Phosphate-buffered Formalin Fixative Under Controlled Fixation Temperature Yielded Excellently Preserved Histomorphology

Aim: Using mouse liver as experimental model, this study attempts to identify a formalin-based fixative and fixation temperature that jointly provides the best balance of preservation of tissue morphology. Methodology: Liver samples from fifty (50) albino mice aged between of 6 to 8 weeks consisting of both male and female was harvested following cervical dislocation and randomly distributed into control and experimental groups. Control samples were fixed in 10mL of 10% formalin at 25oC, 30oC, 35oC, 40oC, 45oC, 50oC, 55oC and 60oC respectively for 24 hours, while experimental samples were each fixed in equal volume of phosphate-buffered 10% formalin (pH 7.2, 7.4, 7.6 and 7.8) at the same temperature and time duration regimen and processed for general tissue morphology. Nuclear, cytoplasm and cell membrane morphology were assessed as evidence of the combined effectiveness of fixative and fixation temperature. Morphology was scored using a four-point grading scale with 1 being poor and 4 being excellent. Results: Nuclear, cytoplasm and cell membrane morphology were excellently preserved in tissue fixed with phosphate-buffered 10% formalin (pH 7.2) at 45oC. Tissue fixed with 10% formalin at 35oC exhibited excellent nuclear and cell membrane morphology, while excellent preservation of cell membrane morphology were observed in tissues fixed with 10% formalin at 40oC, phosphate-buffered 10% formalin (pH 7.4) at 55oC and 60oC, (pH 7.6) at 50oC and 55oC and (pH 7.8) at 55oC respectively. Furthermore, excellent preservation of nuclear morphology was observed in tissue fixed with phosphate-buffered 10% formalin (pH 7.8) at 60oC. Conclusion: Phosphate-buffered 10% formalin at a temperature of 45oC and pH 7.2 provide an excellent formalin-based fixative and fixation temperature that adequately preserves the microanatomy of tissue for histopathology examination.

Effects of fixation temperature and environment on copper speciation in ACQ treated red pine

Red pine (Pinus resinosa Ait.) sapwood samples treated with alkaline (amine) copper quaternary (ACQ) were conditioned at different temperatures and under wet or dry conditions to evaluate the stability of copper (Cu) to leaching and conversion of divalent copper (Cu-II) to monovalent copper (Cu-I). Significant Cu-II was reduced to Cu-I during post-treatment stabilization, especially at elevated temperatures (105°C and 120°C). The percent conversion to Cu-I was higher at lower retentions; in some cases, at low retentions and high fixation temperatures, all of the cupric Cu was reduced to Cu-I, raising the possibility of reduced activity of Cu as a biocide when high temperature conditions are used to fix ACQ treated wood. However, exposure of wood to a leaching procedure (rewetting, re-drying) generally resulted in a significant component of the Cu-I in wood being oxidized back to Cu-II. Steaming of weathered ACQ treated wood at 120°C also converted significant amounts of Cu-II to Cu-I.

Microwave fixation of large tissue

Chemical fixation of large biological tissues in electron microscopy has a disadvantage that fixative doesn’t penetrate well into the deeper part of the tissue. My experience suggests that the poor penetration by chemical fixation can be improved by microwave fixation. Temperature raise caused by microwave irradiation is regulated by the cold bath on the floor of the oven. This paper shows that microwave fixation is readily practicable together with conventional fixation. Well-fixed section with an area as large as 1×5mm is observable and the details of morphological structures are preserved quite well in it.A commercial microwave oven (National Microwave Oven Model NEM-335,2.45GHz 500/180W) was used for the microwave fixation. Irradiation period was controlled by Omuron H5cN-AN. Young mouse kidney and brain were used. The tissue in a glass vial placed in the cold bath was immersed in the mixture of 2% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer (pH7.4),followed by microwave irradiation for 10 sec. twice.

Ultrafast microwave energy fixation for electron microscopy.

We demonstrate that microwave (MW) energy can be used in conjunction with chemical cross-linking agents to fix tissue blocks rapidly for electron microscopy in as brief a time as 26 msec. The optimal ultrafast MW fixation methodology involved immersing tissue blocks up to 2 mm3 in dilute aldehyde fixative and immediately irradiating the specimens in a 7.3 kW MW oven for 26-90 msec, reaching a fixation temperature range of 32-42 degrees C. Ultrastructural preservation of samples irradiated by MW energy was comparable to that of the control samples immersed in aldehyde fixative for 2 hr at 25 degrees C. Potential applications for this new fixation technology include investigation of rapid intracellular processes (e.g., vesicular transport) and preservation of proteins that are difficult to demonstrate with routine fixation methods (e.g., antigens and enzymes).

THE MECHANISM OF COMPLEMENT FIXATION

1. Complement fixation is obtained in every antigen-antibody reaction involving the presence or formation of a heterogeneous phase (red cells, bacteria, precipitate). 2. The physical constants of fixation (temperature coefficient, velocity, quantitative relationships between the reactants) are those commonly associated with adsorption processes, and are the same in the three types of fixation studied. 3. All the in vitro immune reactions involve an aggregation of immune-serum globulins upon the surface of the antigen. It has been shown that the "fixation" of complement is an adsorption by the aggregates so formed; whether these aggregates are visible as a flocculent precipitate (e.g., sheep serum vs. anti-serum) or concentrated as a surface film on a cellular antigen (sensitized cells; agglutinated bacteria), the reaction is fundamentally the same. 4. As yet, it is unknown whether this adsorption is determined by the physical state of the precipitate, and thus, differs only quantitatively from that by Kaolin, charcoal, normal bacteria, heat-denatured proteins, etc.; or whether the comparatively enormous avidity of these aggregates for complement is due to a specific chemical affinity.