Staining Algal Pyrenoids with Carmine After Fixation in an Acidified Hypochlorite Solution

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Abstract
Pyrenoids but not nucleoli are clearly stained with propionocarmine after a special fixation. The pyrenoid is distinguished as the largest dark purple, intracellular body that stains homogeneously. The general procedure is as follows: unicells and filaments are collected by centrifugation and any liquid discarded; cells are fixed 5 rain in a mixture of 10 volumes of 50% ethanol, 10 of concentrated HC1, and 5 of Clorox; washed in 95% ethanol 3 times or more to remove all chlorophyll and fixative; mordanted for 5 min with a suitably adjusted concentration of ferric propionate in 50% propionic acid (reddish orange in color); stained 5 min in 0.5% carmine in 50% propionic acid heated in a boiling water bath. The material is mounted in 50% propionic acid. The results are reproducible when material is processed in centrifuge tubes and when the optimum time in the mordant and stain is determined for a particular species and then repeated accurately. This technique was successful in staining pyrenoids of all of the 8 species tested, representing 6 algal divisions.