Seaweed collection procedure

The collection of seaweeds in the field is done during the low tide. It is necessary to go for collection one or two hours before the time of low tide as per tide tables. This will give more time for seaweed collection and to observe seaweeds in the natural habitat. It is important to make notes on the description of the site
location, topography, associated flora and fauna and other related parameters. Although, there are number methods to collect seaweeds, we consider here two methods which are practical and easy to study.
a) Line transect/belt transect method and b) random sampling method.

Material necessary for seaweed collection
§ Polyethylene bags
§ Knife or scalpel
§ Labeling materials (pen/pencil, labels, marker pens etc.)
§ Rubber bands
§ Field note book
§ Long rope (about 50 m long)
§ Quadrant 0.25 m 2
§ Mono pan balance

Line transect or belt transect method
A line or belt transect is laid perpendicular to the coast from high tide to the low tide with the help of long rope . Sampling points along the rope can be marked depending on the gradient and the expanse of the intertidal area. Incase the intertidal area is small, sampling points can be marked at 5 m intervals along the rope and if intertidal area is quite large the sampling point can be marked at 10 or 20 m along the rope.

§ A quadrant measuring 0.25 m 2 area is places at the sampling points in triplicate covering an area of 5 m 2 on either side of the sampling points.

§ Seaweed species present with in the quadrant are collected (collect complete plant as far as possible along with the hold fast).

§ Seaweed specimen can be removed by hand but those specimen which are closely adhering to the substrate such as crustose and mat forming seaweeds can be removed with the help of knife or scalpel. The specimen that grows close to the rocks can be removed with the rocks using geologist's pick or any other similar tools.

§ All the collected specimen should be counted species wise and number of individuals in each species for quantitative assessment of abundance, density, frequency, species richness, species diversity, percentage cover etc. with statistical consideration.

§ All the collected specimen from the quadrant should be weighed to estimate standing crop biomass.

§ Collected material should be kept in the plastic bags/containers with proper labeling for further preservation and identification at the later stage in the laboratory.

Random sampling method
Samples can be selected at random as per requirement. This can be done by selecting sampling points in the area and using quadrant. Sampling points should be selected in such a manner that every species of the study area has good chance being selected. This type of sampling is usually done in the area where the intertidal expanse is very narrow with steep gradient. It is also employed for qualitative estimation of the seaweed.

Sample preservation

Wet preservation

§ All the adhering materials such as sand particles and other debris as well as epiphytes should be removed from the seaweeds before preservation.

§ A solution of 10 % formaldehyde in seawater should be prepared to preserve the seaweed sample.

§ Before adding the preservative, water from the plastic bags / containers should be drained and sufficient preservative should be added. Fumes of the formaldehyde would help to fix and preserve the seaweed material. Plastic bags should be tied with rubber bands properly to prevent leakage during transportation.

§ All the bags / containers should be properly labeled with date of collection, locality and time and transport to the laboratory for further identification.

Dry preservation (herbarium)
Material required for preparing herbarium is as follows:
§ Plastic trays
§ Forceps
§ Specimen mounting paper (herbarium sheets)
§ Cheese cloth
§ Blotting paper
§ Herbarium wooden press
§ Painting brush
§ Pencils, knife etc.

DRYING SPECIMENS

It is important that dry specimens be prepared carefully so that important morphological characters are displayed as fully and completely as possible. Portions of the specimens (fruiting structures or thallus sections) may be removed and placed in a vial of preservative for microscopic observation, which often is essential for identification. The following procedure, with a bit of practice, should produce good quality dried specimens.

1. Fixing the specimen:
The color of most specimens is best preserved by "fixing" the specimens in 3-5% buffered Formalin seawater away from direct sunlight (overnight fixation is adequate but algae may remain for longer periods of time without damage in this preservative if kept away from light, which causes bleaching). Deterioration may commence upon collection, so it is advantageous to have Formalin handy immediately following collection.

2. Preparing the specimens:

Fleshy specimens

Having been properly fixed, fleshy specimens should be rinsed free of any sand or debris. (Tap water may be used for this.) Remove any artifacts (shells, animals) which are not part of the specimen -- although records of their presence should be made for ecological reference. If the holdfast is too thick and resistant to be pressed, either split it to remove portions, thereby facilitating pressing, or remove the holdfast and dry it separately (properly tagged for reference to the original specimen).

Calcareous specimens
After first fixing the specimens in 3-5 % Formalin, select portions to be liquid preserved in a vial. Soak for several days in a solution of about 40% glycerin in 3% buffered Formalin seawater. Dry and place in small boxes without pressing or, where appropriate, glue carefully to herbarium paper

3. Preparation herbarium

Dry preservation (herbarium)
Material required for preparing herbarium is as follows:
§ Plastic trays
§ Forceps
§ Specimen mounting paper (herbarium sheets)
§ Cheese cloth
§ Blotting paper
§ Herbarium wooden press
§ Painting brush
§ Pencils, knife etc.

Procedure for preparing herbarium
§ Fresh specimen should be cleaned of sand particles, rocks, shells, mud and other adhering materials and epiphytes.
§ A tray containing fresh water (half filled) should be taken and specimen to be mounted be placed in the water.
§ A herbarium sheet, size smaller than the tray to be inserted from below the specimen and then spread specimen on the herbarium sheet with the help of brush in such a way that overlapping of the specimen is minimized.
§ After mounting the specimen on the herbarium sheet, sheet is lifted slowly and tilted to one side to allow water to drain gradually without disturbing the mounted specimen.
§ Remove the sheet and properly arrange the specimen with the help of forceps or needle if required.
§ To blot dry, herbarium sheets are placed on the newspaper sheets or blotting paper to remove the remaining water from the herbarium.
§ A cheesecloth is placed on the top of the specimen in such a way that it covers entire specimen.
§ Now place another sheet of the blotting paper over the herbarium sheet.
§ Once, all the specimen to be preserved are ready, herberia are piled one above the other and then placed between the two sheets of the wooden press. The press is tide tightly with appropriate pressure by a rope.
§ The press is kept at room temperature for 24 hrs. after 24 hrs blotting papers were replaced. The process of replacing blotting papers is repeated till the time specimen is free of moisture.
§ On drying of the specimen, (the specimen get attached to the paper due to the phycocolloid present in the seaweed) the cheese cloth is carefully removed and herbarium sheet is properly labeled containing collection number, name of the specimen, locality, date of collection and other ecological details.

Identification of seaweed species

Although, identification of the seaweed species is time consuming and tedious, it is interesting. Beginners should get familiar, first with herbarium specimen from the museum or reference collection before going for the field collection. Colour and morphological differences between different genera/ species and taxonomic characteristic are required to be carefully studied. Only through
practice of handling and distinghuishing the plants in the natural habitat will help a great deal in learning seaweed identification.
Taxonomic identification key should be followed to identify the seaweed specimen. The taxonomic description of the specimen and anatomical characteristic of the specimen to be identified should be referred from the books. Once you identify the specimen tentetivly following the key, you should compare it with the herbarium form reference center. Later, you may once again get it confirmed from the expert in the field. Some of the seaweed species, particularly, filamentous seaweed are difficult to identify. In such cases chemotaxonomy or genetic approach could be employed.

 

4. Gluing the dried specimen
Many specimens will remain attached to the herbarium sheet following drying due to the presence in the algal walls and intercellular spaces of colloidal "glues". The coarser, non-gelatinous forms (e.g. some Phaeophyta) may not remain attached after drying and may require "glue".

Any good clear-drying glue may be adequate, such as white glue or a white PVA resin. However due to problems with white glue becoming soft / sticky again (under humid conditions), prefers to use "tin" paste applied in spots, to the underside of the specimen. Gummed linen herbarium tapes may also be used to "strap" the specimen(s) to the sheet.

5. Applying specimen label

Using good quality (100% rag acid-free) herbarium label paper, complete the label and affix by means of a clear-drying cement (tin paste), to the lower right-hand corner of the sheet. AVOID gummed labels on poor quality paper.

The completed herbarium sheet should include a label, and may also have museum, as well as annotation notes written directly on the sheet or on spereate labels.

Preserving specimens in liquid preservatives

Any specimens that fit into a jar or vial may be "pickled" by using any of several different preservatives. Do not pack the specimens into the jar. The Formalin fixed, EtOH preserves portions of most specimens, with the exception of Corallines, which are kept in 3-5% Formalin. Most specimens are kept either in 4-dram (21 x 70 ml) shell vials inside canning jars, or in 20-ml scintillation vials with urea/ poly-seal cone caps, filled with the preservative.

Some Standard Algal Preservatives

Formalin - sea water / buffered.

3-5%. A good all-around fixing solution and preservative.

Commercial 37% formaldehyde (= 100% Formalin) is diluted with seawater to make a 3-5% Formalin solution to which baking soda (sodium bicarbonate) is added as a buffer (to prevent unfavorable increases in acidity) using approximately 40 gms. per liter.

Note: Too much buffer may be detrimental. It has been reported that thalli may become brittle and disintegtrate with the excessive addition of buffer.

Fix for 24 - 48 hours for thick, cartilaginous algae.
NOTE: If the specimen is to be stored for very long, it should be kept in the dark, in sealed containers or bags, to prevent bleaching.

Transeau Solution
6:3:1. Recommended by many freshwater phycologists.

Contains 6 parts water, 3 parts ethyl alcohol (95%), 1 part Formalin (commercial). If you figure the approximate proportions, the water may be supplied in the sample and the added preservative need only contain 3 parts alcohol to 1 part Formalin. It is convenient to add the preservative using a squirt bottle.

Alcohol
70% EtOH. A good all-around preservative.

Some herbaria require EtOH, however isopropyl alcohol may be used.

First fixe the specimens in 3-5 % Formalin for at least 24 hours before rinsing with tap water and transferring to 70% EtOH for permanent storage.

F.A.A. (Formalin-acetic acid - alcohol).
A good all-around preservative of particular value for preserving cell structures such as flagella.

DO NOT use F.A.A. on calcified algae, such as Acetabularia. The acid will harm the specimen.

Formula: EtOH (50%), 100 ml + commercial Formalin, 6.5 ml + glacial acetic acid, 2.5 ml.

 

PREVENTING DESICCATION

Standard containers cannot prevent evaporation of preservatives. In time, all samples will go dry unless precautions are taken to refill them or to seal them. Some hints follow:

Addition of glycerin. A few drops of glycerin added to a filled vial greatly aids in salvaging if the contents go "dry". (The glycerin helps to maintain a bit of moisture in what appears to be an otherwise dry vial). However, some siphons and filaments will shrink when glycerin is added.

Sealing with wax. Caps or stoppers may be dipped in paraffin to retard evaporation.

Vials within jars. Vials plugged with cotton may be placed in canning jars, both of which are filled with preservative. This method, using 4-dram (21 x 70 mm) shell vials and 0.5 liter canning jars sealed with a silicon rubber gasket has been successfully used over 30 years.

NOTE: Plastic screw-cap vials are least satisfactory for long-term preservation. The caps will loosen with time, probably due to differences in expansion of glass and plastic with changing temperature. Cork stoppers are more satisfactory, but they are subject to loosening with changes in atmospheric pressure and will become brittle with time, possibly leeching compounds into the preservative.

Herbarium Labels

There are numerous variations in style; however a herbarium label should be on 100% rag paper and should contain the following information:

Geographic area of collection (i.e. name of island, county, state)
Binomial, including author(s)
Where collected, including latitude, and longitude if possible
Depth, substratum type, etc., including how collected (SCUBA, dredge, submersible, etc.)
Specific ecological information
Collector; date of collection
Collector's field number for specimen or collection
Person who identified the specimen

It is important to remember that the information gathered along with the specimen is just as important as the specimen itself. A collection notebook should be kept that details the collections' locality, habitat, water conditions etc. If necessary, it is possible to refer back to information in this notebook.