Preparation of Duckweed fronds for study.
The bleaching and staining precedures are simple, but involve strong chemicals. It is recommended that students use these procedures only under supervision of an instructor.
Rubbing alcohol (70% ethanol or 70% isopropanol)
Household bleach (5% sodium hypochlorite solution)
ammonium alum (aluminum ammonium sulfate dodecahydrate)
distilled or deionized water
Dye solution (Grenacher's Alum Carmine):
Place 1 gram of carmine and 5 grams of ammonium alum in 100 mL of water in a Pyrex flask and swirl or stir. Heat in a boiling water bath for 1 hr, swirling occasionally to dissolve the dye. Allow any undissolved material to settle and then filter. Ordinary alum (aluminum potassium sulfate dodecahydrate) will likely work as well as the ammonium alum but was not tested.Procedure:
|Place fronds in an excess of 70% alcohol solution and boil for 2 min.||Use a bacteriological loop to gently pick up and move fronds without damage. It is convenient to use a heating block or a hot water bath to boil the alcohol.|
|Rinse the fronds in fresh 70% alcohol 2 min at room temperature||Removes any remaining pigment.|
|Place fronds in an excess of the bleach solution (undiluted) until they are colorless and transparent||Oxidiation bleaches any remaining pigments and dissolves insoluble proteins and other cellular constituents.|
|Rinse the fronds in water for 2 min, repeat||Memoves traces of bleach which would interfere with staining.|
|Stain the fronds in the Grencher's stain 12 hr or overnight. Stopper or seal to prevent evaporation.||Stains cell walls. Other dyes may work equally well.|
|Rinse the fronds in several changes of water.||Remove excess dye.|
After this procedure, the frond and root cell walls will be stained as in this figure. I recommend that you use a magnifyer or stereomicroscope to see the arrangement of the cell wall framework.Safety Notes:
This procedure is also suitable for preparation of fronds for embedding and sectioning to view with the optical microscope, but those procedures are beyond this discussion. A book on microtechnique should be consulted.
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Revised: November 18, 2001