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.
The staff of the U.S. National Herbarium prefers to sort the collection by taxa, while still in the field. The specimens are then preserved in separate plastic whirl-pac bags into which waterproof labels have been inserted. All of the specimens from a station are placed in a large labeled plastic bag which is then placed in a light-proof shipping container (liqua-pac).
2. Preparing the 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).
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.
NOTE: Some researchers prefer that glycerin not be used, as it may harm the reproductive structures inside the conceptacles.
3. Pressing the specimen:
Prior to pressing the specimen, a number or note should be written on the lower right-hand corner of the herbarium sheet (unbuffered long-fiber 100% rag mounting paper 11 1/2" x 16 1/2" - 70M; White-Caliper.015). This will aid in identifying specimens and allow for affixing the proper herbarium label after pressing. Also, keep in mind the layout of the herbarium sheet when placing specimens on the sheet with regard to the label location (lower right corner) and herbarium stamp (usually upper right corner).
The larger coarser forms may be placed directly on herbarium paper. However, delicate forms must be placed on the paper while it is submerged in a tray or pan of adequate size containing water (it may be tap water if specimens have been fixed), which will allow for spreading the specimen.
Place specimen (in water) over paper, and using forceps, a pointed instrument, or a small, soft paintbrush for more delicate forms, pull out and separate branches, and spread the specimen to reveal branching patterns and small structures. If necessary, trim away (and note that this was done) an appropriate amount of the specimen where excess material obscures structures.
Remove the paper very carefully from the tray, at an angle so that water flows will tend to spread the branches as the paper is being lifted. A squirt bottle of water may be used at this point to further spread branches. Place the paper containing the specimen(s) on a blotter. Then place a piece of muslin, cheesecloth, a cloth "diaper" or crumpled waxed paper over the specimen, and add another blotter on top.
Place the blotter and specimen sandwich between standard plant press ventilators (corrugated cardboard or aluminum).
Continue adding specimens to the plant press so that each specimen is covered with non-sticking material and between a layer of blotters, enclosed by a layer of ventilators. Finally, firmly tighten the straps of the plant press.
Moderately warm artificial heat should be applied from below the press as it is placed on its side so that warm dry air passes upward through the corrugations of the ventilators. Alternately, the plant press may be placed in a fume hood so that the sash closes on the press, allowing air to be drawn through the corrugations.
Heaters may be devised which include a rack to hold the press and a heating source, such as light bulbs, a heating coil or a hot plate. Note that warm air is all that is required. Too much heat will cause the algae to darken and become brittle. Check specimens at least every 24 hours removing any that are dry; wet blotters and "diapers" should be changed and the straps of the plant press retightened. If a heat source is not available, blotters and diapers must be removed daily and replaced with dry ones. Drying may require many days by this method, so a heat source should be used if at all possible.
Remove press from heat source and allow to cool with the straps tightened before opening. Specimens may curl if exposed to a humid atmosphere before cooling, in which case they must be wetted and re-dried.
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), the U.S. National Herbarium 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 and barcode numbers, as well as annotation notes written directly on the sheet or on separate labels.
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 U.S. National Herbarium attempts to keep Formalin fixed, EtOH preserved (see below) 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.
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.
70% EtOH. A good all-around preservative.
Some herbaria require EtOH, however isopropyl alcohol may be used.
At the U.S. National Herbarium, the staff first fixes 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.
Notes on Hazardous Chemicals
Many of the chemicals used in the preservation and staining processes may be listed as environmentally hazardous. Therefore you need to be aware of possible hazards by consulting the chemical's MSDS* label, and work accordingly. It is suggested that all work with chemical solutions be conducted in a FUME HOOD and that lab personnel wear appropriate eye protection, gloves and a chemical apron.
For example, you should be aware that formaldehyde has been classified as a possible carcinogen, and has a CERCLA rating of:
* MSDS - Material Safety Data Sheet
Data sheet provided by the manufacturer of the chemical which lists the environmental, health and physical information concerning the use and disposal of the chemical, as well as the suggested minimum personal protective equipment to be used. This chemical information sheet typically rates the chemicals (CERCLA - NFPA) by assigning a number for the chemical's respective health hazard, flammability, reactivity and persistence. It is advised that you familiarize yourself with this rating system.
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 by the U.S. National Herbarium for over 30 years.
Scintillation vials with urea/ poly-seal cone caps. The staff of the U.S. National Herbarium has been testing these 20 ml vials for approximately 8 years. Monitoring of material in these vials is ongoing. Several of the vials have now lost some liquid due to evaporation (01/2000).
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.
Karo Corn Syrup / Glycerin - Gel Preparation
I. Karo Corn Syrup
After satisfactory fixation in 3-5% buffered Formalin (about 15 to 30 minutes for more delicate forms and up to 24 hrs. for thick, cartilaginous algae), transfer the specimens to a clean watch glass or slide and rinse in distilled water. At this point, material may be sectioned if necessary. Stain the specimens with a solution approximately 1% v/v aqueous aniline blue (other stains, e.g., acid fuchsin, erythrosin, safranin, etc. are preferred by some investigators) for one (delicate, filamentous forms) to several minutes (thick forms); acidify with a drop of 1% HC1 to fix the stain, then wash with a drop or two of distilled water. Blot away excess water with filter paper.
Add Karo corn syrup diluted with distilled water (20-50%)* to which a preservative has been added (2-3% solution of phenol) to a specimen placed on a clean slide. Carefully cover with a coverslip (No. 1), attempting to avoid trapping bubbles. Add enough syrup to allow for evaporation. Store, flat and face-up, for one to two days; add more syrup (50% - 70%) around the edges of the coverslip, if necessary, to complete the seal. The Karo dries and hardens in a few days (sometimes a week or more). Therefore, slides should be stored flat and should not be allowed to stand on edge during the drying period. Caution should be exercised when cleaning the coverslip, so as not to scratch or dislodge it.
* Note: The concentration of Karo will depend on the alga you are preparing. Some delicate forms (e.g. Liagora) may require a 5-10% solution to begin with, while others (e.g. Centroceras) may tolerate a 50% solution without causing the cells to plasmolyze. After the initial addition of dilute Karo, you may be able to use higher concentrations without damage to the cells.
II. Glycerin - Gel Preparation
This is a relatively rapid method for making semi-permanent preparations, either of sections or of whole mounts.
1. Fixation: Use 2-5% buffered Formalin seawater or an alcoholic fixative (70% EtOH). Allow 30 minutes for fixation, or longer if large cartilaginous specimens are involved.
2. Transfer the specimen to a solution that can be evaporated to leave 100% glycerin. The aim is to extract water from the specimen and replace it by glycerin with a minimum of shrinkage. The details vary according to the fixative and the characteristics of the specimen. For delicate specimens, the procedure should be performed on a pre-cleaned slide; larger, coarser thalli can be placed in small dishes and transferred to a slide for gel addition.
Aqueous fixatives. Transfer the material to a 5% aqueous solution of glycerin, place on a warm plate (50+ °C) and leave until concentrated. This usually will take overnight. Remember that concentration to pure glycerin will diminish the volume of solution to 1/20th of its original volume and that a specimen that is allowed to dry is ruined. Cover to prevent dust accumulation when allowing the glycerin to concentrate.
Alcoholic fixatives. Place in a 5% solution of glycerin in 70% EtOH and allow to concentrate as above.
3. Remove the specimen from the glycerin and blot dry. If the specimen is delicate, do not apply pressure, but simply use patience. Place the material on filter paper, or a paper towel and leave until no excess liquid glycerin is visible. Melt gel/stain mixture (see below) in a beaker placed in a water-bath and apply 3 drops to algal material which has been placed on a clean slide. The speed of staining is increased if the gel is kept molten for a few minutes and the slide rocked gently. Apply a coverslip (preferably No. 0). Bubbles diminish the life of glycerin-gel preparations. For this reason, never allow bubbles to form in the stock gel and never blow out a dropping pipette into molten gel. Should a bubble appear on a slide before applying the coverslip, the easiest way to remove it is to prick it with a red-hot needle.
Some researchers suggest that coverslips should be ringed with varnish or canada balsam. In general this is of no value in that, if the preparation is sealed, it appears to provide no security against drying-up and simply prevents repair. In the event that the gel retracts from the coverslip, add a sufficient amount of gel to fill-in the hole (this is not possible if the preparation has been ringed).
Glycerin - Gel Mixture
Gelatin 50 gm
Glycerin 350 ml
Water 300 ml
Mix, and heat in a beaker immersed in a water-bath until dissolved. Allow to cool and add 1 gm. phenol as a preservative. Add crystal-violet stain drop-wise until the required shade is achieved (usually determined by placing one drop of the solution on a microscope slide and placing a coverslip over it, aiming for a relatively dense color when viewed this way).
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. When possible, the U.S. National Herbarium maintains collectors' notebooks along with the herbarium specimens for its major collections.
See the chapter by Tsuda and Abbott (1985) in the reference section for additional information.