Microscopy fixatives and stains
Fixatives
Lugols Iodine
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 50 mL distilled water
- add 10 g double sublimated iodine
- add 150 mL distilled water
- acid form– add 20 mL glacial acetic acid (CH3COOH).
Fig. 1 Dolichospermum spiroides, CS-546 (Image: I. Jameson)
- basic form– add 10 g sodium acetate (CH3COONa) which makes the solution neutral or slightly basic
- Store e.g. in 200 mL amber glass bottles (or at least foil-wrap borosilicate glass bottles) with screw cap or cap with integrated pipette
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 Dolichospermum culture
stain 4 mL of distilled water with 1 drop or 10 μL Lugols, then add 1 mL of culture. This is far gentler to the cells than adding concentrated Lugols to the sample.
Glutaraldehyde
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.
Fixation is achieved by adjusting 25% EM grade glutaraldehyde to a final concentration of 0.25 – 1% depending on the cell density of the sample