How much formalin is enough to fix tissues?

A total of sixty samples from human breast, uterus, liver, skin and abdominal fat were fixed for 8; 24 and 48 hours at a room temperature of 20 to 22°C with neutral buffered formalin (NBF) with volume to tissue ratios of 1:1; 2:1; 5:1 and 10:1 and manually processed with isopropyl alcohol and mineral oil mixtures. All the slides prepared were evaluated as suitable for diagnostic purposes by nine pathologists from three different Russian histopathology institutions. The microtomy quality differences between the samples was not statistically significant for the different fixation volume ratios tested, but the differences between fixation periods and tissues types were, with 48 hours being the optimum fixation period, with skin and fat the most difficult to infiltrate. Neither the time and volume ratio combinations affected the pH of NBF or the immunostaining for vimentin in uterus or the histochemical periodic acid reaction or reticular demonstration fibers in liver. Fixing tissues with a ratio of NBF volume to tissue volume of 2:1 for 48 hours at 20-22°C was enough to assure a proper fixation and infiltration of the tested tissues and there is no objective reason to expect that other tissues will not behave similarly. It is suggested that in order to obtain good fixation and paraffin wax infiltration in around 10 hours, the fixation with NBF at 2:1 should be at 45°C with pressure and agitation.