Introduction. Ethanol is a commonly used fixative. Fixation of the inner layers of the tissue depends on the ability of the fixative to diffuse into the tissue. It is unknown whether the concentration of ethanol affects its penetration into tissues. This study aimed to compare the penetration rates of 50% and 100% ethanol into bovine heart and liver tissues. Materials and methods. The penetration distance and tissue shrinkage or expansion were measured by analysing the digital images of the heart and liver tissues before and after immersion in ethanol at 20°C for 2, 6, 24 or 30 hours. The penetration coefficients were calculated as the slope of the regression line using the linear regression function between the penetration distance and square root of fixation time. Differences in tissue shrinkage or expansion and penetration distance at various time points between the two concentrations of ethanol were analysed using a mixed design ANOVA followed by Bonferroni's post-hoc test. Results. The penetration distance of 100% ethanol was significantly greater in both heart and liver tissues compared with that of 50% ethanol (n = 4, p < 0.05 for both). 100% ethanol shrank immersed liver tissue significantly more than 50% ethanol (p = 0.002), but the shrinkage of the heart tissue caused by two concentrations of ethanol did not significantly differ (p = 0.054). The greater penetration distance of 100% over 50% ethanol remained unchanged after normalising the penetration distance to the individual tissue's shrinkage (n = 4, p < 0.001). The mean penetration coefficient of 100% ethanol was significantly greater than 50% ethanol in the heart tissue (0.906 vs. 0.442, p = 0.003) and in the liver tissue (0.988 vs. 0.622, p = 0.028). Conclusions. It was proven that in two types of tissue that substantially differ in histological structures, 100% ethanol penetrated tissue significantly faster than 50% ethanol.

Methanol, ethanol and formalin are commonly used as fixatives to preserve biological tissues from decay in the preparation of histological sections. Fixation of the inner layers of the tissue depends on the ability of the fixative to diffuse into the tissue. It is unknown whether methanol penetrates tissues at similar rates to other fixatives. This study aimed to compare the penetration rates of methanol, ethanol and formalin into bovine heart and liver tissues. The penetration distance and tissue shrinkage or expansion were measured by analysing the digital images of tissue before and after immersion in different fixatives for 1, 2, 6 or 10 h. Data were analysed using two-way ANOVA, followed by Bonferroni’s post-hoc test. The penetration distance of methanol was significantly greater in both heart and liver tissues compared with that of ethanol (N=4, P<0.001). Methanol or ethanol immersion led to similar shrinkage of both tissues (P>0.05). The penetration rate of formalin was similar to that of ethanol in both tissues however it was significantly slower than methanol (N=4, P<0.005 in the heart; P<0.001 in the liver). The mean penetration coefficients of methanol, formalin and ethanol in the heart tissue were 2.609, 1.994 and 1.801, respectively, and 3.012, 2.153 and 2.113, respectively, in the liver tissue. The penetration coefficient of methanol was significantly greater than that of ethanol or formalin in both tissues (P<0.001 for each comparison). In conclusion, methanol penetrates tissue significantly faster than ethanol and formalin.