Bensley & Bensley's
Impregnation for Reticulin

Solutions

Reagent	Var I	Var II
Lugol's iodine	+	+
Potassium permanganate, 1% aqu.	+	+
Oxalic acid, 5% aqu.	+	+
Sodium thiosulphate, 3% aqu.	+	+
Tannic acid, saturated in ethanol, 95%.	+	–
Strong ammonium hydroxide (s.g. 0.88)	+	+
Ammoniated water	+	–
Silver nitrate, aqu.	1%	2%
Formalin, 20% aqu.	+	+
Yellow gold chloride, 0.2% aqu.	+	+
Sodium hydroxide, 40% aqu.	+	+
Neutral red, 1% aqu.	+	+

Lugol's iodine
Iodine Potassium iodide Distilled Water	1 g 2 g 300 mL	Mix the iodine and potassium iodide in a 500 mL flask. Add 5 mL of the water. When the iodine has dissolved make up to 300 mL with distilled water.

Ammoniacal silver – Var I

Place 20 mL of 1% silver nitrate in a flask. Add 4 drops of 40% sodium hydroxide Add ammonium hydroxide by drops until the precipitate is almost dissolved. Dilute 1:10 with distilled water.

Ammoniacal silver – Var II

Place 20 mL of 2% silver nitrate in a flask. Add 3 drops of 40% sodium hydroxide Add ammonium hydroxide by drops until the precipitate is just dissolved.

 

Tissue sample
Bensley & Bensley said that sections of tissue fixed in Zenker, Helly, ethanol or formalin are suitable. Although they commented that Var I gave a complete impregnation of paraffin or celloidin embedded tissue, they recommended Var II for paraffin sections because of their tendency to detach from slides. A section adhesive is recommended.

Method

#	Step	Var I	Var II
1	Bring sections to water via xylene and ethanol.
2	Oxidise in 1% potassium permanganate	5 min	5 min
3	Bleach with 5% oxalic acid	+	+
4	Place in Lugol's iodine	+	+
5	Bleach in 3% sodium thiosulphate	+	+
6	Wash with water	+	+
7	a. Place in tannic acid solution at 56°C b. Rinse with ammoniated distilled water Place in 2% silver nitrate	5 min + —	— — 16 hrs
8	Ammoniacal silver solution, VAR I, at 56°C Ammoniacal silver solution, VAR II, at RT	10 min —	— 30 min
9	Wash with water	+	+
10	Reduce with 20% formalin	+	3 min
11	Wash with water	+	+
12	Tone with 0.2% yellow gold chloride	+	+
13	Wash with water	+	+
14	Place in 3% sodium thiosulphate	+	+
15	Wash with water	+	+
16	Place in neutral red	1 min	1 min
17	Wash with water	+	+
18	Dehydrate with ethanol, clear with xylene and mount in a resinous medium

 

Expected results

• Reticulin fibres  –  black
• Nuclei  –  as counterstained
• Background  –  grey or as counterstained

Notes

1. In the method details above several steps do not have times given and are indicated by "+", meaning that the step is required but no other details were given. Common sense should prevail, and the step done for sufficient time to accomplish its obviously intended purpose. If it is a water wash for removal of an excess of the preceding material it would usually be for approximately 1-2 minutes. If it is for toning with gold chloride then see the final note below.
2. The method details also often specify to "Wash in water" without saying whether distilled or tap water should be used. In many cases it does not matter, but common sense should prevail. If tap water is likely to produce a non-specific precipitate of silver, then use distilled water and, when it specifies to "wash", give several changes. Tap water varies in quality and individual laboratory's results may differ due to that. Of course, distilled water could be used throughout, but it is strongly recommended after the silver or gold chloride solutions since these may be affected by tap water contaminants.
3. Bensley & Bensley said that "the silver carbonate solution of Hortega" could be substituted for their own silver oxide solution in Var I. Hortega gave formulas for several such solutions and the authors do not say which one they meant. These formulas differ mainly by the amounts of 10% aqueous silver nitrate added to 5% aqueous sodium carbonate. All redissolve the resulting precipitate with drops of strong ammonium hydroxide.
    
   Hortega's Ammoniacal silver solutions
   
   • Place 50 mL of 5% sodium carbonate in a flask. Add 12 mL of 10% silver nitrate. Let the precipitate settle, then decant the supernatent. Wash, allow to settle and decant several times. Add ammonium hydroxide by drops until the precipitate is almost dissolved. Dilute to 100 mL with distilled water.
   • In addition to the formula above, another adds 12.5 mL silver nitrate, does not decant and wash, but does dilute to 100 mL with distilled water.
   • A third adds 20 mL silver nitrate to 80 mL sodium carbonate, does not decant and wash, and does not dilute with distilled water.
   • A fourth adds 25 mL silver nitrate to 75 mL sodium carbonate, does not decant and wash, and does not dilute with distilled water.
   • A fifth adds 12 mL silver nitrate to 50 mL saturated aqueous lithium carbonate, decants and washes, and dilutes to 100 mL with distilled water.
4. Ensure that the ammonium hydroxide is fresh and full strength. Keep well stoppered when not in use. After removing the amount required immediately restopper the bottle.
5. Improperly made ammoniacal silver solutions can affect the quality of the impregnation. There should be a faint, persistent opalescence, with only a faint smell of ammonia.
6. 20% formalin is made by diluting 20 mL strong formalin with 80 mL water.
7. Bensley & Bensley suggested either Heidenhain's Azan or 1% aqueous acridine red as counterstains. I have substituted neutral red.
8. The formula given for Lugol's iodine is now usually referred to as Gram's iodine.
9. Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.

 

Reference
Bensley, R R, and Bensley, S H, (1938
Handbook of Histological and Cytological technique.
The University of Chicago Press, Chicago, Illinois, USA.

Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.

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