herbarium and staining techniques

DURGESH SIROHI

DEPARTMENT OF BOTANY

C.C.S.UNIV. (MEERUT)

PREPARATION OF HERBARIUM SHEETS,

PRESERVATION, STORAGE &

STAINING OF MATERIALS

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

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.

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.

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.

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.

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

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

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.

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.

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.

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.

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.

Herbarium Cabinets

http://talkingplants.blogspot.in/2010_10_01_archive.html

vasculum

Out in the field, collecting plant material to press

Some collected material, showing labeling

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

Collector on expedition,

writing notes about their

plant

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.

Doing the initial pressing, in the field

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.

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.

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.

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.

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.

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.

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.

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.

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)

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.

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.

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

LABELLING OF SPECIMEN

Name of family

Genus & Species

Locality of collection

Date of collection

Description / remarks / notes

Name of collector & collection number

Vernacular name

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.

Kew’s Herbarium

where over 7 million

pressed plants are

kept

http://www.kew.org/collections/herb_types.html

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.

STAINING TECHNIQUE

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.

STOCK SOLUTION & STRUCTURE

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.

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.



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.


Company, Inc., U.S.A. pp.51-52.

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.

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.


Company, Inc., U.S.A. pp. 59.

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



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

THANK YOU

herbarium and staining techniques

Science