Acta Anatomica

International Archives of Anatomy, Histology, Embryology and Cytology
Archives Internationales d'Anatomie, d'Hlistologie, d'Embryologie et de Cytologie
Internationales Archiv für Anatomie, Histologie, Embryologie und Zellforschung

Basle (Switzerland) S. KARGER New York

Separatum   Printed in Switzerland

Nassar, T. K. and W. M. Shanklin: Acta anat. 25. 188-191, 1955

Department of Histology, School of Medicine, American University of Beirut, Lebanon, and Department of Anatomy, Medical College of Virginia.



This work was presented as Demonstration 46 at the Sixtyseventh Annual Meeting of the American Association of Anatomists in Galveston. April 1954 (see Nassar and Shanklin [1954]).

Textbooks of anatomy and histology give a very inadequate description of the branches of Müller's fibers in layer 9 (layer of nerve fibers) of the retina. Polyak ([1941], pages 343-354) in his excellent volume on the retina describes Müller's fibers in considerable detail, especially in Rhesus Macaque. In spite of his detailed description of these fibers in other layers his description of them in layer 9 is very incomplete.

By the use of a silver impregnation method we have demonstrated in the retina of birds and mammals a much more intricate meshwork of branches from Müller's fibers in the layer of nerve fibers than those described and illustrated by previous investigators.

Our staining method is as follows:

  1. Fix fresh pieces of retina in 10% formal in (Merck blue label) in distilled water for 5 days at room temperature (about 26° C).
  2. Wash in 2 changes of dilute ammonia water (1 ml. concentrated ammonia, 99 ml. distilled water) for 2 hours each, followed by 3 changes of distilled water of 2 hours each.
  3. Dehydrate in graded alcohols, one hour each, and in absolute alcohol for 2 hours.
  4. Clear-in benzene (Merck) for 1 to 2 hours, or in xylene for 2 hours.
  5. Infiltrate with two changes of paraffin for 2 hours, embed and section at 5 micra. Deparaffinize in xylene and hydrate in graded alcohols followed by 3 changes of distilled water.
  6. Place the sections in 10% pure silver nitrate, to which are added 3 drops pyridine per 10 ml. of silver solution, for 24 hours at room temperature. This process may be speeded up by placing the solution in the incubator at about 50° C.
  7. Sections are moved directly to 2% formalin, to which are added 2 drops of pyridine per 10 ml., for 2 minutes.
  8. Wash quickly in distilled water to which 2 drops of pyridine are added per 10 ml.
  9. Impregnate in silver diamminohydroxide. to which are added 3 drops of pyridine per 10 ml, in an incubator for 3 to 5 minutes at about 50° C.

    To prepare silver diamminohydroxide solution place 1 ml. 28% ammonia water in a small flask and add 7 or B ml. 10% silver nitrate rapidly. Continue to add 10% silver nitrate drop by drop, shaking between each addition, to clear the solution until a faint permanent turbidity remains after the last drop added. This takes a total of 9 to 10 ml. silver solution. Dilute the resultant solution with an equal volume of distilled water (Lillie, 1946 p. 70).

  10. Without washing reduce in 2% formalin, to which are added 2 drops pyridine per 10 ml. for 1 minute.
  11. Wash in distilled water and tone in gold chloride (1 gr. gold chloride per 500 ml. water). This step must not be prolonged beyond the exact time needed.
  12. Fix in 5% hypo.
  13. Wash in tapwater, dehydrate in alcohol, clear in xvlene, mount and cover with cover glass.

Observations and Discussion

Our findings in layers 6, 7 and 8 are essentially the same as those of Polyak, hence his description is summarized here. The cell bodies and nuclei of Müller's fibers are located in layer 6. From the body of each cell one or more threads descend giving off secondary, tertiary and additional branches forming a dense neuroglial meshwork which fills layer 7. In the lower zone of this layer some of the main fibers of Miller merge again into a single thick pillar. In layer 8 they form a dainty network around each ganglion cell. Our material confirms the above description.

Shortly after entering the layer of nerve fibers Müller's fibers form heavy, intensely stained pillars. According to the description and illustrations of Polyak (figs. 37, 47, 54, 56, 84, 90 and 96) these pillars only give rise to relatively few branches in this layer. In the cat (fig. 1) Müller's fibers give off numerous branches which form a reticulated network that fills the entire layer of nerve fibers


Fig. 1. Part of retina of the cat showing rich meshwork of branche from Müller's fibers in the layer of optic nerve fibers. The nerve fibers in this layer are unstained. X 600. - Fig.2 Section through retina of the owl (Athena noctua) demonstrating the rich meshwork formed by Müller's fibers in the layer of optic nerve fibers. X 275. 6 = Inner nuclear layer. 7 = Inner plexiform layer, 8 = Layer of ganglion cells, 9 = Layer of optic nerve fibers. 10 = Inner limiting membrane.


thereby providing the individual nerve fibers with insulating sheaths. The longest vitreal ends of Muller's fibers are found in the immediate vicinity of the optic papilla where the ninth layer reaches its maxmum thickness. It is in this region where this branching insulating meshwork reaches its highest development. In the owl (Athena noctua) the branches of Müller's fibers are extremely rich (fig. 2) in the layer of nerve fibers.

Part of this study was made during the appointment of the junior author at the Medical College of Virginia as the A.D. Williams Visiting Professor of Anatomy. This work was aided by a research grant from the A.D. Williams Fluid Fund for Reasearch.


A method for the silver impregnation of Müller's fibers in the retina after paraffin embedding is described. This method demonstrates a rich reticular meshwork of branches from Müller's fibers in the layer of nerve fibers.


Description d'une méthode d'impregnation à l'argent permettant de mettre en évidence les fibres de Müller dans la rétine aprés inclusion à la paraffine. La méthode démontre un riche réseau réticilé formé par les ramifications des fibres de Müller dans la couche des fibres du nerf optique.


Beschreiben wird eine Silberimprägnationsmethode für die Müllerschen Fasern der Restina nach Paraffin-Einbettung. Mit Hilfe der Methode kann ein dichtes Netzwerk von Verästelungen der Müllerschen Fasern in der Nervenfaserschicht sichtbar gemacht werden.


Lillie, R.D.; Stain Techn. 21, 69, 1946.

Nassar, T.K. and W.M. Shanklin: Anat. Rec. 118, 432, 1954

Polyak, S.L.: The Retina. Univ. Chicago Press, Chicago 1900.

Received May20th, 1955

The above article is transcribed from a copy at the AUB's Saab Medical Library.


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