Page 227 - Algae Anatomy, Biochemistry, and Biotechnology

210 Algae: Anatomy, Biochemistry, and Biotechnology

Lugol’s solution is the preferred preservative commonly used for short-term (e.g., a few
months, but possibly a year or more) storage of microalgae. It is excellent for preserving chryso-
phytes but it makes the identification of dinoflagellates difficult, if not impossible. Samples can
be preserved and kept (in dark bottles away from light) for as long as 1 yr in Lugol’s solution
(0.05–1% by volume). The solution is prepared by dissolving 20 g of potassium iodide and 10 g
of iodine crystals in 180 ml of distilled water and by adding 20 ml glacial acetic acid. Note that
Lugol’s solution has a shelf life that is affected by light of about 6 months.
Dried herbarium specimens can be prepared by “floating out” similar to aquatic flowering
plants. Ideally, fresh specimens should be fixed prior to drying. Most algae will adhere to absorbent
herbarium paper. Smaller, more fragile specimens or tangled, mat-forming algae may be dried onto
mica or cellophane. After “floating out,” most freshwater algae should not be pressed but simply
left to air dry in a warm dry room. If pressed, they should be covered with pieces of waxed
paper, plastic or muslin cloth so that the specimen does not stick to the drying paper in the press.
To examine a dried herbarium specimen, a few drops of water are added to the specimen to
make it swell and lift slightly from the paper. This makes it possible to remove a small portion
of the specimen with forceps or a razor blade.
Observations (preferably including drawings or photographs) based on living material are
essential for the identification of some genera and a valuable adjunct to more leisurely observations
on preserved material for others. The simplest method is to place a drop of the water including the
alga onto a microscope slide and carefully lower a coverslip onto it. It is always tempting to put a
large amount of the alga onto the slide but smaller fragments are much easier to view under a micro-
scope. Microalgae may be better observed using the “hanging drop method,” that is, a few drops of
the sample liquid are placed on a coverslip which is turned over onto a ring of paraffin wax, liquid
paraffin or a “slide ring.”
A permanent slide can be prepared with staining such as aniline blue (1% aqueous solution, pH
2.0–2.5), toluidine blue (0.05% aqueous solution, pH 2.0–2.5), and potassium permanganate,
KMnO 4 (2% aqueous solution), which are useful stains for macroalgae (different stains suit
different species) and India Ink, which is a good stain for highlighting mucilage and some
flagella-like structures. Staining time ranges from 30 sec to 5 min (depending on the material),
after which the sample is rinsed in water. Mounting is achieved by adding a drop or two of glycerine
solution (75% glycerine, 25% water) to a small piece of the sample placed on a microscope
slide then carefully lowering the coverslip. Sealing with nail polish is essential. This method is
unsuitable for most unicellular algae which should be examined fresh or in temporary mounts of
liquid-preserved material.
Magnifications of between 40 and 1000 times are required for the identification of all but a few
algal genera. A compound microscope with 10 –12 eyepiece and 4 –10 –40 objectives is
therefore an essential piece of equipment for anyone wishing to discover the world of algal
diversity. An oil immersion 100 objective would be a useful addition, particularly when
aiming at identifying to species level. Phase-contrast or interference (e.g., Nomarski) microscopy
can improve the contrast for bleached or small specimens. A dissecting microscope providing 20 ,
40 up to 60 magnifications is a useful aid but is secondary to a compound microscope. A camera
lucida attachment is helpful for producing accurate drawings while an eyepiece micrometer is
important for size determinations. Formulas for calculating biomass for various phytoplankton
shapes using geometric forms and measurements and shape code for each taxa exists in the litera-
ture, and are routinely used in the procedure for phytoplankton analysis that require biovolume cal-
culations (Table 6.1). Moreover, software packages for image recognition and analysis are
available, which can process phytoplankton images acquired by means of a TV camera mounted
onto a compound microscope. Scanning and transmission electron microscopes are beyond the
reach of all but specialist institutions; however, they are an essential tool for identifying some of
the very small algae and investigating the details of their ultrastructure.