Fixative and staining solutions
The formulations, systems, and methods disclosed herein permit automated preparation of specimens (e.g., biological specimens) for examination. The formulations can include a cytological fixative solution including Azure B, a surfactant, methanol, and ethylene glycol. The disclosed formulations, systems, and methods provide fast, efficient, and highly uniform specimen processing using minimal quantities of fluids. The methods include at least a fixing phase for fixing a specimen to a substrate such as a microscope slide, a staining phase for staining the specimen, and a rinsing phase for rinsing the specimen. One or more of the fixing, staining, and rinsing phases include one or more agitation phases for distributing reagents evenly and uniformly across the specimen. The systems can be implemented as a standalone device or as a component in a larger system for preparing and examining specimens.
1. A single dye containing cytological fixative solution containing a single dye, comprising:
a single dye consisting of Azure B;
a surfactant;
methanol; and
ethylene glycol.
2. The cytological fixative solution of claim 1 , wherein Azure B is present in an amount of from about 0.5 to about 5 g/L.
3. The cytological fixative solution of claim 1 , wherein the surfactant is present in an amount of from about 0.05% to about 0.5% by volume.
4. The cytological fixative solution of claim 1 , wherein the surfactant is present in an amount of from about 0.05% to about 0.3% by volume.
5. The cytological fixative solution of claim 1 , wherein the surfactant is selected from the group consisting of non-ionic, cationic, anionic, and zwitterionic surfactants.
6. The cytological fixative solution of claim 5 , wherein the non-ionic surfactant is polysorbate 20.
7. The cytological fixative solution of claim 6 , wherein the polysorbate 20 is present in an amount of from about 0.5 mL/L to about 2 mL/L.
8. The cytological fixative solution of claim 1 , further comprising a buffering agent.
9. The cytological fixative solution of claim 8 , wherein the buffering agent is selected from the group consisting of bis-tris, phosphate, HEPES, MES, Tris, and any combination thereof.
10. The cytological fixative solution of claim 9 , wherein a 1:10 dilution of the solution in water has a pH of from 6 to 8 and HEPES is present in an amount of from about 0.5 mM to about 10 mM.
11. The cytological fixative solution of claim 9 , wherein a 1:10 dilution of the solution in water has a pH of from 6.7 to 7.3 and HEPES is present in an amount of from about 0.5 mM to about 10 mM.
12. The cytological fixative solution of claim 1 , wherein ethylene glycol is present in an amount of from about 0.5 to about 5% by volume.
13. The cytological fixative solution of claim 1 , wherein a 1:1000 dilution of the fixative solution in water has a UV absorbance of from about 0.1 to about one at a peak wavelength of from about 640 to about 650 nm.
14. The cytological fixative solution of claim 1 , wherein the surfactant is non-ionic.
15. A single dye containing cytological fixative solution containing a single dye, comprising
about 0.5 g/L to about 5.0 g/L of a single dye consisting of Azure B;
about 0.5 mL/L to about 2.0 mL/L polysorbate 20;
about 5 mL/L to about 50 mL/L ethylene glycol;
about 0.10 g/L to bout 10 g/L HEPES sodium salt; and
methanol.
16. A cytological fixative solution, consisting essentially of:
about 0.5 g/L to about 5.0 g/L of Azure B;
about 0.5 mL/L to about 2.0 mL/L polysorbate 20;
about 5 mL/L to about 50 mL/L ethylene glycol;
about 0.10 g/L to about 10 g/L HEPES sodium salt; and
methanol.
17. The cytological fixative solution of claim 16 , consisting of:
about 0.5 g/L to about 5.0 g/L of Azure B;
about 0.5 mL/L to about 2.0 mL/L polysorbate 20 ;
about 5 mL/L to about 50 mL/L ethylene glycol;
about 0.10 g/L to about 10 g/L HEPES sodium salt; and
methanol.