Toluidin Blue Staining Solution — Preparation

Aqueous toluidine blue O solution buffered with sodium tetraborate and stabilised with sucrose. 50 mL batch.

Histology Electron Microscopy Semi-thin sections

Overview

Toluidine blue O is a cationic metachromatic dye that binds strongly to acidic tissue components such as nucleic acids, sulfated proteoglycans, and cartilage matrix, producing deep blue to violet staining depending on the substrate. Sodium tetraborate (Borax) serves as a mild alkaline buffer to maintain the working pH near 7.4 and enhances dye penetration. Sucrose is added as an osmotic stabiliser, particularly beneficial when staining semi-thin resin-embedded sections for light microscopy after electron microscopy processing. The solution is applied directly to sections and rinsed with distilled water.

Safety

⚠ Hazard information — Toluidine Blue O & Sodium Tetraborate

⚠

GHS07
Irritant

🫁

GHS08
Health hazard

🌿

GHS09
Environ. hazard

Toluidine blue O — suspected mutagen/carcinogen (Category 2); avoid prolonged skin contact and inhalation of dust or aerosols.
Sodium tetraborate (Borax) — reproductive toxin Category 1B; do NOT handle if pregnant or planning pregnancy. Wear gloves at all times.
Both dye and borax stain skin, clothing, and bench surfaces immediately — wear nitrile gloves, lab coat, and eye protection throughout preparation.
Work in a well-ventilated area; avoid generating dust when weighing powders.
Do NOT dispose of concentrated dye solution via the drain — toxic to aquatic organisms. Collect in labelled waste container for disposal.
In case of eye contact: rinse immediately with water for ≥ 15 min and seek medical advice.

Reagents & Materials

#	Reagent	CAS	Amount (50 mL)	Final conc.	Supplier	Hazard
1	Toluidine Blue O	92-31-9	0.50 g	1% w/v	Sigma-Aldrich	GHS07 GHS08 GHS09
2	Sodium tetraborate · 10H₂O (Borax)	1303-96-4	0.50 g	1% w/v	Sigma-Aldrich	GHS07 GHS08
3	Sucrose	57-50-1	2.50 g	5% w/v	Sigma-Aldrich	none
4	dH₂O (distilled water)	7732-18-5	ad 50 mL	—	In-house	none

Protocol Steps

Step	Action	Time
01	Weigh 0.50 g Toluidine Blue O, 0.50 g sodium tetraborate, and 2.50 g sucrose into a 100 mL glass beaker. Weigh dye last and keep the balance pan covered between weighings — powder disperses easily and stains surfaces.	5 min
02	Add ~40 mL dH₂O and stir vigorously with a magnetic stirrer until all components are partially dissolved.	5 min
03	Gently heat the solution on a hotplate/stirrer to approximately 60 °C while continuing to stir until fully dissolved and the solution appears clear and homogeneous. Do not exceed 70 °C — overheating may degrade the dye and shift metachromatic properties. Monitor with a thermometer.	10–15 min
04	Allow the solution to cool to room temperature, then adjust the final volume to 50 mL with dH₂O.	15 min
05	Filter the solution through a folded qualitative filter paper (e.g. Whatman No. 1) or a 0.45 µm membrane filter into a clean, labelled amber glass bottle. Filtration removes undissolved dye aggregates that would otherwise deposit as precipitate on sections.	5–10 min
06	Check the pH of the filtered solution with a calibrated pH meter. Target value: pH 7.2–7.4. If pH is too low, add a few drops of 0.1 M NaOH and re-check. If too high, add a few drops of 0.1 M HCl. The borax typically buffers near the target without adjustment.	5 min
07	Label the bottle clearly and store at room temperature in the dark or at 4 °C for extended shelf life. Label must include: name, date, ⚠ BORAX and preparer initials.	—

Notes & Observations

For semi-thin sections (0.5–1 µm) of Epon/Araldite-embedded tissue, apply 1–2 drops of the working solution directly to the dry section on a hotplate (60 °C), allow to stain for 30–60 seconds, then rinse thoroughly with dH₂O and air-dry before coverslipping with a resinous mountant. Staining intensity can be adjusted by diluting the working solution 1:1 with dH₂O. For routine histology on paraffin sections, a working dilution of 0.1–0.5% is often sufficient. Solution is stable for at least 6 months at room temperature when stored in a sealed amber bottle. If a precipitate forms, re-filter before use.