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of C-Fern is supported by the National Science Foundation (NSF-DUE) |
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Copyright
© 1997-2000
Thomas R. Warne and Leslie G. Hickok. All rights reserved. |
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C-Fern
Web Manual - Observation and Analysis of Gametophytes
Preparation of semi-permanent slides Semi-permanent slides suitable for later observations or archival documentation can be prepared by mounting gametophytes in a Hoyer's/Acetocarmine mixture. Hoyer's Medium clears and preserves tissue; acetocarmine is one of the few stains stable in the presence of Hoyer's Medium. Materials:
Prepare mounting mixture fresh by mixing 4 parts Hoyer's Medium and 1 part Acetocarmine Stain. (1 mL of this mixture should be sufficient for 7-8 slides, using 22x22mm coverslips. ) For the acetocarmine to stain properly a small amount of iron oxide should be added to the Hoyer's/Acetocarmine mixture. Do this by dipping an old rusty dissecting tool (e.g. forceps) or rusty iron nail into the mixture until a sufficient amount of iron oxide has leached off. Some experimentation is necessary to obtain the correct amount of iron -- too little gives very weak staining; too much yields excessive precipitation and a strongly colored background. Place 4-10 drops of the Hoyer's/Acetocarmine mixture onto a slide. (For best results be sure slides are very clean.) Transfer gametophytes from the culture dish to the mixture. Submerge gametophytes under the mixture. Put coverslip in place. If gametophytes are large or convoluted it may be difficult to avoid trapping some air bubbles. Place completed slides on a tray to dry undisturbed for several days. Full clearing and staining of gametophytes may take more than several days. Retain the remaining mixture to add to those slides that dry out excessively. Slides prepared with a sufficient amount of Hoyer's/Acetocarmine can keep for well over 10 years. |
