Methods Manual for Managers: 

      Field Techniques for Algal Research

 

These methods were developed for research with the Hawaii Coral Reef Initiative Program and are being compiled for a Methods Manual to be distributed by HCRI.  The manual was written by Linda Preskitt and edited by Celia M. Smith and I.A. Abbott of the University of Hawaii, Botany Department, Honolulu, Hawaii.

Methods:

Algal Collection

     Fixation

     Handling and Pressing

Photoquadrat Survey

     Photoquadrat Framer

Sediment Trap

Light Measurement

Alizarin Stain

Nutrient Enrichment

Algal Collection

Purpose

For many species of algae, identification in the field is impossible. Collecting, fixing and processing algal samples during quantitative or qualitative assessments provide fresh or preserved specimens for laboratory identification and pressed herbarium samples provide voucher specimens for permanent records. Voucher specimens serve as documentation for species lists/collections and provide evidence for species identification.

 

Permits

Local customs and laws may require special permission or permits for the collection of marine algae for many locations in the State of Hawaii. It is your responsibility to be aware of and adhere to these rules. Contact the Department of Land and Natural Resources, Division of Aquatic Resources (808-587-0010) and the local authorities/residents for rules regarding the collection of marine algae.

 

Materials

Ziplock^® bags or plastic vials or containers

Scraper (chisel, small knife, etc.)

Permanent ink pen

Waterproof paper

Mesh or cloth bag

cooler or covered bucket

 

Before collecting, determine who will identify and process samples. Do not collect more material than can be processed. It is important to clearly separate and identify the algae taken from different habitats and/or sites. Mark ziplock bags or vials with date, depth and location, or cut small pieces of waterproof paper to use as labels to be inserted into storage containers.

 

Method

Prior to collecting, mark ziplock bags, vials or containers with date, depth, and location. Use a mesh or cloth bag to carry the chisel or knife and marked bags or vials in the field. If possible, collect an entire specimen by carefully removing the holdfast from the substrate with fingers, chisel or knife. The complete plant, holdfast, rhizoids, and reproductive structures are crucial to proper identification of algae. If the specimen is extremely large, take only a representative portion. (Note: permits may be required to collect the holdfast portion, and it is illegal to collect some reproductive female Gracilaria spp.). Gently place the specimen in the bag or vials with sufficient seawater. Once back on shore or boat, check level of seawater and replenish if needed, then store samples in dark cooler or covered container with ice. Fix or process as soon as possible to avoid tissue degradation/decomposition.

Algal Collection: Fixation

Fixation is the preservation of specimens in a chemical solution to stop the deterioration of tissues. Processing is the handling, sorting and mounting of the specimens on herbarium paper. Fresh or preserving specimens in liquid are best for identification and pressed algae are permanent voucher specimens.

 

Fixation

 

Materials

buckets, trays, bowls

glass jars and vials

tweezers

permanent ink pen

labels or waterproof paper

Formalin

protective gloves

 

Method

If specimens cannot be immediately processed, fixing and storing the samples away from direct sunlight inhibits tissue deterioration and bleaching. A common method of fixation is to preserve the algal samples in a 4% Formalin-seawater solution (commercial 37% Formaldehyde = 100%) in glass jars or vials out of the light. (Note that Formalin is a hazardous chemical and should be handled and discarded properly. Wear protective gloves when handling chemicals.)

The Formalin solution can be prepared ahead of time. Prepare a label or piece of waterproof paper with the date, time and location of collection. Immerse the algae in the Formalin solution in a glass jar or vial and affix label or put waterproof paper inside with algae. The algae should be completely immersed in the Formalin solution for at least 48 hours. Algae may be kept in the solution for longer periods of time (up to 1 to 2 months) if kept out of direct light. Light and fixative solutions will cause pigments to bleach.

 

Formalin Solution

Commercial 37% formaldehyde (= 100% Formalin) is diluted with seawater to make a 4% Formalin solution and buffered with borax or baking soda (sodium bicarbonate) to prevent unfavorable increases in acidity. Use approximately 40 gm (2.75 tablespoons) of baking soda per liter of solution.

 

Alcohol Solution

Samples can be preserved in a 50% ethyl alcohol (or even rubbing alcohol) solution, but it is not recommended. Algal material will lose pigments and become very brittle quickly.

Note: Be cautious with the buffer. Too much buffer may cause the thalli to become brittle and disintegrate.

Disposal: Dispose of properly. A formalin neutralizer is available from American MasterTech Scientific Inc.(209) 308-4031.

Algal Collection: Handling and Pressing

Handling and Sorting

 

Materials

buckets, trays, bowls

glass bottles and vials

tweezers

permanent ink pen

labels or waterproof paper

protective gloves

 

If samples are to be sent off for identification and processing, prepare specimens for transport per instructions of receiving laboratory. If preparing specimens for pressing, if fixed remove from fixative and rinse in tap water. Remove any sand or debris that is not part of the specimen. Separate samples into workable groups that may be grouped on herbarium sheets, such as large and small specimens, samples from same habitat, or taxonomic groups (greens, reds, browns), etc. Carefully track samples with labels and notes for proper records.

 

Pressing Algal Specimens

 

Materials

plant press or two boards and weights

cardboard ventilators

drying blotters or newspapers

herbarium paper

wax paper

tweezers and small paint brushes

paper towels

tray or cookie sheet larger than herbarium paper

 

Method

To facilitate sample tracking, use a numbering system where each specimen has an unique number. Note specimen information on lower right hand corner of the herbarium sheet before pressing specimen. If specimens are only for private vouchers, more than one sample may be put on an herbarium sheet to save on materials. Museum herbaria require one specimen per sheet.

 

Larger, coarser specimens may be laid directly on an herbarium sheet on a flat surface. Spread out branches and holdfast/rhizoids in one plane with tweezers or wet paintbrush. If the holdfast is too thick, remove a portion to ease pressing. For more delicate specimens, place the herbarium sheet on top of a piece of plastic, cloth, grid, etc. the size of the herbarium paper and submerge in a tray 75% filled with water. Floating the specimen in the water, arrange specimen over the herbarium sheet with brushes and tweezers. Slowly raise the base piece with the herbarium sheet and specimen out of the tray and allow to drain.

 

Place herbarium sheet and specimen on dry blotter or newspaper that lies on a cardboard ventilator and cover specimen with sheet of wax paper. Place another blotter layer over the wax paper, top with another cardboard ventilator, and put in plant press. Continue adding specimens to the plant press so that each specimen is covered with wax paper between a layer of blotters, enclosed by a layer of ventilators. Firmly tighten the straps of the plant press. 12-18 hours later replace wax paper and blotters or newspaper (all materials can be dried and reused indefinitely). Reexamine after 24 hours and again replace blotters if needed. Place in dry, well ventilated area. Depending on thickness and water content, specimens may dry in 1 to 10 days. Remove and store herbarium specimens in well ventilated area (Smithsonian Website; Tsuda & Abbott, 1985).

 

Herbarium Pressing Exceptions

Gooey slippery specimens, such as certain species of Predaea, Trichogloeopsis, Liagora, Acrosymphyton, Dudresnaya, etc. are harder to handle. Position plant on dry herbarium sheet using brushes, water and tweezers, place herbarium sheet on flat surface, and let air dry. Do not cover with wax paper or layer.

Small turfs and epiphytes require slide mounting, which is not covered in this manual.

 

Herbarium Supplies

Carolina Biological Supply Company

2700 York Road
Burlington, NC 27215-3398
1-800-334-5551

http://www.carolina.com

 

Pacific Papers

15702 119th NE
Bothell, WA 98011
1-800-676-1151
http://www.pacific-papers.com

 

References

Abbott, Isabella A. and George J. Hollenberg. 1976. Marine Algae of California. Stanford University Press, Stanford, California.

Abbott, Isabella A. 1999. Marine Algae of the Hawaiian Islands. Bishop Museum Press, Honolulu, Hawaii.

Tsuda, Roy T. and Isabella A. Abbott. 1985. Collection, handling, preservation and logistics, pp. 67-68. In: Littler, M.M. and Littler, D.S. (eds.), Ecological Field Methods: Macroalgae. Handbook of Phycological Methods. Cambridge Univ. Press, New York.

 

Web Sites

Smithsonian Institution, National Museum of Natural History, Department of Systematic Biology: http://www.nmnh.si.edu/botany/projects/algae/Alg-CoPr.htm

Cryptogamic Botany Company: http://www.cryptogamicbotany.com/lm_press_seaweed.html