Fixatives: Lugols Formaldehyde Glutaraldehyde
Stains to reveal morphology: Aniline Blue Calcofluor Indian ink
Nuclear stains (fluorescent) DAPI Acridine Orange Hoescht#####
Lugols is a common fixative for phytoplankton
field samples and for stopping growth or immobilizing cells prior to microscopy
counts of microalgal cultures. It is
commonly prepared in an acidic version with glacial acetic acid. However
if it is known or suspected that coccolithophorids may be an important component
of the plankton then the basic form must be employed to prevent dissolution of
the coccoliths. Lugols is light
sensitive and will degrade over several weeks/months so it is not an efficient
preservative. The recommended final
fixation step is to use as much lugols as stains the sample a weak tea colour.
If samples will not be examined for a prolonged interval after collection
there is a tendency to increase the stain. However,
excessive lugols will overstain the cell contents of many flagellate species and
in some instances can lead to flagella being shed.
●
on
a magnetic stirrer dissolve 20 g potassium iodide (KI) in 50mL distilled water
For counting dense cultures where a dilution step
is appropriate prepare the diluent (media, seawater or freshwater depending on
the species) and stain this first
eg for a 1 in 5 dilution of a freshwater Anabaena
culture
stain 4 mL of distilled water with 1drop or 10 uL
Lugols, then add 1 mL of culture. This
is far gentler to the cells than adding concentrated lugols to the sample.
Glutaraldehyde
is a common fixative for preparation of whole cells and tissues in electron
microscopy but it is also an excellent fixative for light microscopy.
It preserves the fluorescent emission of chlorophyll allowing
epifluorescent examination post fixation. Its major drawback is that it emits
toxic vapours that have been associated with nasal-pharangeal cancer so
efficient fume extraction at the microscope is recommended if extended use is
anticipated.