STAINTECHNOLOGY
Vol. 50, No. 5 %lodin U.S.A.
Copyright 0 1975 by The Williams & Wilkins Co
NOTES ON TECHNIC
Biotech Histochem Downloaded from informahealthcare.com by University of Toronto on 12/25/14 For personal use only.
AN ULTRAMICROTOME ILLUMINATORFOR EPOXYRESINEMBEDDED SINGLE CELL SPECIMENS JUHA
NICKELS, Central Laboratory ojPathology, University of Helsinki, Helsinki, Finland
By fibre optics it is possible to transmit light over considerable distances to a small area without loss of light intensity. The technique has been used to facilitate the trimming of single cell specimens in epoxy resin blocks (Nickels 1974), and is also applicable for illumination of single cell specimens in the ultramicrotome. Procedure. Commercially available fibre optics are used (Faseroptik, Schott, Mainz). A fibre bundle 2 mm in diameter is suitable for epoxy blocks cast in 6 mm
FIG. 1 (I+). Trimmed epoxy resin block containing fibre optic bundle. FIG. 2 (righf). Epoxy resin block illuminated with a fibre optic bundle in specimen holder
diameter gelatin capsules. A suitable length can be made by cutting the bundle and molding the individual fibre cores together with plastic (e.g. Plasticraft, Turner Research Ltd., Leeds). The end of the bundle is placed in the epoxy resin block in a hole drilled just under the cell specimen (fig. 1); the rest of the bundle is protected by a plastic tube. Light conduction between the epoxy resin block and the fibres is facilitated by using a drop of immersion oil around the head of the bundle in the hole. The epoxy block is placed in an ordinary Sorvall specimen holder, the fibre bundle running through a hole drilled on the side of the holder (fig. 2). The light source is an ordinary microscope light placed so that the free end of the fibre optic cable passes the illuminator at the moment when the block face as well as 359
Biotech Histochem Downloaded from informahealthcare.com by University of Toronto on 12/25/14 For personal use only.
360
STAIN TECHNOLOGY
the knife edge are in focus under the stereomicroscope. The cable must not touch the light source. The ordinary spot light of the ultramicrotome completes the illumination. Dzsmsszon. T h e intense fibre optic and spotlight illumination gives good contrast to the specimen in the epoxy resin block. Loss of tissue is avoided during final trimming and microsectioning with the ultramicrotome, and the cutting of useless “empty” sections can be controlled. Scratches on the edge of the knife and the block face itself are clearly visible. Realigning the block when recutting is facilitated. The commercially available equipment is easy to handle. REFERENCES Nickels,J . 1974. Fibre optics in the illumination of epoxy resin embedded cells. Microsc. Acta 76: 48-50.
STAINING OF CLEAVAGE CELLSIN YOLK RICHEGGS OF ACHETADOMESTICUS WITH TOLUIDINE BLUEAND PYRONIN B (ORTHOPTERA)
FRITZE. SCHWALM, Department of Biological Sciences, Illinois State University, Normal, Illinois 61761
In eggs of Orthoptera the amount of yolk obscures what little cytoplasm is present in the early stages of development. Detailed observation of cells is limited to sectioned material despite the fact that only few cells or single cell layers inhabit the egg at these stages. A stain mixture of toluidine blue and pyronin B, as described by Ito and Winchester (1963), had been found to reveal considerable detail in sections of Araldite-embedded insect embryos (Schwalm and Bender 1973). The distinct differences in staining properties of yolk and various cytoplasmic components suggested that this stain should also be useful for whole mount preparations. Eggs of the house cricket were prepared by mechanically removing the chorion in 5 % sodium hypochlorite with watchmaker’s forceps. The vitelline membrane around the egg should stay intact. After two rinses in tap water, eggs were fixed in Carnoy’s solution (ethano1:chloroform:glacialacetic acid 6:3:1) at 60 C for 5 min, then washed 5 min each in 100%alcohol and in descending steps of 8O,50,30, and 10%alcohol. To 0.5 ml 10% alcohol one drop of stain was added. This stain consisted of 4 parts toluidine blue 0 (Fisher Scientific, Cert. NO.22, C.I. 52040) 1% w/v in 1% sodium borate, mixed with 1 part pyronin B (Allied Chemical, Cert. No. 29, C.I. 45010), 1% in aqueous solution. The mixture was filtered through Whatman No. 1 filter paper. Eggs were stained from 10 sec to 2 min, then dehydrated through 50% into 80% alcohol. At this step the alcohol was repeatedly renewed until all stain was removed from the yolk. Dehydration was completed by quickly transferring through 95%and 100% alcohol. Specimens were then cleared in xylene and mounted on microscope slides in Permount (Figs. 1 & 2).
Vol. 50, No. 5 %lodin U.S.A.
Copyright 0 1975 by The Williams & Wilkins Co
NOTES ON TECHNIC
Biotech Histochem Downloaded from informahealthcare.com by University of Toronto on 12/25/14 For personal use only.
AN ULTRAMICROTOME ILLUMINATORFOR EPOXYRESINEMBEDDED SINGLE CELL SPECIMENS JUHA
NICKELS, Central Laboratory ojPathology, University of Helsinki, Helsinki, Finland
By fibre optics it is possible to transmit light over considerable distances to a small area without loss of light intensity. The technique has been used to facilitate the trimming of single cell specimens in epoxy resin blocks (Nickels 1974), and is also applicable for illumination of single cell specimens in the ultramicrotome. Procedure. Commercially available fibre optics are used (Faseroptik, Schott, Mainz). A fibre bundle 2 mm in diameter is suitable for epoxy blocks cast in 6 mm
FIG. 1 (I+). Trimmed epoxy resin block containing fibre optic bundle. FIG. 2 (righf). Epoxy resin block illuminated with a fibre optic bundle in specimen holder
diameter gelatin capsules. A suitable length can be made by cutting the bundle and molding the individual fibre cores together with plastic (e.g. Plasticraft, Turner Research Ltd., Leeds). The end of the bundle is placed in the epoxy resin block in a hole drilled just under the cell specimen (fig. 1); the rest of the bundle is protected by a plastic tube. Light conduction between the epoxy resin block and the fibres is facilitated by using a drop of immersion oil around the head of the bundle in the hole. The epoxy block is placed in an ordinary Sorvall specimen holder, the fibre bundle running through a hole drilled on the side of the holder (fig. 2). The light source is an ordinary microscope light placed so that the free end of the fibre optic cable passes the illuminator at the moment when the block face as well as 359
Biotech Histochem Downloaded from informahealthcare.com by University of Toronto on 12/25/14 For personal use only.
360
STAIN TECHNOLOGY
the knife edge are in focus under the stereomicroscope. The cable must not touch the light source. The ordinary spot light of the ultramicrotome completes the illumination. Dzsmsszon. T h e intense fibre optic and spotlight illumination gives good contrast to the specimen in the epoxy resin block. Loss of tissue is avoided during final trimming and microsectioning with the ultramicrotome, and the cutting of useless “empty” sections can be controlled. Scratches on the edge of the knife and the block face itself are clearly visible. Realigning the block when recutting is facilitated. The commercially available equipment is easy to handle. REFERENCES Nickels,J . 1974. Fibre optics in the illumination of epoxy resin embedded cells. Microsc. Acta 76: 48-50.
STAINING OF CLEAVAGE CELLSIN YOLK RICHEGGS OF ACHETADOMESTICUS WITH TOLUIDINE BLUEAND PYRONIN B (ORTHOPTERA)
FRITZE. SCHWALM, Department of Biological Sciences, Illinois State University, Normal, Illinois 61761
In eggs of Orthoptera the amount of yolk obscures what little cytoplasm is present in the early stages of development. Detailed observation of cells is limited to sectioned material despite the fact that only few cells or single cell layers inhabit the egg at these stages. A stain mixture of toluidine blue and pyronin B, as described by Ito and Winchester (1963), had been found to reveal considerable detail in sections of Araldite-embedded insect embryos (Schwalm and Bender 1973). The distinct differences in staining properties of yolk and various cytoplasmic components suggested that this stain should also be useful for whole mount preparations. Eggs of the house cricket were prepared by mechanically removing the chorion in 5 % sodium hypochlorite with watchmaker’s forceps. The vitelline membrane around the egg should stay intact. After two rinses in tap water, eggs were fixed in Carnoy’s solution (ethano1:chloroform:glacialacetic acid 6:3:1) at 60 C for 5 min, then washed 5 min each in 100%alcohol and in descending steps of 8O,50,30, and 10%alcohol. To 0.5 ml 10% alcohol one drop of stain was added. This stain consisted of 4 parts toluidine blue 0 (Fisher Scientific, Cert. NO.22, C.I. 52040) 1% w/v in 1% sodium borate, mixed with 1 part pyronin B (Allied Chemical, Cert. No. 29, C.I. 45010), 1% in aqueous solution. The mixture was filtered through Whatman No. 1 filter paper. Eggs were stained from 10 sec to 2 min, then dehydrated through 50% into 80% alcohol. At this step the alcohol was repeatedly renewed until all stain was removed from the yolk. Dehydration was completed by quickly transferring through 95%and 100% alcohol. Specimens were then cleared in xylene and mounted on microscope slides in Permount (Figs. 1 & 2).
