1) Aqueous Eosin Y, Acidified (0.
5% w/v) —
standard H&E
Reagents
Eosin Y, disodium salt (CI 45380) … 0.5 g
Distilled water … up to 100 mL
Glacial acetic acid … 0.5–1.0 mL (to pH ~4.6–5.0)
Preparation
1. Warm ~80 mL water to room temp; dissolve 0.5 g eosin Y with stirring.
2. Make up to 100 mL with water.
3. Add 0.5 mL glacial acetic acid; check pH. Adjust with a few extra drops to pH 4.6–5.0
(brisk, selective staining).
4. Filter through Whatman No. 1 (or 0.45 μm) into an amber bottle. Label & date.
Use
Typical counterstain: 30 sec–2 min (depends on tissue and hematoxylin strength).
Brief dip in 95% ethanol differentiates if too intense.
Eriochrome Black T Indicator Solution
Reagents
Eriochrome Black T … 0.5 g
Triethanolamine or Ethanolamine … 100 mL
(or sometimes 95% ethanol is used, but triethanolamine gives better stability)
Preparation
1. Weigh 0.5 g Eriochrome Black T.
2. Dissolve in 100 mL triethanolamine, mix thoroughly.
3. Store in a tightly stoppered amber glass bottle (room temp).
tandard aqueous methyl orange (0.1% w/v)
Reagents
Methyl orange (CI 13025) … 0.10 g
Distilled water … to 100 mL
Preparation
1. Dissolve 0.10 g methyl orange in ~80 mL water with stirring.
2. Make up to 100 mL with water, mix.
3. Filter if hazy. Store in an amber bottle at room temp.
Methylene Blue Indicator (0.1% w/v,
aqueous)
Use: general redox indicator (blue when oxidized; fades to colorless when reduced).
Methylene blue (as chloride) … 0.10 g
Distilled water … to 100 mL
Prep: Dissolve MB in ~80 mL water, make up to 100 mL, mix, filter if needed.
Use: 2–3 drops in ~100 mL test solution.
Storage: Amber bottle, RT (15–25 °C). Stable ~6–12 months.
Phenolphthalein Indicator Solution (1% w/v
in ethanol)
Use: Acid–base titrations (strong acid vs. strong base; weak acid vs. strong base).
Reagents
Phenolphthalein … 1.0 g
95% Ethanol (or denatured ethanol) … 100 mL
Preparation
1. Dissolve 1.0 g phenolphthalein in ~80 mL of ethanol.
2. Make up the volume to 100 mL with ethanol.
3. Mix well; filter if cloudy.
4. Store in tightly stoppered amber glass bottle.
Starch Indicator Solution (1% w/v)
Reagents
Soluble starch (potato or corn starch, analytical grade) … 1.0 g
Distilled water … 100 mL
Optional stabilizer: Mercuric chloride (HgCl₂, toxic) or salicylic acid / thymol crystals (a
safer modern choice) — prevents microbial growth.
🔸 Preparation
1. Suspend 1 g starch in a small volume (~5 mL) of cold distilled water (to make a slurry).
2. Bring ~95 mL distilled water to a gentle boil.
3. Add the starch slurry slowly into the boiling water with stirring.
4. Boil for 2–3 minutes until solution clears (turns translucent).
5. Cool to room temp.
6. Add 0.02 g salicylic acid (or a few crystals of thymol) as preservative.
7. Transfer to a clean amber glass bottle, label, and store refrigerated.
Phenol Red Indicator Solution (0.02% w/v,
aqueous)
Reagents
Phenol Red sodium salt … 0.02 g
Distilled water … to 100 mL
Preparation
1. Dissolve 0.02 g phenol red in ~80 mL distilled water.
2. Make up to 100 mL with distilled water.
3. Mix well and filter if needed.
4. Store in an amber glass bottle at room temperature.
Preparation of Methyl Red Indicator Solution (0.1% w/v):
1. Weigh 0.1 g of methyl red indicator powder.
2. Dissolve in 300 mL of 95% ethanol (or sometimes in 600 mL of ethanol + 200 mL of
water).
3. Make up to 100 mL total volume with ethanol.
4. Store in a tightly stoppered amber bottle (light sensitive).
Preparation of K₂Cr₂O₇ standard solution
(example: 0.1 N)
This is the standard laboratory recipe for a 0.1 N dichromate solution (common for
standardization of Fe²⁺ etc.):
Materials
Potassium dichromate, dried (primary standard grade)
Distilled water
1 L volumetric flask, analytical balance, glass rod
Calculation / weigh
Equivalent weight approach: 1.0 N K₂Cr₂O₇ ≈ 49.03 g/L → for 0.1 N weigh 4.903 g and
dilute to 1.000 L.
(If you prefer molarity: Molar mass ≈ 294.18 g·mol⁻¹ → 0.01704 M ≈ 0.1 N in terms of
6-electron change for Cr₂O₇²⁻; the normality approach above is the commonly used lab
shortcut.)
Procedure
1. Accurately weigh 4.903 g K₂Cr₂O₇ (dried).
2. Transfer to a beaker, add ~200–300 mL distilled water and stir until dissolved (warm
slightly if needed).
3. Transfer quantitatively to a 1 L volumetric flask and rinse beaker several times into the
flask.
4. Make up to the mark with distilled water, invert/mix thoroughly.
5. Store in amber glass bottle, tightly stoppered, labelled with concentration, date, and
hazard.
Preparation of Bromophenol Blue Indicator Solution (0.04% w/v,
common strength)
Materials:
Bromophenol Blue powder
95% Ethanol (or sometimes 50% ethanol + water mixture)
Distilled water
Volumetric flask
Procedure:
1. Weigh 0.04 g bromophenol blue powder.
2. Dissolve in 25 mL of 95% ethanol.
3. Dilute to 100 mL with distilled water (or 50% ethanol, depending on method).
4. Mix well, transfer to an amber bottle, and label.
Preparation of Dithizone Indicator Solution
1) Stock Solution (commonly used in assays, e.g. Pb limit test):
Dissolve 0.025 g dithizone in 100 mL chloroform.
Shake well, filter if necessary.
Store in a dark, amber-stoppered bottle (light sensitive).
2) Saturated Aqueous Solution (rarely used directly):
Shake an excess of dithizone with distilled water containing a little ethanol, filter.
Not very stable, so prepared fresh.
3) Buffer Solution for Use with Dithizone:
Often dithizone is used along with ammonium citrate / ammonia buffer or acetate
buffer to control pH for selective metal complexation.
Dithizone solutions are unstable and decompose on exposure to light and air. Prepare
fresh frequently.
Store in amber bottles, in refrigerator if possible.
Always wash chloroform solution with dilute ammonia and then water before use to
remove oxidized products.
