each plant as it is collected in the field then & there.
 To keep all the specimens collected in a vasculum & bring them to
camp. They are then pressed one by one.

20. Doing the initial pressing, in the field

21. PRESSING & DRYING
 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. Arrange plant material on blotting paper retaining the character
of the plant. Remove leaves and flowers of congested specimens to
reduce the bulk without losing the character of the plant.
 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 .
 Inspect the material 24 hours later, replacing the top layer of blotting
paper with dry card. 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.

23.  The purpose is to extract the moisture, so that plants do not rot .
Pressing and drying preserve the morphological integrity of the
plants, which can be then mounted on herbarium paper and
stored for a long time. Pressing plants immediately after
collection results in the best herbarium specimens. Samples that
are let to wilt prior to pressing will produce inferior specimens. A
plant press consists of a wooden frame (for rigidity),blotter paper
(to absorb moisture), and folded newspaper (to contain the plant
material). The plant press is tightened using straps with buckles
or bolts with wing nuts.

24.  Plants should be carefully arranged as they are placed in the press
to maximize preservation of diagnostic features. Leaves, flowers,
and fruits should be spread out so that they do not overlap and
can be observed from different perspectives. The collection
number should be clearly written on the outside of the newspaper
containing each plant specimen. The plant press must be kept
tight; this prevents shrinkage and wrinkling of the plant material
and yields specimens that are easier to mount securely on
herbarium paper.

25.  The pressed plants must be thoroughly dried prior to storage and
mounting. Best results are obtained with the use of an electric
drier that holds the presses and provides steady bottom heat
between 95°F and 113°F. In the absence of a drier , make sure to
replace the blotter paper periodically until the specimens are
dry. Rapid drying promotes the best retention of plant colour, but
excessively high temperatures or long drying periods can result in
blackened, discoloured, and brittle specimens.

26. MICROWAVE OVENS
 Small numbers of specimens can be dried using a microwave oven. The
technique recommended in the literature is to place the specimens
between unprinted absorbent paper, for example butcher's paper, not
newspaper, which is unsuitable because the chemicals present in the ink
may cause a fire. The specimens should be put in a special press which
should be of a microwave-safe material (wood, acrylic or polycarbonate
sheets e.g. plexiglass or perspex, NO metal components). If such a press is
not available, sheets of cardboard can be placed above and below the
specimens and then weighted down. Drying time depends on the power
of oven.
 It should be noted that microwave treatment damages seeds and the
cellular structure of the plants which may reduce the long-term value of
the specimens.

27. ALTERNATIVE DRYING TECHNIQUES
 Silica gel/other desiccants & freeze drying
 Alternative methods of drying plant specimens have been used for some
time, but are mostly restricted to special purpose collections. The main
alternatives are freeze-drying and drying in a desiccant powder such as
desiccant silica gel. In general these techniques are used where it is
essential to preserve the shape of a delicate plant or organ of the plant
such as the flower. Freeze-drying has also been used to preserve the
chemical composition of a plant as accurately as possible for later study.

28. Disadvantages and special conservation problems of specimens
dried in these manners are that they are particularly
susceptible to damage. The dried parts are fragile, lack support
and often catch on packing materials. They must, therefore, be
packed especially carefully and stored in small boxes or tubes
with some appropriate packing material that does not snag
and break small projections. Acid-free tissue paper is often
used. Drying in desiccant silica gel crystals or powder can also
have the disadvantage that it is difficult to remove all traces
of the silica gel after drying.

29. SPECIAL PRESERVATION &
PROCESSING TECHNIQUES
 Wet or spirit collections
 Very fleshy or delicate structures, including small algae and
orchid flowers, are best preserved in an air-tight glass or plastic
jar with a liquid preservative rather than by drying. The type of
preservative used should be clearly labelled in the jar. Such
material is often referred to as a spirit collection or wet collection.
Most material can be satisfactorily preserved in 70% ethyl
alcohol (or 70% methylated spirit) with 30% water. Colours will
fade quickly in spirit, however, so it is a good idea to keep
comprehensive notes and photographs.

30. POISONING
 For avoiding attack by fungus or pests
 Mercuric chloride – dip the whole plant in saturated solution of
mercuric chloride in ethyl alcohol for 15 – 20 minutes. The plant
is put in dryers & pressed till completely dried. Mercuric chloride
is corrosive for metals therefore enamel coating trays should be
used. Gloves should be used. Mounted specimens can be brushed.
 Formalin – 10% used during collection tours.
 Fumigation : killing pests from mounted or unmounted
specimens.
 All fumigators are harmful to human beings too.
 Ethylene chloride : C Cl4 (3:1)

31. MOUNTING
 Mounting is the process of attaching a dried pressed plant and its label to a sheet of
heavy paper. This provides physical support that allows the specimen to be handled and
stored with a minimum of damage. Prior to attachment, the specimen and its label are
laid out on the paper to allow maximum observation of diagnostic (usually
reproductive) features as well as the range of variation in vegetative structures,
including both sides of the leaves. Plants are generally positioned in a life-like
arrangement (that is, with roots or lower stem toward the bottom of the sheet and
flowers toward the top). When laying out the plant, be sure to leave space on the sheet
for the specimen label. A paper envelope or packet should also be attached to the sheet
to contain any fragments of the specimen that break off over time. Once the optimum
arrangement of the specimen has been determined, it is attached to the sheet using a
combination of glue. Glue is used sparingly to attach the larger portions of the plant,
such as stems, large leaves, and fruits. Gummed linen mounting strips are then applied
to reinforce portions of the plant that might be torn loose as the specimen is used. Large
or bulky items may need to be sewn onto the sheet with a sturdy linen thread.

32.  Excessive application of glue that embed flowers and seeds on the
sheet may make it impossible to observe diagnostic features or to
remove samples, thus rendering the specimen useless for scientific
study. The best way to learn proper mounting procedures is
through hands-on training and practice with a variety of plant
specimens. Because herbarium specimens are intended for long-
term study and storage, it is critical that all supplies used for
mounting be both durable and archival. Archival denotes
materials that are free of acids and other compounds that may
cause them or the specimen to degrade or discolour over time.
Consequently, the mounting paper, label paper, packet paper, ink,
glue, mounting strips, and storage folders should all be acid free
and designed for long-term stability.

34. Layout of a herbarium specimen
• Label in bottom right hand corner, with other labels and capsule
above.
• Plant to left of label. If the specimen is large you can attach the
label on the right hand side to flap over the specimen
• Turn specimen to fit on page

35. LABELLING OF SPECIMEN
 Name of family
 Genus & Species
 Locality of collection
 Date of collection
 Description / remarks / notes
 Name of collector & collection number
 Vernacular name

36. STORING OF SPECIMENS
 Place the prepared specimen in a sealed plastic bag and freeze for
72 hours. Ideally the temperature should be -320C, although most
domestic freezers have a minimum of -180C. 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.

37. Kew’s Herbarium
where over 7 million
pressed plants are
kept
http://www.kew.org/collections/herb_types.html

38. USES
 Herbaria are essential for the study of plant taxonomy, the study of
geographic distributions, and the stabilizing of nomenclature. Thus, it
is desirable to include in a specimen as much of the plant as possible
(e.g., flowers, stems, leaves, seed, and fruit).
 Specimens housed in herbaria may be used to catalogue or identify
the flora of an area. A large collection from a single area is used in
writing a field guide or manual to aid in the identification of plants
that grow there..
 Herbaria also preserve a historical record of change in vegetation over
time. In some cases, plants become extinct in one area or may become
extinct altogether. In such cases, specimens preserved in an herbarium
can represent the only record of the plant's original distribution.
Environmental scientists make use of such data to track changes
in climate and human impact.

39. STAINING TECHNIQUE

40. DYES TYPE COMMON NAME SOLUBILITY STAINS
Safranin
(Basic)
Coal tar dye
(Artificial )
Azo dye 5.45% in water
3.41% in alcohol
Lignified, cutinized,
suberized structure &
chromosomes , nucleoli
& centrosomes
Janus Green B
(Basic)
Artificial Diazin 5.18% in water
1.12% in alcohol
Used as vital stain for
fungi, flagella of the
protozoa, mitochondria.
Crystal Violet
(Basic)
Coal tar dye
(Artificial)
Gentian violet 1.68% in water
13.87% in alcohol
Nuclear stain
Fast Green
( Acidic )
Coal tar dye
(Artificial )
- 16.04% in water
0.33% in alcohol
Cytoplasm , cellulose
cell walls
Cotton Blue
(acidic)
Artificial Aniline blue/ china
blue
- Cellulose, cell wall,
achromatic figure,
filamentous
chlorophyta.

41. STOCK SOLUTION & STRUCTURE

42. SAFRANIN  2.25 g sample in 225ml of
95% ethyl alcohol, & part of
this stock solution is diluted.
 Diluted with an equal volume
(1:1) of water for use.
 If strong, then further diluted
with 50% alcohol.
 Excess stain should be
washed with water.
Johansen , 1940
• Dissolve 4g safranin in 200 ml methyl cellosolve; when
solution is complete, 100 ml each of 95% alcohol & 100 ml
distilled water are added, followed by 4g sodium acetate
& 8ml formalin.
•Johansen , D.A. (1940). “Plant Microtechnique”, 1st edition. McGraw-Hill Book
Company, Inc., U.S.A. pp.62.

43. FAST GREEN
 The clove oil- absolute alcohol solution is
recommended as the one giving the optimum
results.
 Add dry dye to a mixture of equal parts of methyl
cellosolve, absolute alcohol, & clove oil to give a
dark greenish solution.
 If too strong, dilute with clove oil.
•Johansen , D.A. (1940). “Plant Microtechnique”, 1st edition. McGraw-Hill Book
Company, Inc., U.S.A. pp.59.

44. HEMATOXYLIN
 A relatively stable hematoxylin solution may be prepared by adding
5 ml of a 10% absolute alcohol solution to 100 ml methyl
cellosolve, 50 ml distilled water & 50 ml tap water that contain
calcium compound in solution.
It is a chromogen derived from logwood,
hematoxylin campechianum. The dye
solution itself has little or no affinity for
tissues, unless iron or aluminium is
present in the latter, consequently
mordanting in some form is necessary.
•Johansen , D.A. (1940). “Plant Microtechnique”, 1st edition. McGraw-Hill Book
Company, Inc., U.S.A. pp.51-52.

45. DELAFIELD’S HEMATOXYLIN
 Hematoxylin crystals – 5g
 Aluminium ammonium sulphate – 3g
 50% ethyl alcohol – 1000ml
 Dissolve the dye & the alum with the aid of heat, then add 6g
mercuric oxide & boil for 30 min. Filter, then bring up again to
the original volume with 50% alcohol. Acidify in the proportion
of 1 drop hydrochloric acid to each 100ml of solution.

46. JANUS GREEN B
 Janus Green B is a basic dye and vital stain used in histology.
 The vital stain is prepared by dissolving from 0.001 to 0.41g in
100 ml of physiological saline solution.
 Used in various high dilutions as a vital stain for fungi & for the
flagella of the protozoa.
•Johansen , D.A. (1940). “Plant Microtechnique”, 1st edition. McGraw-Hill Book
Company, Inc., U.S.A. pp. 59.

47. GRAM’S IODINE
 Iodine is well known as a specific colour indicator for starch,
when made up in combination with potassium iodide.
 0.3g in 3% potassium iodide acts as a complexing agent.
 The colour reactions of iodine on sections of fresh material are as
follows:
BLUE BROWN YELLOW
Starch Cellulose Pectin
saponin Proteins Cutin
Insulin deposits Callose
Alkaloids Cork
•Johansen , D.A. (1940). “Plant Microtechnique”, 1st edition. McGraw-Hill Book
Company, Inc., U.S.A. pp.63.

48. REFERENCES
• Johansen , D.A. (1940). “Plant Microtechnique”, 1st edition.
McGraw-Hill Book Company, U.S.A. pp.49-64.
• Saxena , N.B. and Shamindra Saxena (2009). “Plant Taxonomy”,
6th edition. Pragati Prakashan, Meerut. pp. 101-108.
• http://www.rbgsyd.nsw.gov.au/plant_info/identifying_plants/processi
ng_plant_specimens/Preserving_plant_specimens
• http://www.rhs.org.uk/Plants/Plant-science/RHS-
Herbarium/techniques

49. THANK YOU