The penetration of methanol into bovine cardiac and hepatic tissues is faster than ethanol and formalin

Abstract

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.

Keywords: Ethanol; formalin; methanol; penetration coefficient; penetration rate..

Figure 1.

The normalised penetration distance of different fixatives in heart tissue. A) A typical image of tissue before fixation; two dots were marked on a face of the tissue and were used for orientation, making sure that the tissue was immersed in the fixative with the marked face always facing up. B) A typical image of the marked face after fixation. C) The shrinkage or expansion of the heart tissue was calculated after exposure to different fixatives for 1, 2, 6 or 10h at 37ºC. N=4; the change in the marked surface area (area) was calculated. The square root of the absolute value of the ratio of Δarea/area before fixation was then calculated as the shrinkage or expansion of the tissue; error bars represent standard deviation; positive values represent tissue expansion, whereas negative values represent tissue shrinkage. D) The penetration distance was normalised to the tissue shrinkage or expansion of each individual sample. n=4; *P<0.001, using two-way ANOVA followed by Bonferroni post-hoc tests. NS, not significant.

Figure 2.

The penetration distance of different fixatives in heart tissue. A) Heart cubes (size: 2x2x2 cm) were immersed in ethanol, methanol and 10% neutral-buffered formalin at 37ºC with the marked face always facing up; the tissue was taken out of the fixatives at certain time points (1, 2, 6, or 10 h) and cut into two halves along the middle plane parallel to the marked face and the newly cut face was then imaged with a digital camera. B) Representative image for the measurement of penetration distance; the penetration distance (the distance of discoloured region) was measured 10 times on each side using PhotoShop software and a total of 40 measurements were recorded for each tissue; the mean of the 40 measurements was calculated as the penetration distance of the fixative in this sample. C) Penetration distance over time for three fixatives; n=4; error bars represent standard deviation; the difference between fixatives was analysed using twoway ANOVA followed by Bonferroni post-hoc tests.

Figure 3.

The depth of penetration of different fixatives into heart tissue relative to the square root of the fixation time. The penetration distance in millimetres was the mean of the measurements from four heart tissues. The x-axis represents the square root of the fixation time in hours. For example, the value 3 on the x-axis represents 9 h of fixation.

Figure 4.

The penetration distance of different fixative in liver tissue. A) Penetration distance of ethanol (▲), formalin (▼) and methanol (○) in the liver tissue; liver tissue cubes (size: 2x2x2 cm) were immersed in fixative for 1, 2, 6, or 10 h at 37°C; the tissue was taken out of the fixative at certain time points and the penetration distance was then measured using PhotoShop software; n=4; error bars represent standard deviation; *P<0.001, using two-way ANOVA followed by Bonferroni post-hoc tests; NS, not significant. B) The shrinkage or expansion of the liver tissue. The shrinkage or expansion was calculated after exposure to the fixatives for 1, 2, 6 or 10 h at 37°C; n=4; the change in the marked surface area (area) was calculated; the square root of the absolute value of the ratio of Δarea/area before fixation was then calculated as the shrinkage or expansion of the tissue; error bars represent standard deviation; positive values represent tissue expansion, whereas negative values represent tissue shrinkage. C) The penetration distance in the liver tissue was normalised to the tissue shrinkage or expansion of each individual sample; n=4; error bars represent standard deviation; *P<0.001, using two-way ANOVA followed by Bonferroni post-hoc tests. D) Depth of penetration of fixatives into the heart tissue relative to the square root of the fixation time; the penetration distance in millimetres was the mean of the measurements from four liver tissues; the x-axis represents the square root of the fixation time in hours.