Mix the saffron stigmata with the ethanol and seal tightly. Place in an oven at 56°C for 1-2 weeks.
Tissue sample Brasil's fixative was specified. 5µ paraffin sections of neutral buffered formalin fixed tissue are suitable but results may be improved by refixing in Bouin's fixative or saturated aqueous picric acid at 56°C for one hour prior to staining, then washing the sections in tap water to remove all yellow discolouration.
Bring sections to water with xylene and ethanol.
Treat with Bouin's fluid if desired.
Stain nuclei with hemalum, differentiate and blue.
Wash well with water.
Place in solution A for 2-20 minutes.
Wash with tap water until red stain stops being removed, 1-5 minutes.
Differentiate phloxine with 80% ethanol up to 1½ minutes.
Thoroughly dehydrate with absolute ethanol.
Place into solution B for 2-20 minutes until a suitable red-yellow balance is achieved.
Rinse well with absolute ethanol.
Clear with xylene and mount with a synthetic resinous medium.
Nuclei – blue
Cytoplasm – red shades
Muscle – pink
Elastic fibres – bright red
Collagen – orange-yellow
Any hemalum may be used, but Bencosme specified a particular formula.
Saffron is expensive. It may be available in East Indian grocery stores or health food stores as its
most common use today is as a spice and food colouring. Usually, whole stigmata are more effective than ground saffron.
If the saffron solution is too strong it may be diluted with absolute ethanol. Store and use at room temperature.
It has a limited life of about a month and is best when freshly made.
It is important that there be no water in the saffron solution and that sections be thoroughly dehydrated before it is applied.
If contaminated with moisture the solution must be discarded.
Reference Medical Laboratory Technology, 2nd ed. (1969) Lynch M.J., Raphael S.S., Mellor L.D., Spare P.D. and Inwood M.J.H.,
W. B. Saunders Co., Toronto, On., Canada