|Chromium trioxide||0.25 g||0.75 g|
|Osmium tetroxide, 1% aqueous||10 mL||40 mL|
|Acetic acid, glacial||0.1 mL||5 mL|
|Distilled water||90 mL||60 mL|
Flemming gave formulas for both a weak and a strong solution, but the strong solution is more commonly used than the weak. Due to the expense of the osmium tetroxide it is customary to use a fixative volume of 5-10 times the volume of the tissue rather than the usually recommended 20 times. The acetic acid is optional and may be omitted for the preservation of mitochondria. For chromosomes it should be included.
Penetration of Flemming's fluid is slow and uneven, so tissue may be variably fixed. Thin pieces of tissue are required because of the poor penetration and 2 millimetres or less is recommended. Thicker pieces may be improperly fixed in the center.
Pieces less than 2 millimetres should be fixed within 12 hours.
Wash in running tap water overnight.
Note that these solutions do not store well, and enough should be made for a few days only.
Davenport, H.A., (1960),
Histological and histochemical techniques,
W. B. Saunders Co., London, UK., Oxford, UK.
Handbook of Histopathological and Histochemical Techniques, Ed. 3,
Butterworths, London, UK.