MEDIA, REAGENTS AND SOLUTIONS

MEDIA

The following media are used in the Microbiological Analyses Section of this volume and are referenced in that Section. The listing is alphabetical.

Baird-Parker Medium

Tryptone: 10 g
Beef extract: 5 g
Yeast extract: 1 g
Sodium pyruvate: 10 g
Glycine: 12 g
Lithium chloride 6H2O: 5 g
Agar: 20 g
Distilled water: 1 litre

Autoclave 15 min at 121°. Final pH is 7.0 ± 0.2. If desired for immediate use, maintain melted medium at 48-50° before adding enrichment. Otherwise, store solidified medium at 4 ± 1 ° up to one month. Melt medium before use.

Bismuth Sulfite Agar (Wilson and Blair)

Polypeptone (or peptone): 10 g
Beef extract: 5 g
Dextrose: 5 g
Na2HPO4 (anhydrous): 4 g
FeSO4 (anhydrous): 0.3 g
Bismuth sulfite (indicator): 8 g
Brilliant green: 0.025 g
Agar: 20 g
Distilled water: 1 litre

Mix thoroughly and heat with agitation. Boil about 1 min to obtain uniform suspension. (Precipitate will not dissolve.) Cool to 45-50°. Suspend precipitate by gentle agitation, and pour 20 ml portions into sterile 15 x 100 mm petri dishes. Let plates dry about 2 h with lids partially removed; then close plates. Final pH, 7.7 ± 0.2. Do not autoclave. Prepare plates on day before streaking and store in dark. Selectivity decreases in 48 h. Store plates in dark.

Brain Heart Infusion (BHI) Broth and Agar

Medium 1

Calf brain infusion: 200 g
Beef heart infusion: 250 g
Proteose peptone or gelysate: 10 g
NaC1:5 g
Na₂HPO₄-12 H₂O:2.5 g
Dextrose: 2 g
Distilled water: 1 litre

Dissolve ingredients in distilled water with gentle heat. Dispense broth into bottles or tubes for storage. Autoclave 15 min at 121°. Final pH, 7.4 ± 0.2.

Medium 2

Brain heart infusion : 6 g
Peptic digest of animal tissue: 6 g
NaCl: 5 g
Dextrose: 3 g
Pancreatic digest of gelatine: 14.5 g
Na₂HPO₄: 2.5 g
Distilled water: 1 litre

Suspend ingredients of Medium 2 in distilled water and boil for 1 min to completely dissolve.

For both Medium 1 and Medium 2, dispense broth into bottles or tubes for storage. Autoclave 15 min at 121°. Final pH, 7.4 ± 0.2. Commercially available BHI is acceptable.

To prepare brain heart infusion agar, add 15 g agar to 1 litre BHI broth. Heat to dissolve agar before dispensing into bottles or flasks. Autoclave 15 min at 121°.

Brilliant Green Lactose Bile Broth

Peptone: 10 g
Lactose: 10 g
Oxgall: 20 g
Brilliant green: 0.0133 g
Distilled water: 1 litre

Dissolve peptone and lactose in 500 ml distilled water. Add 20 g dehydrated oxgall dissolved in 200 ml distilled water. The pH of this solution should be 7.0-7.5. Mix and add water to make 975 ml. Adjust pH to 7.4. Add 13.3 ml 0.1% aqueous brilliant green in distilled water. Add distilled water to make 1 litre. Dispense into fermentation tubes, making certain that fluid level covers inverted vials. Autoclave 15 min at 121°. Final pH, 7.2 ± 0.1.

Buffered Peptone Water (BPW)

Peptone: 10g
NaCl: 5 g
Na₂HPO₄: 3.5 g
KH₂PO₄: 1.5 g
Distilled water: 1 litre

Autoclave at 121° for 15 min. Final pH 7.2 ± 0.2.

Dichloran 18% Glycerol (DG18) Agar

Glucose: 10 g
Bacteriological peptone: 5 g
KH₂PO₄: 1 g
MgSO₄-7H₂O: 0.5 g
Dichloran (0.2% in ethanol, w/v): 1 ml
Chloramphenicol: 0.1 g
Agar: 15 g
Distilled water: 1 litre

Mix above items and steam to dissolve agar, then bring volume to 1000 ml with distilled water. Add 220 g glycerol and sterilize by autoclaving at 121° for 15 min. The final pH should be 5.6 and the final a_w, 0.955.

This medium is used as a general purpose mould enumeration medium and is preferred when the a_w of the analyzed food is 0.95 or lower. The low water activity of this medium reduces interference by bacteria and fast-growing fungi. When both yeasts and moulds must be enumerated, DRBC agar should be used (see next media).

Dichloran Rose Bengal Chloramphenicol (DRBC) Agar

Glucose: 10 g
Bacteriological peptone: 5 g
KH₂PO₄: 1 g
MgSO₄-7H₂O: 0.5 g
Rose bengal (5% soln., w/v): 0.5 ml
Dichloran (2,6-dichloro-4-nitroaniline) solution (0.2%(w/v) in ethanol): 1 ml
Chloramphenicol: 0.1 g
Agar: 15 g
Distilled water: 1 litre

Final pH should be 5.6. Mix ingredients, heat to dissolve agar and sterilize by autoclaving at 121° for 15 min. Temper to 45 ± 1° in a water bath and pour plates.

EC Broth

Trypticase or tryptose: 20 g
Bile salts No. 3: 1.5 g
Lactose: 5 g
K₂HPO₄: 4 g
KH2PO4: 1.5 g
NaC1:5 g
Distilled water: 1 litre

Distribute 8 ml portions to 16 x 150 mm test tubes containing inverted 10 x 75 mm fermentation tubes. Autoclave 15 min at 121°. Final pH 6.9 ± 0.2.

Hektoen Enteric (HE) Agar

Peptone: 12 g
Sodium thiosulfate: 5 g
Yeast extract: 3 g
Ferric ammonium citrate: 1.5 g
Bile salts: 9 g
Bromthymol blue: 0.064 g
Lactose: 12 g
Acid fuchsin: 0.1 g
Sucrose: 12 g
Agar: 13.5 g
Salicin: 2 g
Distilled water: 1 litre
NaC1:5g

Heat to boiling with frequent agitation to dissolve. Boil no longer than 1 min. Do not overheat. Cool in water bath. Pour 20 ml portions into sterile 15 x 100 mm petri dishes. Let dry 2 h with lids partially removed. Final pH, 7.6 ± 0.2. Do not store more than 1 day.

Koser's Citrate Broth

NaNH₄HPO₄-4H₂O: 1.5 g
K₂HPO₄: 1 g
MgSO₄-7H₂O: 0.2 g
Sodium citrate-2H₂O: 3 g
Distilled water: 1 litre

Dispense into screw-cap tubes as desired. Autoclave 15 min at 121°. Final pH, 6.2 ± 0.2. This formulation is listed in Official Methods of Analysis of the AOAC and Standard Methods for the Examination of Water and Wastewater of the APHA. It differs from the composition of commercially available dehydrated media. The latter have been found to be satisfactory.

Lactose Broth

Beef extract: 3 g Peptone: 5 g Lactose: 5 g Distilled water: 1 litre

• For E. coli: Dissolve ingredients and dispense 10 ml portions into 20 x 150 mm tubes containing inverted 10 x 75 mm fermentation vials. Autoclave 15 min at 121°. Final pH 6.9 ± 0.2.
• For Salmonella: Dispense 225 ml portions into 500 ml Erlenmeyer flasks. After autoclaving 15 min at 121° and just before use, aseptically adjust volume to 225 ml. Final pH, 6.9 ± 0.2.

Lauryl Tryptose (LST) Broth

Tryptose or trypticase: 20 g
Lactose: 5 g
K₂HPO₄: 2.75 g
KH₂PO₄: 2.75 g
NaC1:5 g
Sodium lauryl sulfate: 0.1 g
Distilled water: 1 litre

Dispense 10 ml portions to 20 x 150 mm tubes containing inverted 10 x 75 mm fermentation tubes. Autoclave 15 min at 121°. Final pH, 6.8 ± 0.2.

Levine's Eosin-Methylene Blue (L-EMB) Agar

Peptone: 10 g
Lactose: 10 g K₂HPO₄: 2 g
Agar: 15 g
Eosin Y: 0.4 g
Methylene blue: 0.065 g
Distilled water: 1 litre

Boil to dissolve peptone, phosphate, and agar in 1 liter of water. Add water to make original volume. Dispense in 100 or 200 ml portions and autoclave 15 min at not over 121°. Final pH, 7.1 ±0.2.

Before use, melt, and to each 100 ml portion add:

1. 5 ml sterile 20% lactose solution;
2. 2 ml aqueous 2% eosin Y solution; and
3. 4.3 ml 0.15% aqueous methylene blue solution.

When using complete dehydrated product, boil to dissolve all ingredients in 1 liter water. Dispense in 100 or 200 ml portions and autoclave 15 min at 121°. Final pH, 7.1 ± 0.2.

Lysine Decarboxylase Broth (Falkow) (for Salmonella)

Gelysate or peptone: 5 g
Yeast extract: 3 g
Glucose: 1 g
L-Lysine: 5 g
Bromcresol purple: 0.02 g
Distilled water: 1 litre

Heat until dissolved. Dispense 5 ml portions into 16 x 125 mm screw-cap tubes. Autoclave loosely capped tubes 15 min at 121°. Screw the caps on tightly for storage and after inoculation. Final pH, 6.8 ± 0.2 .

Lysine Iron Agar (Edwards and Fife)

Gelysate or peptone: 5 g
Yeast extract: 3 g
Dextrose: 1 g
L-Lysine hydrochloride: 10 g
Ferric ammonium citrate: 0.5 g
Sodium thiosulfate (anhydrous): 0.04 g
Bromcresol purple: 0.02 g
Agar: 15 g
Distilled water: 1 litre

Heat to dissolve ingredients. Dispense 4 ml portions into 13 x 100 mm screw-cap tubes. Autoclave 12 min at 121°. Let solidify in slanted position to form 4 cm butts and 2.5 cm slants. Final pH, 6.7 ± 0.2.

MacConkey Agar

Proteose peptone or polypeptone: 3 g
Peptone or gelysate: 17 g
Lactose: 10 g
Bile salts No. 3 (or bile salts mixture): 1.5 g
NaC1:5 g
Neutral red: 0.03 g
Crystal violet: 0.001 g
Agar: 13.5 g
Distilled water: 1 litre

Suspend ingredients and heat with agitation to dissolve. Boil 1-2 min. Autoclave 15 min at 121°, cool to 45-50°, and pour 20 ml portions into sterile 15 x 100 mm petri dishes. Dry at room temperature with lids closed. Do not use wet plates. Final pH, 7.1 ± 0.2.

Malonate Broth

Yeast extract: 1 g
(NH₄)₂SO₄: 2 g
K₂HPO₄: 0.6 g
KH2PO4: 0.4 g
NaC1:2g
Sodium malonate: 3 g
Dextrose: 0.25 g
Bromthymol blue: 0.025 g
Distilled water: 1 litre

Dissolve by heating, if necessary. Dispense 3 ml portions into 13 x 100 mm test tubes. Autoclave 15 min at 121°. Final pH, 6.7 ± 0.2.

Malt Agar

Malt extract, powdered: 20 g
Agar: 20 g
Distilled water: 1 litre

Mix ingredients, steam to dissolve agar and sterilize for 15 min at 121°. Temper medium to 45° and pour plates under aseptic conditions.

Malt Extract Agar

Malt extract: 30 g
Agar: 20 g
Distilled water: 1 litre

Boil to dissolve ingredients. Autoclave 15 min at 121°. Dispense 20-25 ml into sterile 15 x 100 mm petri dishes. Final pH, 5.5 ± 0.2.

MR-VP Broth

Medium 1

Buffered peptone-water powder: 7 g
Glucose: 5 g
K2HPO4: 5 g
Distilled water: 1 litre

Dissolve ingredients in 800 ml water with gentle heat. Filter, cool to 20°, and dilute to 1 litre. Autoclave 12-15 min at 121°. Final pH, 6.9 ± 0.2.

Medium 2

Pancreatic digest of casein: 3.5 g
Peptic digest of animal tissue: 3.5 g
Dextrose: 5 g
Potassium phosphate: 5 g
Distilled water: 1 litre

Dissolve ingredients in water with gentle heat if necessary. Dispense 10 ml into 16 x 150 mm test tubes and autoclave 15 min at 118-121°. Final pH, 6.9 ± 0.2.

Medium 3

Peptone: 5 g
Glucose: 5 g
Phosphate buffer: 5 g
Distilled water: 1 litre

Dissolve ingredients in water. Dispense 10 ml into 16 x 150 mm test tubes and autoclave 15 min at 121°. Final pH, 7.5 ± 0.2.

For Salmonella: Dispense 10 ml into 16 x 150 mm test tubes, and autoclave 12-15 min at 121°.

Motility Test Medium (semisolid)

Beef extract: 3 g
Peptone or gelysate: 10 g
NaCl: 5 g
Agar: 4 g
Distilled water: 1 litre

Heat with agitation and boil 1-2 min to dissolve agar. Dispense 20 ml portions into 20 x 150 mm screw-cap tubes, replacing caps loosely. Autoclave 15 min at 121°. Cool to 45° after autoclaving. Tighten caps, and refrigerate at 5-8°. To use, re-melt in boiling water or flowing steam, and cool to 45°. Aseptically dispense 20 ml portions into sterile 15 x 100 mm petri plates. Cover plates and let solidify. Use same day as prepared. Final pH, 7.4 ± 0.2.

Nutrient Broth

Beef extract: 3 g
Peptone: 5 g
Distilled water: 1 litre

Heat to dissolve. Dispense 10 m1 portions into tubes or 225 ml portions into 500 ml Erlenmeyer flasks. Autoclave 15 min at 121°. Final pH, 6.8 ± 0.2.

Phenol Red Carbohydrate Broth

Trypticase or proteose peptone No. 3: 10 g
NaC1: 5 g
Beef extract (optional): 1 g
Phenol red (7.2 ml of 0.25% phenol red solution): 0.018 g
Distilled water: 1 litre
Carbohydrate^*

^*Dissolve either 5 g dulcitol, 10 g lactose, or 10 g sucrose (as specified in the Salmonella test) in this basal broth. Dispense 2.5 ml portions into 13 x 100 mm test tubes containing inverted 6 x 50 mm fermentation tubes. Autoclave 10 min at 118°. Final pH, 7.4 ± 0.2. Alternatively, dissolve ingredients, omitting carbohydrate, in 800 ml distilled water with heat and occasional agitation. Dispense 2.0 ml portions into 13 x 100 mm test tubes containing inverted fermentation tubes. Autoclave 15 min at 118° and let cool. Dissolve carbohydrate in 200 ml distilled water and sterilize by passing solution through bacteria-retaining filter. Aseptically add 0.5 ml sterile filtrate to each tube of sterilized broth after cooling to less than 45°. Shake gently to mix. Final pH, 7.4 ±0.2.

For viable yeasts and moulds: Dispense 20-25 ml portions into sterile 15 x 100 mm petri dishes.

Plate Count Agar (Standard Methods)

Tryptone: 5 g
Yeast extract: 2.5 g
Dextrose: 1 g
Agar: 15 g
Distilled water: 1 litre

Heat to dissolve ingredients. Dispense into suitable tubes or flasks. Autoclave 15 min at 121°. Final pH 7.0 ± 0.2.

Potassium Cyanide (KCN) Broth

Potassium cyanide: 0.075 g
Proteose peptone No. 3 or polypeptone: 3 g
NaC1:5g
KH₂PO₄: 0.225 g
Na₂HPO₄: 5.64 g
Distilled water: 1 litre

Dissolve above ingredients except potassium cyanide and autoclave 15 min at 121°. Cool and refrigerate at 5-8°. Final pH, 7.6 ± 0.2. Prepare KCN stock solution by dissolving 0.5 g KCN in 100 ml sterile distilled water cooled to 5-8°C. Using bulb pipetter, add 15 ml cold KCN stock solution to 1 litre cold, sterile base. DO NOT PIPET BY MOUTH. Handle with gloves.

Mix and aseptically dispense 1.0-1.5 ml portions to 13 x 100 mm sterile tubes. Using aseptic technique, stopper tubes with No. 2 corks impregnated with paraffin. Prepare corks by boiling in paraffin about 5 min. Place corks in tubes so that paraffin does not flow into broth but forms a seal between rim of tubes and cork. Store tubes at 5-8° no longer than 2 weeks before use.

Potato Dextrose Agar

Potato infusion: 200 ml
Dextrose: 20 g
Agar: 20 g
Distilled water: 1 litre

To prepare potato infusion, boil 200 g sliced, unpeeled potatoes in 1 litre distilled water for 30 min. Filter through cheesecloth, saving effluent, which is potato infusion. Mix in other ingredients and boil to dissolve. Autoclave 15 min at 121°. Dispense 20-25 ml portions into sterile 15 x 100 mm petri dishes. Final pH, 5.6 ± 0.2. Medium should not be re-melted more than once.

For potato dextrose salt agar, prepare potato dextrose agar, as above, and add 75 g NaC1 per litre.

Purple Carbohydrate Broth

Proteose peptone No. 3 : 10 g
Beef extract (optional): 1 g
NaC1:5 g
Bromcresol purple: 0.02 g
Distilled water: 1 litre

Prepare as for phenol red carbohydrate broth (M109). Final pH, 6.8 ± 0.2.

Rappaport-Vassiliadis Medium

Tryptone: 5 g
NaCl: 8 g
KH₂PO₄: 1.6 g
Distilled water: 1 litre

Magnesium chloride solution

MgCl₂-6H₂O: 400 g
Distilled water: 1 litre

Malachite green oxalate solution

Malachite green oxalate: 0.4 g
Distilled water: 100 ml

To prepare the complete medium, combine 1000 ml broth base, 100 ml magnesium chloride solution, and 10 ml malachite green oxalate solution (total volume of complete medium is 1110 ml). Broth base must be prepared on same day that components are combined to make complete medium. Magnesium chloride solution may be stored in dark bottle at room temperature up to 1 year. To prepare solution, dissolve entire contents of MgCl₂.6H₂O from newly opened container according to formula, because this salt is very hygroscopic. Malachite green oxalate solution may be stored in dark bottle at room temperature up to 6 months. Merck analytically pure malachite green oxalate is recommended because other brands may not be equally effective. Dispense 10 ml volumes of complete medium into 16 x 150 mm test tubes. Autoclave 15 min at 115°. Final pH, 5.5 ± 0.2. Store in refrigerator and use within 1 month.

This medium must be made from its individual ingredients. Use of commercially available dehydrated media is not recommended. Users of this medium should be aware that there are formulations and incubation temperatures for this medium other than those recommended in this volume.

Selenite Cystine Broth

Medium 1

Tryptone or polypeptone: 5 g
Lactose: 4 g
Sodium selenite (NaHSeO₃): 4 g
Na₂HPO₄: 10 g
L-Cystine: 0.01 g
Distilled water: 1 litre

Heat to boiling to dissolve. Dispense 10 ml portions into sterile 16 x 150 mm test tubes. Heat 10 min in flowing steam. Do not autoclave. Final pH, 7.0 ± 0.2. The medium is not sterile. Use same day as prepared.

Medium 2 (North-Bartram modification)

Polypeptone: 5 g
Lactose: 4 g
Sodium selenite (NaHSeO₃): 4 g
Na₂HPO₄: 5.5 g
KH₂PO₄: 4.5 g
L-Cystine: 0.01 g
Distilled water : 1 litre

Heat with agitation to dissolve. Dispense 10 ml portions to sterile 16 x 150 mm test tubes. Heat 10 min in flowing steam. Do not autoclave. Use same day as prepared.

Simmons Citrate Agar

Sodium citrate-2H₂O: 2 g
NaC1:5 g
K₂HPO₄: 1 g
NH₄H₂PO₄: 1 g
MgSO₄: 0.2 g
Bromthymol blue: 0.08 g
Agar: 15 g
Distilled water: 1 litre

Heat gently with occasional agitation. Boil 1-2 min until agar dissolves. Fill 13 x 100 or 16 x 150 mm screw-cap tubes 1/3 full. Autoclave 15 min at 121°. Before medium solidifies, incline tubes to obtain 4-5 cm slants and 2-3 cm butts. Final pH, 6.8 ± 0.2.

Tetrathionate Broth

Polypeptone: 5 g
Bile salts: 1 g
Calcium carbonate: 10 g
Sodium thiosulfate-5H₂O: 30 g
Distilled water: 1 litre

Suspend ingredients in 1 litre distilled water, mix, and heat to boiling. (Precipitate will not dissolve completely.) Do not autoclave. Cool to less than 45°. Store at 5-8°. Final pH, 8.4 ± 0.2.

Toluidine Blue - DNA Agar

Deoxyribonucleic acid (DNA): 0.3 g
Agar: 10 g
Calcium chloride (anhydrous): 1.1 mg
NaCl: 10 g
Toluidine blue O: 83 mg
Tris(hydroxymethyl)aminomethane: 6.1 g
Distilled water: 1 litre

Dissolve the Tris(hydroxymethyl)aminomethane in 1 litre distilled water. Adjust the pH to 9.0. Add the remaining ingredients except the toluidine blue O and heat to boiling to dissolve.

Dissolve toluidine blue O in the medium. Dispense to rubber-stopped flasks. Sterilization is not necessary if used immediately. The sterile medium is stable at room temperature for 4 months and is satisfactory after several melting cycles.

Triple Sugar Iron (TSI) Agar

These two media are interchangeable for general use. For use with V. parahaemolyticus, add 25 g NaC1 per litre to either formula.

Suspend ingredients of Medium 1 in distilled water, mix thoroughly, and heat with occasional agitation. Boil about 1 min to dissolve ingredients. Fill 16 x 150 mm tubes 1/3 full and cap or plug to maintain aerobic conditions. Autoclave Medium 1 for 15 min at 118°. Prepare Medium 2 in the same manner as Medium 1, except autoclave 15 min at 121°. Before the media solidify, incline tubes to obtain 4-5 cm slant and 2-3 cm butt. Final pH, 7.3 ± 0.2 for Medium 1 and 7.4 ± 0.2 for Medium 2.

Trypticase (Tryptic) Soy Agar

Trypticase peptone: 15 g
Phytone peptone: 5 g
NaCl: 5 g
Agar: 15 g
Distilled water: 1 litre

Heat with agitation to dissolve agar. Boil 1 min. Dispense into suitable tubes or flasks. Autoclave 15 min at 121°. Final pH, 7.3 ± 0.2.

Trypticase (Tryptic) Soy Broth

Trypticase peptone: 17 g
Phytone peptone: 3 g NaCl: 5 g
K₂HPO₄: 2.5 g
Glucose: 2.5 g
Distilled water: 1 litre

Heat with gentle agitation to dissolve. Dispense 225 ml into 500 ml Erlenmeyer flasks. Autoclave 15 min at 121°C. Final pH, 7.3 ± 0.2. For trypticase soy broth without glucose, prepare as above, but omit 2.5 g glucose.

Trypticase (Tryptic) Soy Broth containing 10% NaCl and 1% Na pyruvate

Trypticase or tryptose (pancreatic digest of casein): 17 g
Phytone peptone (pancreatic or enzymatic digest of casein): 3 g
NaCl: 100 g
K₂HPO₄: 2.5 g
Dextrose: 2.5 g
Na pyruvate: 10 g
Distilled water: 1 litre

Adjust to pH7.3. Heat gently if necessary. Dispense 10 ml into 16 x 150 mm tubes. Autoclave 15 min at 121°. Final pH, 7.3 ± 0.2. Store up to one month at 4 ± 1°.

Trypticase (Tryptic) Soy Broth with ferrous sulphate

Trypticase peptone: 17 g
Phytone peptone: 3 g NaCl: 5 g
K₂HPO₄: 2.5 g
Glucose: 2.5 g
Ferrous sulphate: 35 mg
Distilled water: 1 litre

Heat with gentle agitation to dissolve. Dispense 225 ml into 500 ml Erlenmeyer flasks. Autoclave 15 min at 121°. Final pH, 7.3 ± 0.2.

Trypticase Soy-Tryptose Broth

Trypticase soy broth (commercial, dehydrated): 15 g
 Tryptose broth (commercial, dehydrated): 13.5 g
Yeast extract: 3 g
Distilled water: 1 litre

Dissolve ingredients in 1 litre water. Heat gently to dissolve. Dispense 5 ml portions into 16 x 150 mm test tubes. Autoclave 15 min at 121°. Final pH, 7.2 ± 0.2.

Tryptone Yeast Extract Agar

Tryptone: 10 g
Yeast extract: 1 g
Carbohydrate: 10 g (glucose and mannitol are the carbohydrates used for identification of Staphylococcus aureus)
Bromcresol purple: 0.04 g
Agar: 2 g
Distilled water: 1 litre

Dissolve agar with heat and gentle agitation. Adjust pH to 7.0 ± 0.2. Fill 16 x 125 mm tubes 2/3 full. Autoclave 20 min at 115°. Before use, steam medium 10-15 min. Solidify by placing tubes in ice water.

Tryptone (Tryptophane) Broth, 1%

Tryptone or trypticase: 10 g
Distilled water: 1 litre

Dissolve and dispense 5 ml portions into 16 x 125 or 16 x 150 mm test tubes. Autoclave 15 min at 121°. Final pH, 6.9 ± 0.2. For use with V. parahaemolyticus, add 30 gNaC1.

Tryptose Blood Agar Base

Tryptose: 10 g
Beef extract: 3 g
NaCl: 5 g
Agar: 15 g
Distilled water: 1 litre

Suspend ingredients in distilled water, mix thoroughly, and heat with occasional agitation. Boil about 1 min. Fill 16 x 150 mm tubes 1/3 full and cap or plug to maintain aerobic conditions. Autoclave 15 min at 121°. Before media solidify, incline tubes to obtain 4-5 cm slant and 2-3 cm butt.

Universal Preenrichment Broth

Tryptone: 5 g
Protease peptone: 5 g
KH₂PO₄: 15 g
Na₂HPO₄:7 g
NaCl: 5 g
Dextrose: 0.5 g
MgSO₄: 0.25 g
Ferric ammonium sulphate: 0.1 g
Sodium pyruvate: 0.2 g
Distilled water: 1 litre

Heat with gentle agitation to dissolve. Autoclave 15 min at 121°. Final pH, 6.3 ± 0.2.

Urea Broth

Urea: 20 g
Yeast extract: 0.1 g
K₂HPO₄ : 9.1 g
Na₂HPO₄: 9.5 g
Phenol red: 0.01 g
Distilled water: 1 litre

Dissolve ingredients in distilled water. Do not heat. Sterilize by filtration through 0.45 μm membrane. Aseptically dispense 1.5-3.0 ml portions to 13 x 100 mm sterile test tubes. Final pH, 6.8 ±0.2.

Urea Broth (Rapid)

Urea: 20 g
Yeast extract: 0.1 g
KH₂PO₄: 0.091 g
Na₂HPO₄: 0.095 g
Phenol red: 0.01 g
Distilled water: 1 litre

Prepare as for urea broth, above.

Violet Red Bile Agar (VRBA)

Yeast extract: 3 g
Peptone or gelysate: 7 g
NaCl: 5 g
Bile salts or bile salts No. 3: 1.5 g
Lactose: 10 g
Neutral red: 30 mg
Crystal violet: 2 mg
Agar: 15 g
Distilled water: 1 litre

Suspend ingredients in distilled water and let stand for a few min. Mix thoroughly and adjust to pH 7.4 ± 0.2. Heat with agitation and boil for 2 min. Do not sterilize. Before use, cool to 45° and use as a plating medium. After solidification, add a cover layer above the agar of approximately 3.0 to 4.0 ml to prevent surface growth and spreading of colonies.

Xylose Lysine Desoxycholate (XLD) Agar

Yeast extract: 3 g
Ferric ammonium citrate: 0.8 g
L-lysine: 5 g
Sodium thiosulfate: 6.8 g
Xylose: 3.75 g
NaC1: 5 g
Lactose: 7.5 g
Agar: 15 g
Sucrose: 7.5 g
Phenol red : 0.08 g
Sodium desoxycholate: 2.5 g
Distilled water: 1 litre

Heat with agitation just until medium boils. Do not overheat. Pour into plates when medium has cooled to 50°. Let dry about 2 h with covers partially removed. Then close plates. Final pH, 7.4 ± 0.2. Do not store more than 1 day.

REAGENTS

The following reagents are used in the Microbiological Analyses Section of this Volume and are referenced in that Section. The listing is alphabetical.

Brilliant green solution

Brilliant green dye, sterile: 0.1 g
Distilled water, sterile: 100 ml

On day of use, add 20 ml I2-KI solution and 10 ml brilliant green solution to 1 litre base. Re-suspend precipitate by gentle agitation and aseptically dispense 10 ml portions into 20 x 150 or 16 x 150 mm sterile test tubes. Do not heat medium after addition of I2-KI and dye solutions.

Bromcresol Purple Dye Solution (0.2%)

Bromcresol purple dye: 0.2 g
Sterile distilled water: 100 ml

Butterfield's Phosphate-Buffered Dilution Water

KH2PO4: 34 g
Distilled water: 500 ml

Adjust pH to 7.2 with 1 N NaOH. Bring volume to 1 liter with distilled water. Sterilize 15 min at 121°. Store in refrigerator.

Dilution blanks

Take 1.25 ml of above stock solution and bring volume to 1 litre with distilled water. Dispense into bottles to 90 or 99 ± 1 ml. Sterilize 15 min at 121°.

Cellulase Solution

Dissolve 1 g cellulase in 99 ml sterile distilled water. Filter sterilize through a 0.45 μm filter. Cellulase solution may be stored at 2-5° for 2 weeks.

Chlorine Solution (200 ppm)

Commercial bleach (5.25% sodium hypochlorite): 8 ml
Distilled water containing 1 g sodium dodecyl sulphate: 992 ml

Dissolve 1 g sodium dodecyl sulfate in 992 ml distilled water. Add 8 ml commercial bleach and mix well. Make immediately before use.

Ethanol Solution (70%)

Ethanol (95%): 700 ml
Distilled water: add to final volume of 950 ml

Formalinized Physiological Saline Solution

Formaldehyde solution (36-38%): 6 ml
NaCl: 8.5 g
Distilled water: 1 litre

Dissolve 8.5 g NaCl in 1 liter distilled water. Autoclave 15 min at 121°. Cool to room temperature. Add 6 ml formaldehyde solution. Do not autoclave after addition of formaldehyde.

Hydrochloride Solution (1 N)

HCl (concentrated): 89 ml
Distilled water to make 1 litre

Kovac's Reagent

p-Dimethylaminobenzaldehyde: 5 g
Amyl alcohol (normal only): 75 ml
HCl (concentrated): 25 ml

Dissolve p-dimethylaminobenzaldehyde in normal amyl alcohol. Slowly add HC1. Store at 4°. To test for indole, add 0.2-0.3 ml reagent to 5 ml of 24 h bacteria culture in tryptone broth. Dark red colour in surface layer is positive test for indole. For enteropathogenic E. coli, also test at 72 h if negative at 24 h.

Lysostaphin Solution

Dissolve 2.5 mg of lysostaphin in 0.02M phosphate-saline buffer containing 1 % NaCl.

Methyl Red Indicator

Methyl red: 0.1 g
Ethanol (95%): 300 ml
Distilled water to make 500 ml

Nonfat dry milk

Nonfat dry milk: 100g
Distilled water: 1 litre

For Salmonella:

Suspend 100 g dehydrated nonfat dry milk in 1 liter distilled water. Swirl until dissolved. Autoclave 15 min at 121°.

Papain

Papain: 5 g
Distilled water: 1 litre

Add papain to sterile, distilled water and swirl to dissolve completely. Dispense 100 ml portion into bottles.

Physiological Saline Solution Sterile (0.85%)

NaCl: 8.5 g
Distilled water: 1 litre

Dissolve 8.5 g NaCl in water. Autoclave 15 min at 121°. Cool to room temperature.

Potassium Hydroxide Solution (40%)

KOH: 40 g
Distilled water to make 100 ml

Sodium Hydroxide Solution (1 N)

NaOH: 40 g
Distilled water to make 1 litre

Voges-Proskauer (VP) Test Reagents

Solution 1

alpha-Naphthol: 5 g
Alcohol (absolute): 100 ml

Solution 2

Potassium hydroxide: 40 g
Distilled water to make 100 ml

Voges-Proskauer (VP) test. At room temperature, transfer 1 ml of 48 h culture to test tube and add 0.6 ml solution 1 and 0.2 ml solution 2. Shake after adding each solution. To intensify and speed reaction, add a few creatine crystals to mixture. Read results 4 h after adding reagents. Development of eosin pink colour is a positive.

BUFFER SOLUTIONS

Buffer Test Solutions

Buffer TS (pH 2)

Combine 11.90 ml of 0.2 M hydrochloric acid and 88.10 ml of 0.2 M potassium chloride, and dilute to 200 ml with water.

Buffer TS (pH 5)

Add 51.5 ml of 0.2 M disodium hydrogen phosphate to 48.5 ml of 0.1 M citric acid.

Buffer TS (pH 5.45)

Dissolve 1.8360 g of citric acid and 3.198 g of disodium hydrogen phosphate in carbon dioxide-free water to make 200 ml.

Buffer TS (pH 6.5)

Combine 50 ml of 0.2 M potassium dihydrogen phosphate and 15.2 ml of 0.2 M sodium hydroxide, and dilute to 200 ml with water.

Buffer acetate TS (pH 5.0)

Add 4.6 g of anhydrous sodium acetate to 11.6 ml of 2 M acetic acid and dilute to 200 ml with water. Adjust the pH to 5.0 ±0.1 with glacial acetic acid or 10% sodium hydroxide solution.

Barbital buffer solution (pH 7.6)

Dissolve 4.3 g of barbital sodium in 200 ml of water, adjust the pH to 7.6 with dilute hydrochloric acid, and filter.

Citric acid buffer solution

Dissolve 21 g of citric acid in water to make 1,000 ml (Solution A). Dissolve 28.4 g of disodium hydrogen phosphate in water to make 1,000 ml (Solution B). Combine 11 volumes of Solution A and 389 volumes of Solution B.

Formic acid buffer solution (pH 2.5)

Add 18 ml of water to 0.8 ml of formic acid, adjust the pH to 2.5 with strong ammonia TS, and dilute to 200 ml with water.

Phosphate buffer solution (pH 7.0)

Combine 50 ml of 0.2 M potassium dihydrogen phosphate and 29.54 ml of 0.2 M sodium hydroxide, and dilute to 200 ml with water.

Phosphate buffer solution (pH 7.3-7.4) (0.02M)

Stock solution 1:

Sodium phosphate dibasic anhydrous: 28.4 g
NaCl: 85 g
Distilled water: 1 litre

Stock solution 2:

Sodium phosphate monobasic monohydrate: 27.6 g
NaCl: 85 g
Distilled water: 1 litre

Make 1:10 dilutions of each stock solution. For example:

Using a pH meter, titer diluted solution1 to pH 7.3-7.4 by adding about 65 ml of solution 2. Use the resulting 0.02 M phosphate saline buffer solution in the lysostaphin susceptibility test on S. aureus.

Note: Do not titer 0.2 Mphosphate buffer to pH 7.3-7.4 and then dilute to 0.02 Mstrength. This results in a drop in pH of approximately 0.25. Addition of 0.85% salt after pH adjustment also results in a drop of approximately 0.2.

Phosphate buffer solution (pH 7.5)

Dissolve 53.7 g of disodium hydrogen phosphate in water to make 1,000 ml (Solution A). Dissolve 20.4 g of potassium dihydrogen phosphate in water to make 1,000 ml (Solution B). Combine 21 volumes of Solution A and 4 volumes of Solution B, and adjust the pH to 7.5 with either Solution A or Solution B.

Standard Buffer Solutions

Reagent Solutions

Previously dry the crystalline reagents (except for boric acid), at 110° to 120°, and use water that has been previously boiled and cooled to prepare the solutions. Store the prepared reagent solutions in chemically resistant glass or polyethylene bottles, and use within 3 months. Discard if moulding is evident.

• Boric acid/potassium chloride, 0.2 M

  Dissolve 12.366 g of boric acid (H₃BO₃) and 14.911 g of potassium chloride (KC1) in water to make 1,000 ml.

• Hydrochloric acid, 0.2 M

  Dilute 19 ml of hydrochloric acid with water to make 1,000 ml and standardize the solution as follows: dissolve about 0.3 g, accurately weighed, of primary standard anhydrous sodium carbonate (Na₂CO₃), previously dried at about 270° for 1 h in 100 ml of water. Titrate with the hydrochloric acid using 2 drops of methyl red TS. When the solution becomes faintly pink, boil to expel carbon dioxide, cool, and continue the titration until the faint pink colour is no longer affected by continued boiling. Each 10.60 mg of Na₂CO₃ is equivalent to 1 ml of 0.2 M hydrochloric acid.

• Potassium chloride, 0.2 M

  Dissolve 14.911 g of potassium chloride (KC1) in water to make 1,000 ml.

• Potassium hydrogen phthalate, 0.2 M

  Dissolve 40.844 g of potassium hydrogen phthalate [KHC6H4(COO)2] in water to make 1,000 ml.

• Potassium dihydrogen phosphate, 0.2 M

  Dissolve 27.218 g of potassium dihydrogen phosphate (KH2PO4) in water to make 1,000 ml.

• Sodium hydroxide, 0.2 M

  Dissolve about 9 g of sodium hydroxide (NaOH) in about 950 ml of water, and add a freshly prepared saturated solution of barium hydroxide until no more precipitate forms. Shake the mixture thoroughly, and allow it to stand overnight in a stoppered bottle. Decant or filter the solution, and standardize the clear liquid as follows: Dissolve about 1 g, accurately weighed, of primary standard potassium hydrogen phthalate [KHC₆H₄(COO)₂], previously dried at 105° for 3 h in 75 ml of carbon dioxide-free water, and titrate with the sodium hydroxide solution to a permanent pink colour using 2 drops of phenolphthalein TS, as indicator. Each 40.84 mg of KHC6H4(COO)2 is equivalent to 1 ml of 0.2 M sodium hydroxide.

Composition of Standard Buffer Solutions

To prepare a standard buffer solution having a pH within the range 1.2 to 10.0, combine the appropriate solutions, prepared above, as shown in the following table, and dilute with water to make 200 ml. The standard pH values given in this table are considered to be reproducible to within ± 0.02 of the pH unit specified at 25°.

STANDARD SOLUTIONS

Ammonium Standard Solution

Dissolve 296.0 mg of ammonium chloride, NH4C1, in sufficient water to make 100 ml. Transfer 10.0 ml of this solution into a 1,000-ml volumetric flask, dilute to volume with water. Each ml of this solution contains 0.01 mg of NH⁺₄.

Barium Standard Solution

Dissolve 177.9 mg of barium chloride, BaCl₂.2H₂0, in water in a 1,000-ml volumetric flask, dilute to volume with water, and mix. Each ml of this solution contains 0.1 mg of Ba.

Barium Chloride Standard Solution

Dissolve 4.3 g of barium chloride in sufficient water to make 1,000 ml. Perform gravimetric analysis on the solution, and calculate the quantity of sodium sulfate (Na₂SO₄) corresponding to 1 ml of the solution. Each ml of this solution corresponds to about 2.5 mg of Na₂SO₄.

Chromium Standard Solution

To 0.934 g of potassium chromate, add 1 drop of 10% sodium hydroxide solution and water to 1,000 ml. To a 1.0 ml portion of the solution, add 1 drop of 10% sodium hydroxide solution and water to 1,000 ml. Each ml of this solution contains 0.25 μg of Cr.

Condensed Formaldehyde Standard Solution

Dilute 8.1 g of formalin (containing 37% of HCHO) with water to 1,000 ml. To a 10.0 ml portion of the solution, add water to 1,000 ml. Each ml of this solution contains 0.03 mg of HCHO. Prepare freshly before use.

Dithizone Standard Solution

Dissolve 10 mg of dithizone in 1,000 ml of chloroform. Store in a stoppered bottle lead free and in a cold place.

Formaldehyde Standard Solution

Dilute 2.7 g of formalin (containing 37% of HCHO) with water to 1,000 ml. To a 10 ml portion of the solution, add water to 1,000 ml. Each ml of this solution contains 0.01 mg of HCHO. Prepare the solution fresh.

Iron Standard Solution

Dissolve 8.63 g of ferric ammonium sulfate in 20 ml of dilute nitric acid, and add water to 1,000 ml. To 10 ml of the solution add 20 ml of dilute nitric acid and water to 1,000 ml. Each ml of this solution contains 0.01 mg of Fe. Store in a dark bottle.

Lead Standard Solution

Dissolve 159.8 mg of lead nitrate in 10 ml of dilute nitric acid, and add water to 1,000 ml. Prepare and store this solution in lead-free glassware. Dilute 10 ml of the solution with water to 100 ml. Each ml of this solution contains 0.01 mg of Pb. Prepare the solution fresh.

Lead Standard Solution for Dithizone test

To 10 ml of lead standard solution, add 1% nitric acid to 100 ml. Each ml of this solution contains 1 μg of Pb. Prepare the solution fresh.

Magnesium Standard Solution

Dissolve 50.0 mg magnesium metal, Mg, in 1 ml of hydrochloric acid in a 1,000-ml volumetric flask, dilute to volume with water, and mix. Each ml of this solution contains 0.05 mg Mg.

Mercury Standard Solution

Dissolve 0.135 g of mercuric chloride in 10 ml of dilute nitric acid and sufficient water to make 1,000 ml. Dilute 10 ml of the solution with 10 ml of dilute nitric acid and water to make 1,000 ml. Dilute the second solution in same manner. Each ml of this final solution contains 0.1 μg of Hg in 1 ml. Prepare the solution fresh.

Methanol Standard Solution

To 5 ml of 0.1% methanol, add 2,5 ml of ethyl alcohol not containing methanol, and add water to 50 ml. Each ml of this solution contains 0.1 mg of CH₃OH.

Nitrate Standard Solution

Dissolve 1.63 g of potassium nitrate in water to make 1,000 ml. To a 10 ml portion of the solution, add water to 100 ml. Each ml of this solution contains 0.1 mg of NO₃.

Phosphate Standard Solution

Dissolve 143.3 mg of monobasic potassium phosphate, KH2PO4, in water in a 100 ml volumetric flask, dilute to volume with water, and mix. Transfer 10.0 ml of this solution into a 1,000-ml volumetric flask, dilute to volume with water, and mix. Each ml of this solution contains 10 μg phosphate.

Potassium Phosphate, Monobasic, Standard Solution

Dissolve 4.394 g of potassium phosphate monobasic in sufficient water to make 1,000 ml. Each ml of this solution contains 1 mg of phosphate.

Selenium Standard Solution

Add 10 ml of dilute sulfuric acid (1 in 2) to 1 g of selenium. Heat to dissolve, and evaporate to dryness on a water bath. Dissolve the residue in sufficient water to make 1,000 ml. To a 10

ml portion of the solution, add water to 1,000 ml. Each ml of this solution contains 0.01 mg of Se.

Thiamine Hydrochloride Standard Solution

Dissolve 0.1 g of vitamin B₁ hydrochloride reference standard previously dried at 105° for 2 h, in water to make 1,000 ml. To a 10 ml portion of the solution, add water to 1,000 ml. Each ml of this solution contains 1 μg of vitamin B₁ hydrochloride reference standard.

Zinc Standard Solution

Dissolve 4.4 g of zinc sulfate in water to make 1,000 ml. To a 10 ml portion of the solution, add water to 1,000 ml. Each ml of this solution contains 0.01 mg of Zn.

TEST SOLUTIONS

For the preparation of Test Solutions (TS), analytical grade reagents are to be used.

Certain of the following Test Solutions are intended for use as acid-base indicators in volumetric analyses. Such solutions should be adjusted so that when 0.15 ml of indicator solution is added to 25 ml of carbon dioxide-free water, 0.25 ml of 0.02 N acid or alkali, respectively, will produce the characteristic colour change.

The notation "PbT" indicates a lead-free solution.

In general, the directive to use a freshly prepared solution indicates that the solution is of limited stability and must be prepared on the day of use.

Acetic Acid TS

A solution containing approximately 30% w/v of CH3COOH in water (approximately 5N).

Acetic Acid TS, Dilute

A solution containing approximately 6% w/v of CH3COOH (approximately N).

Acetic Acid TS, Strong

See acetic acid TS.

Acetic Anhydride/Benzol TS

To 10 ml of acetic anhydride add sufficient benzol to make 100 ml.

Acetic Anhydride/Pyridine TS

To 25 g of acetic anhydride add sufficient dehydrated pyridine to make 100 ml. Prepare freshly before use.

Acetic Periodic Acid TS

Dissolve 5.4 g of periodic acid in 100 ml of distilled water and then add 1900 ml of glacial acetic acid and mix thoroughly. Store the solution in a dark glass-stoppered bottle or store in the dark in a clear glass-stoppered bottle.

Alcoholic Potassium Hydroxide TS

See potassium hydroxide TS, ethanolic.

Alizarin Yellow GG TS

Dissolve 0.1 g of alizarin yellow GG in 100 ml ethanol. Filter if necessary.

Alizarin Yellow GG/Thymolphthalein TS

Prepare by mixing 10 ml of alizarin yellow GG TS with 20 ml of thymolphthalein TS.

Alkaline Cupric Tartrate TS

(Fehling's TS). See cupric tartrate TS, alkaline.

Alkaline Mercuric-Potassium Iodide TS

(Nessler's TS). See mercuric-potassium iodide TS, alkaline.

Alkaline Tartrate Solution TS

See tartrate solution TS, alkaline.

1-Amino-2-Naphthol-4-Sulfonic Acid TS

Dissolve 0.2 g of 1-amino-2-naphthol-4-sulfonic acid in 195 ml of sodium bisulfite solution (3 in 20) and 5 ml of anhydrous sodium sulfite solution (1 in 5), and filter if necessary. Stopper tightly, and store in a dark, cold place. Use within 10 days of preparation.

Ammonia TS

A solution containing between 9.5% and 10.5% of NH3 (approximately 6 N). Prepare by diluting 400 ml of ammonium hydroxide (28%) with sufficient water to make 1,000 ml.

Ammonia TS, Dilute

See ammonia TS.

Ammonia TS, Strong

A solution containing approximately 25% w/v of NH3 in water (approximately 15 N).

Ammonia TS, Dilute (PbT)

Dilute ammonia TS, which complies with the following test: to 20 ml of ammonia TS add 1 ml of potassium cyanide TS (PbT), dilute to 50 ml with water and add 2 drops of sodium sulfide TS (PbT); no darkening should be produced.

Ammonia TS, Ethanolic

A 9 to 11 % w/v solution of NH3 in ethanol. A transparent, colourless liquid having a strong odour of ammonia. Specific gravity is about 0.80. Store in a rubber-stoppered container and in a cold place.

Ammonia/Ammonium Chloride Buffer TS

(Approx. pH 10). Dissolve 67.5 g of ammonium chloride (NH4C1) in water, add 570 ml of ammonium hydroxide (28%) and dilute with water to 1,000 ml.

Ammoniacal Silver Nitrate TS

Add ammonia TS, dropwise, to a 1 in 20 solution of silver nitrate until the precipitate that first forms is almost, but not entirely, dissolved. Filter the solution, and store in a dark bottle.

(Note: Ammoniacal silver nitrate TS forms explosive compounds on standing. Do not store this solution, but prepare a fresh quantity for each series of determination. Neutralize the excess reagent and rinse all glassware with hydrochloric acid immediately after completing a test.)

Ammonium Acetate TS

A 10% w/v solution of ammonium acetate (CH3COONH4) in water.

Ammonium Acetate Citrate TS (PbT)

Dissolve 12.5 g of ammonium acetate (CH3COONH4) and 12.5 g of ammonium citrate [C3H4OH(COOH)(COONH4)2] in water, add strong ammonia TS until the solution is alkaline to thymol blue paper and add water to 100 ml. Purify with a 0.002% w/v solution of dithizone in chloroform, and finally shake the solution with chloroform to remove excess of dithizone.

Ammonium Carbonate TS

Dissolve 20 g of ammonium carbonate and 20 ml of ammonia TS in sufficient water to make 100 ml.

Ammonium Chloride TS

10.5% w/v of ammonium chloride in water (approximately 2 N).

Ammonium Chloride/Ammonium Hydroxide TS

Mix equal volumes of water and strong ammonia TS, and saturate with ammonium chloride.

Ammonium Citrate TS (PbT)

Dissolve 40 g of citric acid in 90 ml of water. Add 2 or 3 drops of phenol red TS, then cautiously add strong ammonia TS until the solution acquires a reddish colour. Remove any lead that may be present by extracting the solution with 20-ml portions of dithizone extraction TS until the dithizone solution retains its orange-green colour.

Ammonium Molybdate TS

Dissolve 6.5 g of finely powdered molybdic acid (85%) in a mixture of 14 ml of water and 14.5 ml of strong ammonia TS. Cool the solution, and add it slowly, with stirring, to a well-cooled mixture of 32 ml of nitric acid and 40 ml of water. Allow to stand for 48 h, and filter through glass wool. This solution deteriorates upon standing and is unsuitable for use if, upon the addition of 2 ml of sodium phosphate TS to 5 ml of the solution, an abundant yellow precipitate does not form at once or after slight warming. Store it in the dark. If a precipitate forms during storage use only the clear, supernatant solution.

Ammonium Molybdate/Sulfuric Acid TS

Dissolve 18.8 g of ammonium molybdate in 300 ml of water, and add 150 ml of sulfuric acid and sufficient water to make 500 ml.

Ammonium Oxalate TS

A 3.0% w/v solution of ammonium oxalate [(COONH4)2] in water (approximately 0.5 N).

Ammonium Sulfanilate TS

To 2.5 g of sulfanilic acid add 15 ml of water and 3 ml of ammonia TS and mix. If necessary, add with stirring, more ammonia TS, until the acid dissolves. Adjust the pH of the solution to about 4.5 with dilute hydrochloric acid TS, using bromocresol green TS as an outside indicator, and dilute to 25 ml.

Ammonium Sulfide TS

Saturate ammonia TS with hydrogen sulfide (H2S), and add two-thirds of its volume of ammonia TS. Residue on ignition: not more than 0.05%. The solution is not rendered turbid either by magnesium sulfate TS or by calcium chloride TS (carbonate). This solution is unsuitable for use if an abundant precipitate of sulfur is present. Store it in a small, well-filled, dark amber-coloured bottle, in a cold, dark place.

Ammonium Thiocyanate TS

A 7.6% w/v solution of ammonium thiocyanate (NH4SCN) in water (approximately N).

Ammonium Thiocyanate/Cobalt Nitrate TS

Dissolve 17.4 g of ammonium thiocyanate and 2.8 g of cobalt nitrate in sufficient water to make 100 ml.

Amylase TS

To 0.2 g of amylase (crystal), add 100 ml of water, shake well and filter. Prepare freshly before use.

Anthrone TS

Dissolve about 0.1 g of anthrone in 100 g of sulfuric acid. Prepare freshly before use.

Antimony TS, Standard

Dissolve 2.742 g of antimony potassium tartrate in water, and dilute to 100 ml; dilute 5 ml of this solution to 500 ml with water. Each ml of the solution contains 0.001 mg of Sb.

Antimony Trichloride TS

Wash the surface of antimony trichloride with anhydrous chloroform until the washings become transparent. Add anhydrous chloroform to antimony trichloride to make a saturated solution. Store in a tight container shaded from light and in a cold place. Prepare freshly before use.

Arsenic TS, Dilute

Mix 1 ml of strong arsenic TS with sufficient water to produce 100 ml. The dilute solution of arsenic must be freshly prepared. 1 ml contains 0.01 mg of arsenic.

Arsenic TS, Strong

Dissolve 0.132 g of arsenic trioxide in 50 ml of hydrochloric acid (a 25% w/v solution of HC1 in water) and add sufficient water to 100 ml.

Arsenous Acid TS

Dissolve 1 g of arsenous acid in 30 ml of sodium hydroxide solution (1 in 40), and heat. Cool, and slowly add sufficient glacial acetic acid to 100 ml.

Barium Chloride TS

A 12% w/v solution of barium chloride (BaCl₂.2H₂O) in water (approximately N).

Barium Diphenylamine Sulfonate TS

A 0.3% w/v solution of p-diphenylamine sulfonic acid barium salt in water.

Benedict's Qualitative Reagent

See cupric citrate TS, alkaline.

Benzidine TS

Dissolve 50 mg of benzidine in 10 ml of glacial acetic acid, dilute to 100 ml with water and mix. (Caution: benzidine is toxic.)

Bertrand's TS, A

Dissolve 40 g of fine cupric sulfate in sufficient water to make 1,000 ml. Fill a glass-stoppered container almost to the top, and store.

Bertrand's TS, B

Dissolve 200 g of potassium sodium tartrate and 150 g of sodium hydroxide in sufficient water to make 1,000 ml. Store in a rubber-stoppered container.

Bertrand's TS, C

Dissolve 50 g of ferric sulfate (shall not reduce potassium permanganate solution) in sufficient water. Add 200 ml of sulfuric acid, and add sufficient water to make 1,000 ml.

Bertrand's TS, D

Dissolve 5 g of potassium permanganate in sufficient water to make 1,000 ml.

(Standardization: Dissolve 0.25 g of ammonium oxalate in 100 ml of water, and add 2 ml of sulfuric acid. Heat this solution to the temperature of 60° to 70°, and titrate with Bertrand's TS, D. If the volume of Bertrand's TS, D consumed is designated as a ml, each 1 ml of Bertrand's TS, D is equivalent to (0.2238/a)g of Cu).

2,2'-Bipyridine TS

Dissolve 0.100 g of 2,2'-bipyridine in 50 ml of purified absolute ethanol TS. 

Bismuth Nitrate TS (I)

Reflux 5 g of bismuth nitrate (Bi(NO₃)₃.5H₂O), in 7.5 ml of nitric acid and 10 ml of water until dissolved, cool, filter and dilute the solution to 250 ml.

Bismuth Nitrate TS (II)

Dissolve 5 g of bismuth nitrate (Bi(NO₃)₃.5H₂O) in 25 ml of water and 25 ml of glacial acetic acid and dilute to 250 ml.

Borax Buffer (PbT)

Dissolve 3.0 g of borax in 90 ml of water, and extract with successive portions, each of 5 ml of 1 volume of diphenylthiocarbazone solution PbT and 4 volumes of chloroform, with vigorous shaking, until the extract is blue or purple in colour; continue the extraction with successive portions, each of 10 ml of chloroform, until the extract is colourless; reject the extracts, and dilute the solution to 100 ml with water.

Boric Acid TS

Dissolve 5 g boric acid in 500 ml distilled water in a 1,000-ml measuring flask. Add 25 ml alcoholic indicator solution (67 mg methyl red and 33 mg bromocresol green in 100 ml 96% ethanol) and 200 ml ethanol. Make up to volume with distilled water. The boric acid indicator solution is red. 5 ml must turn green with not more than 3 drops of 0.01 N NaOH.

Bromide/Bromate TS

(About 0.1 N bromine) (7.991 g Br per litre). Dissolve 3 g of potassium bromate (KBrO₃) and 15 g of potassium bromide (KBr) in sufficient water to make 1,000 ml and standardize the solution as follows: transfer about 25 ml of the solution, accurately measured, into a 500-ml iodine flask and dilute with 120 ml of water. Add 5 ml of hydrochloric acid, stopper the flask and shake it gently. Then add 5 ml of potassium iodide TS, re-stopper, shake the mixture, allow it to stand for 5 min and titrate the liberated iodine with 0.1 N sodium thiosulfate, adding starch TS near the end of the titration. Calculate the normality. Store this solution in a dark amber coloured, glass-stoppered bottle.

Bromine TS

(Bromine water). A saturated solution of bromine, prepared by agitating 2 to 3 ml of bromine (Br2) with 100 ml of cold water in a glass-stoppered bottled, the stopper of which should be lubricated with petrolatum. Store it in a cold place, protected from light.

Bromine/Acetic Acid TS

Dissolve 5 ml of bromine in 145 ml of potassium acetate in acetic acid TS. Prepare this solution fresh daily.

Bromine/Bromide TS

Add 1 ml of bromine to 300 ml of glacial acetic acid saturated with dry potassium bromide (5 g). 15 ml of this solution require about 50 ml of 0.05 N sodium thiosulfate. This solution is stored in a dark bottle and kept in the dark. It is standardized at least once a day during use.

Bromine/Glacial Acetic Acid TS

Dissolve about 1.5 g of bromine in sufficient glacial acetic acid to make about 100 ml. Each 1 ml of this solution is equivalent to about 2 ml of 0.1 N sodium thiosulfate.

Bromine/Hydrochloric Acid TS

Mix 1 ml of bromine/potassium bromide TS with 100 ml of hydrochloric acid, arsenic-free.

Bromine/Potassium Bromide TS

Dissolve 30 g of bromine and 30 g of potassium bromide in sufficient water to make 100 ml.

Bromocresol Blue TS

Use bromocresol green TS.

Bromocresol Green TS

Dissolve 0.05 g of bromocresol green in 100 ml of ethanol, and filter if necessary. For pH determinations, dissolve 0.05 g in 1.4 ml of 0,05 N sodium hydroxide, and dilute with carbon dioxide-free water to 100 ml.

Bromocresol Green/Methyl Red TS

Mix equal volumes of bromocresol green TS and methyl red TS.

Bromocresol Purple TS

Dissolve 0.25 g of bromocresol purple in 20 ml of 0.05 N sodium hydroxide, and dilute with water to 250 ml.

Bromophenol Blue TS

Dissolve 0.1 g of bromophenol blue in 100 ml of dilute ethanol (1 in 2), and filter if necessary. For pH determinations, dissolve 0.1 g in 3.0 ml of 0.05 N sodium hydroxide, and dilute with carbon dioxide-free water to 200 ml.

Bromophenol Blue TS

(For citric acid). Mix bromophenol blue TS with equal volume of ethanol, adjust pH to 7.0 by adding 0.01 N sodium hydroxide solution.

Bromophenol Blue/Sodium Hydroxide TS

Dissolve 0.1 g of bromophenol blue in 3 ml of 0.05 N sodium hydroxide by mixing well, and add sufficient water to 25 ml.

Bromothymol Blue TS

Dissolve 0.1 g of bromothymol blue in 100 ml of dilute ethanol (1 in 2), and filter if necessary. For pH determinations, dissolve 0.1 g in 3.2 ml of 0.05 N sodium hydroxide, and dilute with carbon dioxide-free water to 200 ml.

Buffer TS

See Buffer Test Solutions.

Calcium Chloride TS

A 7.5% w/v solution of calcium chloride (CaCl₂.2H₂O) in water (approximately N).

Calcium Hydroxide TS

A solution containing approximately 0.14 g of Ca(OH)2 in each 100 ml. To prepare, add 3 g of calcium hydroxide [Ca(OH)2] to 1,000 ml of water, and agitate the mixture vigorously and repeatedly during 1 h. Allow the excess calcium hydroxide to settle, and decant or draw off the clear supernatant liquid.

Carr-Price TS

Weigh an unopened (100 g) bottle of antimony trichloride. Open the bottle and empty the contents into a wide-mouthed, glass-stoppered amber bottle containing approximately 100 ml of chloroform. By difference, obtain the weight of antimony trichloride and then add sufficient chloroform to supply 100 ml for each 25 g. Dissolve by warming or shaking for several hours and filter through sodium sulfate into a clean, dry, amber bottle with ground glass stopper. This solution may be stored at room temperature but should be kept in the dark when not in use. The reagent is apparently stable for long periods of time, but it is convenient to make up sufficient amounts to last for one month. Rinse all glassware coming in contact with this reagent with chloroform, a mixture of ethanol and ether or dilute or concentrated hydrochloric acid before washing, since the antimony oxychloride which forms is insoluble in water.

Ceric Ammonium Nitrate TS

Dissolve 6.25 g of ceric ammonium nitrate [(NH₄)₂Ce(NO₃)₆] in 100 ml of 0.25 N nitric acid. Prepare the solution fresh every third day.

Chloral Hydrate TS

Dissolve 50 g of chloral hydrate in a mixture of 15 ml of water and 10 ml of glycerol.

Chlorine TS

(Chlorine water). A saturated solution of chlorine in water. Place the solution in a small, completely filled, light-resistant container. Chlorine TS, even when kept from light and air, is apt to deteriorate. Store it in a cold, dark place. For full strength, prepare this solution fresh.

Chromate TS, Standard

Dissolve 0.0566 g potassium dichromate (K₂Cr₂O₇) in 1,000 ml of water. Each ml contains 0.02mgofCr.

Chromic Acid TS

See Chromium trioxide TS.

Chromium Trioxide TS

A 3% w/v solution of chromium trioxide in water.

Chromotropic Acid TS

Dissolve 2.0 g of chromotropic acid (4,5-dihydroxy-2,7-naphthalene-disulfonic acid, disodium salt) in 40 ml of water in a 1 -litre volumetric flask. Dilute to volume with 15 M sulfuric acid.

Citric Acid Buffer Solution

See standard buffer solutions.

Cobaltous Chloride TS

Dissolve 2 g of cobaltous chloride (C0CI₂.6H₂O) in 1 ml of hydrochloric acid and sufficient water to make 100 ml.

Cobaltous Chloride TSC

Dissolve about 65 g cobaltous chloride (C0CI₂6H₂O) in enough of a mixture of 25 ml of hydrochloric acid and 975 ml of water to make 1,000 ml. Place exactly 5 ml of this solution in a 250-ml iodine flask, add 5 ml of hydrogen peroxide TS and 15 ml of 20% sodium hydroxide solution. Boil for 10 min, cool, and add 2 g of potassium iodide and 20 ml of 25% sulfuric acid. When the precipitate has dissolved, titrate the liberated iodine with 0.1 N sodium thiosulfate, using starch TS as indicator. Each ml of 0.1 N sodium thiosulfate is equivalent to 23.8 mg of C0CI₂6H₂O). Adjust the final volume of the solution by adding enough of the hydrochloric acid and water mixture so that each ml contain 59.5 mg of CoCl₂-6H₂O.

Cobalt-Uranyl Acetate TS

Dissolve, with warming, 40 g of uranyl acetate [UO₂(C₂H₃O₂)₂.2H₂O] in a mixture of 30 g glacial acetic acid and sufficient water to make 500 ml. Similarly, prepare a solution containing 200 g of cobaltous acetate [Co(C₂H₃O₂)₂.4H₂O] in a mixture of 30 g of glacial acetic acid and sufficient water to make 500 ml. Mix the two solutions while still warm, and cool to 20°. Maintain the temperature at 20° for about 2 h to separate the excess salts from solution, and then filter through a dry filter.

Congo Red TS

Dissolve 0.10 g of congo red (sodium diphenyl-diazo-bis-alpha-naphthylaminesulfonate) (C₃2H22N6O6S₂Na2) in 20 ml of 90% ethanol and add sufficient water to make 100 ml.

Copper Sulfate Solution TS

Dissolve 34.639 g of CUSO₄5H₂O in water, dilute to 500 ml, and filter through glass wool or paper. Determine the Cu content of the solution (preferably by electrolysis), and adjust the content to 440.9 mg Cu/25 ml.

Cresol Red TS

Triturate 0.10 g of cresol red in a mortar with 26.2 ml of 0.01 N sodium hydroxide until solution is complete, then dilute the solution with water to 250 ml.

Cresol Red/Thymol Blue TS

Add 15 ml of thymol blue TS to 5 ml of cresol red TS, and mix.

Crystal Violet TS

A 1 % solution of methyl violet (methyl-rosaniline chloride; crystal violet) in glacial acetic acid.

Cupric Acetate TS, Strong

Dissolve 13.3 g of cupric acetate in 5 ml of acetic acid and 195 ml of water.

Cupric Citrate TS, Alkaline

(Benedict's qualitative reagent). With the aid of heat, dissolve 173 g of sodium citrate (C₆H₅Na₃O₇.2H₂O) and 117 g of sodium carbonate (Na₂CO₃.H₂O) in about 700 ml of water, and filter through paper, if necessary. In a separate container dissolve 17.3 g of cupric sulfate (CuSO₄.5H₂O) in about 100 ml of water, and slowly add this solution, with constant stirring, to the first solution. Cool the mixture, dilute to 100 ml, and mix.

Cupric Nitrate TS

A 2.4% w/v solution of cupric nitrate [Cu(NO₃)₂.3H₂O] in water.

Cupric Sulfate TS

A 12.5% w/v solution of cupric sulfate (CuSO₄5H₂O) in water.

Cupric Sulfate TSC

Dissolve about 65 g of cupric sulfate (CuSO₄5H₂O) in enough of a mixture of 25 ml of hydrochloric acid and 975 ml of water to make 1,000 ml. Pipet 10.0 ml of this solution in a 250-ml iodine flask, add 40 ml of water, 4 ml of acetic acid, and 3 g of potassium iodide. Titrate the liberated iodine with 0.1 N sodium thiosulfate, using starch TS as indicator. Each ml of 0.1 N sodium thiosulfate is equivalent to 24.97 mg of CUSO45H2O. Adjust the final volume of the solution by adding enough of the mixture of hydrochloric acid and water to make each ml contain 62.4 mg of CuSO₄5H₂O.

Cupric Sulfate/Ammonia TS

Dissolve 0.4 g of cupric sulfate in 50 ml mixture of ammonia TS and solution of citric acid (1 in 5) in the ratio of 2:3.

Cupric Tartrate TS, Alkaline

(Fehling's TS). [The Copper Solution (A)]. Dissolve 34.66 g of carefully selected, small crystals of cupric sulfate (CuSO₄.5H₂O) showing no trace of efflorescence or of adhering moisture, in sufficient water to make 500 ml. Store this solution in a small, tight container. [The Alkaline Tartrate Solution (B)]. Dissolve 173 g of crystallized potassium sodium tartrate (KNaC₄H₄O₆.4H₂O) and 50 g of sodium hydroxide (NaOH) in sufficient water to make 500 ml. Store this solution in a small, alkali-resistant container. For use, mix exactly equal volumes of Solutions A and B at the time required.

Cyanogen Bromide TS

Dissolve 5 g of cyanogen bromide in water to make 50 ml. (Caution: Prepare this solution under a hood, as cyanogen bromide volatilizes at room temperature and the vapour is highly irritating and poisonous).

Denigès' Reagent

See mercuric sulfate TS.

4,4'-Diaminodiphenylamine TS

Triturate 4,4'-diaminodiphenylamine sulfate with a small amount of ethanol, and add ethanol again. Transfer this solution to a flask connected to a reflux condenser, heat on a water bath, and prepare a saturated solution.

Di-ß-Naphthylthiocarbazone/Chloroform TS

Add 0.1 g of di-ß-naphthylthiocarbazone to 100 ml of carbon tetra-chloride. Dilute this solution 1:40 with chloroform.

2,6-Dichlorophenol-Indophenol TS

Warm 0.1 g of 2,6-dichlorophenol-indophenol sodium (C12H6C12NNaO2) with 100 ml of water and filter. The solution must be used within 3 days of preparation.

2,7-Dihydroxynaphthalene TS

Dissolve 0.1 g of 2,7-dihydroxynaphthalene in 1,000 ml of sulfuric acid and allow the solution to stand until the initial yellow colour disappears. If the solution is very dark, discard it and prepare a new solution from a different supply of sulfuric acid. This solution is stable for approximately one month if stored in a dark bottle.

p-Dimethylaminobenzaldehyde TS

Dissolve 0.125 g of p-dimethylaminobenzaldehyde in a cooled mixture of 65 ml of sulfuric acid and 35 ml of water, and add 0.05 ml of ferric chloride TS. Use within 7 days after preparation.

Dimethylglyoxime TS

A 1 % w/v solution of dimethylglyoxime in ethanol.

2,4-Dinitrophenylhydrazine TS

Dissolve 0.2 g of 2,4-dinitrophenylhydrazine in 100 ml of 85% sulfuric acid. Filter through a glass-filter (G3) if necessary. Store in a light-shaded bottle and in a dark, cold place. Use within 2 weeks of preparation.

Diphenylamine TS

A 1 % w/v solution of diphenylamine [(C₆H₅)₂NH] in sulfuric acid. The solution should be colourless.

Diphenylcarbazide TS

Dissolve 0.125 g of diphenylcarbazide [(C₆H₅.NH.NH)₂CO] in a mixture of 25 ml acetone and 25 ml water. To be prepared immediately before use.

Diphenylcarbazone TS

An approximately 1% w/v solution of diphenylcarbazone (C₁₃H₁₂N₄O) in ethanol. Store this solution in a brown bottle.

Diphenylthiocarbazone Solution (PbT)

Extract 15 ml of a 0.1% w/v solution of diphenylthiocarbazone (C₆H₄.N:N-CS-NH-NH-C₆H₅) in chloroform, with 2 successive portions, each of 50 ml of water containing 5 ml of dilute ammonia TS; acidify the combined extracts with dilute hydrochloric acid PbT, and extract with 100 ml of chloroform; wash the extract with 2 successive portions, each of 10 ml of water, and filter through a dry filter. Determine the approximate strength of this solution by the method for determination of zinc (see titanium dioxide monograph), using 5 ml of standard zinc sulfate TS diluted to 25 ml with water in place of the 25 ml of acid solution used in the determination; dilute with chloroform so that 3 ml is approximately equivalent to each ml of standard zinc sulfate TS. This solution must be freshly prepared.

oua-Dipyridyl TS

A 0.2% w/v solution of alpha,alpha-dipyridyl (C10H8N2) in absolute ethanol.

Dithizone TS

Dissolve 25.6 mg of dithizone in 100 ml of ethanol.

Dithizone TS, Extraction

Dissolve 30 mg of dithizone in 1,000 ml of chloroform, and add 5 ml of ethanol. Store the solution in a refrigerator. Before use shake a suitable volume of the dithizone extraction solution with about half its volume of 1 % nitric acid, discarding the nitric acid. Do not use more than 1 month old.

Dithizone TS, Standard

Dissolve 10 mg of dithizone in 1,000 ml of chloroform. Keep the solution in a glass- stoppered, lead-free bottle, suitably wrapped to protect it from light, and store in a refrigerator.

Dragendorff TS

Solution 1: Weigh 0.85 g of basic bismuth nitrate, and dissolve in 10 ml of acetic acid and 40 ml of water.

Solution 2: Weigh 8 g of potassium iodide, and dissolve in 20 ml of water. Mix 5 ml of Solution 1, 5 ml of Solution 2, 20 ml of acetic acid, and 100 ml of water before use.

Eosin Y TS

(Adsorption indicator) A 0.5% solution of eosin Y in water.

Eriochrome Black TS

Dissolve 0.2 g of eriochrome black T and 2 g of hydroxylamine hydrochloride (NH₂OH.HC1) in sufficient methanol to make 50 ml, and filter. Store the solution in a light-resistant container and use within 2 weeks.

Ethanol, Aldehyde-free

To 1,000 ml of ethanol, add 5 ml of sulfuric acid and 20 ml of water, and distil. Add 10 g of silver nitrate and 1 g of potassium hydroxide to a 1,000 ml portion of this distillate, boil for 3 h by connecting a reflux condenser, and recover the ethanol by distillation.

Ethanol TS, Purified Absolute

Add about 0.1 % potassium permanganate and 0.1 % potassium hydroxide to absolute ethanol and distil in an all-glass apparatus.

Ethanol TS, 72%

Mix 360 ml of purified absolute ethanol TS with 150 ml of water.

Ethanolic Potassium Hydroxide TS

See potassium hydroxide TS, ethanolic.

p-Ethoxychrysoidin TS

Dissolve 50 mg of p-ethoxychrysoidin monohydrochloride in a mixture of 25 ml of water and 25 ml of ethanol, add 3 drops of hydrochloric acid, stir vigorously, and filter if necessary to obtain a clear solution.

Fehling's TS

See cupric tartrate TS, alkaline.

Ferric Ammonium Sulfate TS

An 8% w/v solution of ferric ammonium sulfate [FeNH₄(SO₄)₂.12H₂O] in water.

Ferric Ammonium Sulfate/Hydrochloric Acid TS

A 0.1% w/v solution of ferric ammonium sulfate [FeNH₄(SO₄)₂.12H₂O] in hydrochloric acid.

Ferric Chloride TS

A 9% w/v solution of ferric chloride (FeCl₃.6H₂O) in water (approximately N).

Ferric Chloride TSC

Dissolve about 55 g of ferric chloride (FeCl₃.6H₂O) in sufficient of a mixture of 25 ml of hydrochloric acid and 975 ml of water to make 1,000 ml. Pipet 10 ml of this solution in a 250-ml iodine flask, add 15 ml of water and 3 g of potassium iodide, and allow the mixture to stand for 15 min. Dilute with 100 ml of water, and titrate the liberated iodine with 0.1 N sodium thiosulfate, adding starch TS. Each ml of 0.1 N sodium thiosulfate is equivalent to 27.03 mg of FeCl₃.6H₂O. Adjust the final volume of the solution by the addition of enough of the mixture of hydrochloric acid and water, so that each ml contains 45.0 mg of FeCl₃.6H₂O.

Ferric Chloride TS, Ethanolic

A 0.2% w/v solution of ferric chloride (FeCl₃.6H₂O) in absolute ethanol. Prepare this solution fresh.

Ferric Chloride TS, Dilute

To 2 ml of ferric chloride TS, add sufficient water to make 100 ml. Prepare freshly before use.

Ferric Chloride/Hydrochloric Acid TS

Dissolve 5 g of ferric chloride (FeCl₃.6H₂O) in 5 ml of hydrochloric acid and sufficient water to make 100 ml.

Ferric Sulfate TS

Add 500 ml of water to 50 g of ferric sulfate, and mix thoroughly. To this mixture, add 200 ml of sulfuric acid, dissolve by shaking well, and add sufficient water to make 1,000 ml.

Ferric Sulfate TS, Acid

Add 7.5 ml of sulfuric acid to 100 ml of water, and dissolve 80 g of ferrous sulfate in the mixture with the aid of heat. Mix 7.5 ml of nitric acid and 20 ml of water, warm, and add to this the ferrous sulfate solution. Concentrate the mixture until, with the sudden emission of a red coloured vapour, the black colour of the liquid changes to red. Test for the absence of ferrous iron, and, if necessary, add a few drops of nitric acid and boil again. When the solution is cold, add sufficient water to make 110 ml.

Ferrous Sulfate TS

Dissolve 8 g of clear crystals of ferrous sulfate (FeSO₄.7H₂O) in about 100 ml of recently boiled and thoroughly cooled water. Prepare this solution fresh.

Ferrous Sulfate TS, Acid

Dissolve 7 g of ferrous sulfate crystals in 90 ml of recently boiled and thoroughly cooled water, and add sufficient sulfuric acid to make 100 ml. Standardize frequently with 0.1 N potassium permanganate (approximately 0.25 N).

Fluorescein TS

A 0.1% w/v solution of sodium fluorescein in 50% ethanol.

Folin-Ciocalteu TS

Into a 150-ml flask introduce 10 g of sodium tungstate (Na₂WO₄.2H₂O), 2.5 g of sodium molybdate (Na₂MoO₄.2H₂O), 70 ml of water, 5 ml of phosphoric acid, and 10 ml of hydrochloric acid. Reflux the mixture gently for about 10 h, and add 15 g of lithium sulfate (Li₂SO₄.H₂O), 50 ml of water, and few drops of bromine. Boil the mixture, without the condenser, for 15 min or until the excess bromine is expelled. Cool, dilute with water to 100 ml, and filter. The filtrate has no greenish tint. Before use, dilute 1 part of filtrate with 1 part of water.

Formaldehyde TS

A solution containing approximately 37.0% w/v of HCHO. It may contain methanol to prevent polymerization.

Formalin/Sulfuric Acid TS

Mix 0.2 ml of formaldehyde TS with 10 ml of sulfuric acid. Prepare freshly before use.

Fuchsin/Sulfurous Acid TS

Dissolve 0.5 g of basic fuchsin in 300 ml of hot water, and cool. Add a solution of 5 g anhydrous sodium sulfite dissolved in 50 ml of water while stirring, and add 5 ml of hydrochloric acid with shaking. Dilute with water to 500 ml, and allow to stand for 5 h. Store in a light-shaded bottle, and in a cold place.

Hydriodic Acid TS

Distil hydriodic acid over red phosphorus, passing carbon dioxide through the apparatus during the distillation. The constant-boiling mixture distilling over a 126°-127°, which is colourless or nearly colourless, is used. Place the acid in a small, brown, glass-stoppered bottle previously swept out with carbon dioxide, seal with paraffin, and store in a cool, dark place.

Hydrochloric Acid TS, Brominated

Mix 1 ml of solution of bromine with 100 ml of hydrochloric acid.

Hydrochloric Acid TS, Dilute

A solution containing 10% w/v of HC1. Prepare by diluting 266 ml of hydrochloric acid (36%) with sufficient water to make 1,000 ml.

Hydrochloric Acid, Dilute (PbT)

A solution containing approximately 10% w/v of HCl; it complies with the following test: make 10 ml alkaline with ammonia dilute, PbT add 1 ml of potassium cyanide solution PbT, dilute to 50 ml with water, and add 2 drops of a 10% sodium sulfide solution in water. No darkening is produced.

Hydrochloric Acid TS, Stannated

Mix 1 ml of a solution of stannous chloride TS with 100 ml of hydrochloric acid (a 25% w/v solution of HC1 in water).

Hydrogen Peroxide TS

A solution containing between 2.5 and 3.5 g of H2O2 in each 100 ml. It may contain suitable preservatives, totalling not more than 0.05%.

Hydrogen Sulfide TS

A saturated solution of hydrogen sulfide made by passing H2S into cold water. Store it in a small, dark, amber-coloured bottle, filled nearly to the top. It is unsuitable unless it possesses a strong odour of H2S, and unless it produces at once a copious precipitate of sulfur when added to an equal volume of ferric chloride TS. Store in a cold dark place.

Hydroxylamine Hydrochloride TS

Dissolve 20 g of hydroxylamine hydrochloride (HONH₂-HC1) in sufficient water to make approximately 65 ml. Transfer to a separatory funnel, add a few drops of thymol blue pH indicator, then add strong ammonia TS until the solution assumes a yellow colour. Add 10 ml of a 4% solution of sodium diethyldithiocarbamate, mix well, and allow to stand for 5 min. Extract this solution with successive 10 to 15-ml portions of chloroform until a 5-ml portion of the chloroform extract does not assume a yellow colour when shaken with a dilute cupric sulfate solution. Add diluted hydrochloric acid PbT until the solution is pink, and then dilute with sufficient water to make 100 ml.

8-Hydroxyquinoline TS

A 5% w/v solution of 8-hydroxyquinoline (oxine) in ethanol.

Indigo Carmine TS

Dissolve a quantity of indigo carmine (sodium indigotindisulfonate) equivalent to 0.18 g of C₁₆H₈O₈N₂S₂Na₂, in sufficient water to make 100 ml. This solution should be used within 60 days of preparation.

Iodine TS

Dissolve 14 g of iodine in a solution of 36 g of potassium iodide in 100 ml of water, add 3 drops of hydrochloric acid, and dilute with water to 1,000 ml.

Iron Indicator TS

Place 62.5 g of ferric ammonium sulfate in a one-litre bottle, dissolve in 500 ml of water, add 450 ml of concentrated nitric acid, and mix.

Iron TS, Standard

Dissolve 0.70 g of Fe(NH₄)₂(SO₄)₂-6H₂O in 50 ml of water and add 20 ml of dilute H2SO4(1:15). Dilute to 1,000 ml with water and mix thoroughly. Dilute 10 ml of this solution to 100 ml with water. Each ml contains 0.01 mg of Fe.

Lead TS, Standard

Dissolve 0.1598 g of lead nitrate [Pb(NO3)2] in water to which has been added 1 ml of nitric acid and dilute to 1,000 ml. Then dilute 10 ml of this solution to 100 ml. Each ml contains 0.01 mg of lead. This solution must be freshly prepared.

Lead Acetate TS

Dissolve 9.5 g of clear, transparent crystals of lead acetate [Pb(COOCH₃)₂.3H₂O] in sufficient recently boiled water to make 100 ml. Store in a well-stoppered bottle.

Lead Acetate TS, Basic

Mix 10 parts of finely pulverized lead oxide (PbO) with 30 parts of lead acetate [Pb(COOCH₃)₂.3H₂O] and 5 parts of water and heat gently in a closed vessel shaking repeatedly, until the mixture is white. Add 95 parts of water, heat for 1 h, shaking repeatedly, allow to cool and filter. Add water if necessary to obtain a solution of specific gravity 1.225-1.230.

Lead Subacetate TS

Triturate 14 g of lead monoxide (PbO) to a smooth paste with 10 ml of water, and transfer the mixture to a bottle, using an additional 10 ml of water for rinsing. Dissolve 22 g of lead acetate [Pb(COOCH₃)₂.3H₂0] in 70 ml of water, and add the solution to the lead oxide mixture. Shake it vigorously for 5 min, then set it aside, shaking it frequently over 7 days. Finally filter, and add enough recently boiled water through the filter to make 100 ml.

Lead Subacetate TS, Dilute

Dilute 3.25 ml of lead subacetate TS with sufficient water, recently boiled and cooled, to make 100 ml. Store in a small, well-filled, tight container.

Litmus TS

Boil 10 g of litmus of reagent purity with 40 ml of ethanol (90%) for 1 h and pour away the clear liquid, repeat this operation twice with 30 ml of ethanol (90%). Digest the washed litmus with 100 ml of boiling water for 1 h, cool and filter.

Manganese Sulfate TS

Dissolve 90 g of manganese sulfate in 200 ml of water, 175 ml of phosphoric acid and 350 ml of diluted sulfuric acid (1 in 2). Add sufficient water to 1,000 ml.

Magnesia Mixture TS

Dissolve 5.5 g of magnesium chloride (MgCl₂.6H₂O) and 7 g of ammonium chloride (NH4Cl) in 65 ml of water, add 35 ml of ammonia TS, set the mixture aside for a few days in a well-stoppered bottle, and then filter. If the solution is not perfectly clear, filter again before use.

Magnesium Sulfate TS

Dissolve 12 g of crystals of magnesium sulfate (MgSO₄7H₂O), selected for freedom from efflorescence, in water to make 100 ml.

Malachite Green TS

A 1% w/v solution of malachite green oxalate in glacial acetic acid.

Mayer's TS

See mercuric-potassium iodide TS.

Mercuric Acetate TS

A 6% w/v solution of mercuric acetate [Hg(COOCH3)2] in glacial acetic acid. Store in a tight container protected from direct sunlight.

Mercuric Chloride TS

A 6.5% w/v solution of mercuric chloride (HgCl₂) in water (approximately 0.5 N).

Mercuric-Potassium Iodide TS

(Mayer's TS). Dissolve 1.358 g of mercuric chloride (HgCl2) in 60 ml of water. Dissolve 5 g of potassium iodide (KI) in 10 ml of water. Mix the two solutions, and add water to make 100 ml.

Mercuric-Potassium Iodide TS, Alkaline

(Nessler's TS). Dissolve 10 g of potassium iodide (KI) in 10 ml of water, and add slowly with stirring, a saturated solution of mercuric chloride until a slight red precipitate remains undissolved. To this mixture add an ice-cold solution of 30 g of potassium hydroxide (KOH) in 60 ml of water, then add 1 ml more of the saturated solution of mercuric chloride. Dilute with water to 200 ml. Allow the precipitate to settle, and draw off the clear liquid. A 2-ml portion of this reagent, when added to 100 ml of a 1 in 300,000 solution of ammonium chloride in ammonia-free water, produces at once a yellowish brown colour.

Mercuric Nitrate TS

Dissolve 40 g of yellow mercuric oxide (HgO) in a mixture of 32 ml of nitric acid and 15 ml of water. Store in a light-shaded, glass-stoppered bottle (approximately 4 N).

Mercuric Sulfate TS

(Denigès' TS). Mix 5 g of yellow mercuric oxide (HgO) with 40 ml of water, and while stirring slowly add 20 ml of sulfuric acid, then add another 40 ml of water, and stir until completely dissolved (approximately 0.5 N).

Mercurous Nitrate TS

Dissolve 200 g of mercury in nitric acid and add sufficient water to produce 1,000 ml. Mercurous nitrate TS should be kept in a bottle containing a little metallic mercury.

p-Methylaminophenol Sulfate TS

Dissolve 2 g of p-methylaminophenol sulfate [(HO.C₆H₄.NHCH₃)₂.H₂SO₄] in 100 ml of water. To 10 ml of this solution add 90 ml of water and 20 g of sodium bisulfite. Confirm the suitability of this solution by the following test: add 1 ml of the solution to each of four tubes containing 25 ml of 0.5 N sulfuric acid and 1 ml of ammonium molybdate TS. Add 5 μg of phosphate (PO4) to one tube, 10 μg to a second, and 20 μg to a third, using 0.5, 1.0, and 2.0 ml, respectively, of Phosphate Standard Solution, and allow to stand for 2 h. The solutions in the three tubes should show readily perceptible differences in blue colour corresponding to the relative amounts of phosphate added, and the one to which 5 μg of phosphate was added should be perceptibly bluer than the blank.

Methylene Blue TS

Dissolve 0.125 g of methylene blue in 100 ml of ethanol, and dilute with ethanol to 250 ml.

Methylene Blue TS, Diluted

To 1 ml of methylene blue TS, add sufficient water to make 100 ml.

Methyl Orange TS

Dissolve 0.1 g of methyl orange in 100 ml of water and filter if necessary.

Methyl Orange/Xylencyanol FF TS

Dissolve 1 g of methyl orange and 1.4 g of xylencyanol FF in 500 ml of 50% v/v ethanol.

Methyl Red TS

Dissolve 0.1 g of methyl red in 100 ml of ethanol, and filter if necessary. For pH determinations, dissolve 0.1 g in 7.4 ml of 0.05 N sodium hydroxide, and dilute with carbon dioxide-free water to 200 ml.

Methyl Red/Methylene Blue TS

Add 10 ml of methyl red TS to 10 ml of methylene blue TS, and mix.

Methylrosaniline Chloride TS

See crystal violet TS.

Methyl Violet TS

See crystal violet TS.

Millon's TS

To 2 ml of mercury in an Erlenmeyer flask add 20 ml of nitric acid. Shake the flask under a hood to break up the mercury into small globules. After about 10 min add 35 ml of water and, if a precipitate or crystals appear, add sufficient dilute nitric acid (1 in 5, prepared from nitric acid free from the oxides which have been removed by blowing air through it until it is colourless) to dissolve the separated solid. Add sodium hydroxide solution (1 in 10), dropwise, with thorough mixing, until the curdy precipitate that forms after the addition of each drop no longer redissolves but is dispersed to form a suspension. Add 5 ml more of the dilute nitric acid, and mix well. Prepare this solution fresh.

Murexide Indicator Preparation

Add 0.4 g of murexide to 40 g of powdered potassium sulfate, and grind in a glass mortar to a homogeneous mixture. (Tablets containing 0.4 mg of murexide admixed with potassium sulfate or potassium chloride are available commercially.)

Naphthalenediol TS

Dissolve 0.1 g of 2,7-dihydroxynaphthalene in 1,000 ml of sulfuric acid and allow the solution to stand in the dark until the yellow colour has disappeared (at least 18 h).

1-Naphthol TS

Dissolve 1 g of 1-napthol in 25 ml of methanol. Prepare this solution freshly.

Naphthol Green TS

A 0.05% w/v solution of naphthol green in water.

alpha-Naphtholbenzein TS

A 1 % w/v solution of alpha-naphtholbenzein in benzol.

Nessler's TS

See mercuric-potassium iodide TS, alkaline.

Neutral Red TS

A 0.1% w/v solution of neutral red in 50% ethanol.

Ninhydrin TS

A 0.2% w/v solution of ninhydrin (triketohydrindene hydrate, C9H4O3H2O) in water. Prepare this solution fresh.

Nitric Acid TS, Dilute

A solution containing about 10% w/v of HNO3. Prepared by diluting 105 ml of nitric acid (70%) with water to make 1,000 ml.

Nitric Acid/Sulfuric Acid TS

Prepare about 1,000 ml of nitric acid (32-35% w/v of HNO₃) by diluting 420 ml of nitric acid (70%) with 580 ml of distilled water, and add 30 ml of sulfuric acid.

Nitrite Standard TS

Dissolve 1.5 g of sodium nitrite (NaNO2) in 1,000 ml of carbon dioxide-and ammonia-free water. Each contains 1 mg of NO₂.

o-Nitrobenzaldehyde TS

Saturate a 2 N sodium hydroxide solution with o-nitrobenzaldehyde (NO2C6H4CHO).

Orthophenanthroline TS

Dissolve 0.15 g of orthophenanthroline (C₁₂H₈.N₂.H₂O) in 10 ml of a solution of ferrous sulfate, prepared by dissolving 1.48 g of clear crystals of ferrous sulfate (FeSO₄.7H₂O) in 100 ml water. The ferrous sulfate solution must be prepared immediately before dissolving the orthophenanthroline. Store the solution in well-closed containers.

Oxalic Acid TS

A 6.3% w/v solution of oxalic acid (C₂H₂O₄2H₂O) in water (approximately N).

Oxalic Acid/Sulfuric Acid TS

Add an equal volume of sulfuric acid to water, and cool. To a 500 ml portion of the solution, add 25 g of oxalic acid.

8-Oxyquinoline TS

Dissolve 2 g of 8-oxyquinoline in 6 ml of glacial acetic acid. Add sufficient water to 100 ml. Prepare freshly before use.

Phenol Red TS

(Phenolsulfonphthalein TS). Dissolve 0.1 g of phenolsulfonphthalein in 100 ml of ethanol, and filter if necessary. For pH determinations, dissolve 0.1 g in 5.7 ml of 0.05 N sodium hydroxide, and dilute with carbon dioxide-free water to 200 ml.

Phenolphthalein TS

Dissolve 0.2 g of phenolphthalein (C20H14O4) in 60 ml of 90% ethanol and add sufficient water to make 100 ml.

Phenolphthalein/Thymol Blue TS

Dissolve 2 g of phenolphthalein and 0.1 g of thymol blue in 100 ml of absolute ethanol, and filter if necessary. Prepare freshly before use.

Phenolsulfonphthalein TS

See phenol red TS.

Phenylhydrazine Hydrochloride/Sodium Acetate TS

Dissolve 0.5 g of phenylhydrazine hydrochloride in 10 ml of sodium acetate TS, and filter if necessary. Prepare freshly before use.

p-Phenylphenol TS

On the day of use, dissolve 0.75 g of p-phenylphenol in 50 ml of sodium hydroxide TS.

Phloroglucin/Hydrochloric Acid TS

Dissolve 0.1 g of phloroglucin in 1 ml of ethanol, add 9 ml of hydrochloric acid, and mix well. Store in a dark place.

Phosphomolybdic Acid TS

Dissolve 5 g of phosphomolybdic acid (20MoO₃.2H₃PO₄'48H₂O) in water, filter and dilute to 100 ml with water.

Phosphotungstic Acid TS

A 1% w/v solution of phosphotungstic acid (approximately 24WO₃.2H₃PO₄.48H₂O) in water.

Picric Acid TS

See trinitrophenol TS.

Platinic Chloride TS

A 13% w/v solution of platinic chloride in water (approximately 0.5 N).

Platinum/Cobalt TSC

Transfer 1.246 g of potassium chloroplatinate (K₂PtC1₆), and 1.00 g of crystallized cobaltous chloride, (C0Cl₂6H₂O), into a 1,000-ml volumetric flask, dissolve in about 200 ml of water and 100 ml of hydrochloric acid, dilute to volume with water, and mix. (Use this solution only when specified in an individual monograph.)

Potassium Acetate TS

A 10% w/v solution of potassium acetate (KCOOCH3) in water.

Potassium Acetate in Acetic Acid TS

Dissolve 10 g of potassium acetate in 100 ml of a solution consisting of 90 ml of glacial acetic acid and 10 ml of acetic anhydride.

Potassium Bichromate TS

See potassium dichromate TS.

Potassium Bromate/Potassium Bromide TS

Dissolve 1.4 g of potassium bromate and 8.1 g of potassium bromide in sufficient water to make 100 ml.

Potassium Chloride/Hydrochloric Acid TS

Dissolve 25 g of potassium chloride in 0.85 ml of hydrochloric acid and 75 ml of water.

Potassium Chromate TS

A 10% w/v solution of potassium chromate (K₂CrO₄) in water.

Potassium Cyanate TS

Dissolve 1 g of potassium cyanate in 9 ml of water. Prepare freshly before use.

Potassium Cyanide TS (PbT)

Dissolve 50 g of potassium cyanide in sufficient purified water to make 100 ml. Remove the lead by shaking with portions of the dithizone extraction TS. Part of the dithizone remains in the aqueous phase but can be removed, if desired, by washing with chloroform. The strong potassium cyanide solution is then diluted to a concentration of 10 g per 100 ml.

Potassium Dichromate TS

A 7.5% w/v solution of potassium dichromate (K₂Cr₂O₇) in water.

Potassium Ferricyanide TS

Dissolve 1 g of potassium ferricyanide [K₃Fe(CN)₆] in 10 ml of water. Prepare this solution fresh.

Potassium Ferrocyanide TS

Dissolve 1 g of potassium ferrocyanide [K₄Fe(CN)₆.3H₂O] in 10 ml of water. Prepare this solution fresh.

Potassium Hydroxide TS

A 6.5% w/v solution of potassium hydroxide (KOH) in water (approximately N).

Potassium Hydroxide TS, Ethanolic

Place a few g (5 to 10) of potassium hydroxide in a 2-litre flask, add 1 to 1.5 L of 95% ethanol and boil on a water bath under reflux condenser from 30 to 60 min. Distil and collect the ethanol. Dissolve 40 g of potassium hydroxide, low in carbonate, in 1,000 ml of the distilled ethanol keeping the temperature below 15.5° while the alkali is being dissolved. This solution should remain clear.

Potassium Iodate TS

A 0.71% w/v solution of potassium iodate in water. Preserve in the dark.

Potassium Iodide TS

A 16.5% w/v solution of potassium iodide (KI) in water (approximately N). Store in a light-resistant container.

Potassium Permanganate TS

A 1.0% w/v solution of potassium permanganate (KMnO₄) in water.

Potassium Permanganate/Phosphoric Acid TS

To 75 ml of phosphoric acid, add sufficient water to 500 ml, and dissolve 15 g of potassium permanganate in the solution.

Potassium Pyroantimonate TS

To 2 g of potassium pyroantimonate, add 100 ml of water. Boil the solution for about 5 min, cool quickly, and add 10 ml of 15% potassium hydroxide solution. Allow to stand for one day, and filter.

Potassium Sodium Tartrate TS

A 14.1% w/v solution of potassium sodium tartrate (KNaC₄H₄O₆.4H₂O) in water.

Potassium Sulfate TS

A 1% w/v solution of potassium sulfate (K₂SO₄) in water.

Pyridine/Acetic Anhydride TS

Mix 3 volumes of pyridine with 1 volume of acetic anhydride. Prepare freshly before use.

Pyridinium Chloride/Chloroform TS

Place 75 g of anhydrous pyridine (C₅H₅N) and approximately 400 ml of chloroform in a 2-litre graduated cylinder. Weigh the cylinder and cool it in an ice water bath. Bubble dry hydrogen chloride slowly through the solution. At intervals of several min interrupt the flow of gas, remove, dry and weigh the cylinder and its contents to determine the rate of flow of the gas. When approximately 35 g have been added, stop the flow of gas, warm the mixture to room temperature and expel the vapours with a stream of dry air. Add 100 ml of anhydrous pyridine and dilute to 1,000 ml with chloroform. When most of the reagent has been used, discard the last 100 ml.

Quimociac TS

Dissolve 70 g of sodium molybdate (Na₂MoO₄.2H₂O) in 150 ml of water (Solution A). Dissolve 60 g of citric acid in a mixture of 85 ml of nitric acid and 150 ml of water, and cool (Solution B). Gradually add Solution A to Solution B, with stirring, to produce Solution C. Dissolve 5.0 ml of synthetic quinoline in a mixture of 35 ml of nitric acid and 100 ml of water (Solution D). Gradually add Solution D to Solution C, mix well, and allow to stand overnight. Filter the mixture, add 280 ml of acetone to the filtrate, dilute to 1,000 ml with water, and mix. Store in a polyethylene bottle. Caution. This reagent contains acetone. Do not use near an open flame. Operations involving heating or boiling should be conducted in a well-ventilated hood.

Quinaldine Red TS

A 0.1% w/v solution of quinaldine red in glacial acetic acid.

Salicylaldehyde TS

A 20% v/v solution of salicylaldehyde in ethanol.

Schiff s TS

Aqueous solution of 0.125 g of crystalline rosaline chlorohydrate in 1,000 ml and discolourized with sulfurous acid.

Schiff s TS, Modified

Dissolve 0.2 g of rosaniline hydrochloride (C₂₀H₂₀ClN₃) in 120 ml of hot water. Cool, add 2 g of sodium bisulfite (NaHSO3) followed by 2 ml of hydrochloric acid, and dilute to 200 ml with water. Store in a brown bottle at 15° or lower.

Silicotungstic Acid TS

Dissolve 10 g of silicotungstic acid (SiO₂.12WO₃.26H₂O) in water and neutralize with 10% sodium hydroxide solution to a methyl red endpoint. Dilute to approximately 100 ml.

Silver Ammonionitrate TS

See Silver ammonium nitrate TS.

Silver Ammonium Nitrate TS

Dissolve 1 g of silver nitrate in 20 ml of water. Add ammonia TS, dropwise, with constant stirring, until the precipitate is almost but not entirely dissolved. Filter, and store in a tight, light-resistant container.

Silver Nitrate TS

A 4.2% w/v solution of silver nitrate (AgNO3) in water (approximately 0.25 N).

Silver Nitrate TS, Acid

Dissolve 15 g of silver nitrate in 50 ml of water, add 400 ml of ethanol and several drops of concentrated nitric acid. This solution is standardized against 0.05 N ammonium thiocyanate by the Volhard method. The solution is very stable.

Silver Nitrate Spray TS

Prepare the following two solutions:

1. Dissolve 50 g of silver nitrate in 450 ml of distilled water. Store in an amber bottle.
2. Add 120 ml of concentrated ammonium hydroxide to 330 ml of distilled water. When required combine equal volumes of solutions (a) and (b) for use as spray reagent.

Sodium Acetate TS

A 13.6% w/v solution of sodium acetate in water (approximately N).

Sodium Azide TS

A 5% w/v solution of sodium azide in water.

Sodium Bisulfite TS

Prepare a solution of sodium bisulfite in water (approximately 0.5 N). Check the pH and if necessary, adjust to the range 3.0 to 4.5 with dilute sulfuric acid or sodium hydroxide.

Sodium Bitartrate TS

A 1% w/v solution of sodium bitartrate (NaHC₄H₄O₆H₂O) in water (approximately N). Prepare this solution fresh.

Sodium Borate TS

A 2% w/v solution of sodium borate (Na₂B₄O₇.10H₂0) in water.

Sodium Carbonate TS

A 10.6% w/v solution of anhydrous sodium carbonate (Na₂CO₃) in water.

Sodium Chloride TS

A 10% w/v solution of sodium chloride in water.

Sodium Cobaltinitrite TS

Dissolve 10 g of sodium cobaltinitrite [Na₃Co(NO₂)6] in water to make 50 ml and filter if necessary.

Sodium Ethoxide TS

Dissolve 10 g of sodium in 120 ml of absolute ethanol, using the following method: remove surplus oil from the sodium metal with filter paper, weigh in benzol and again dry on a filter paper. Cut the weighed metal into small pieces about the size of a pea and carefully add one or two pieces at a time to a 500-ml conical flask which is fitted with a water-cooled reflux condenser and contains the 120 ml of ethanol.

Sodium Fluorescein TS

A 0.1% w/v solution of sodium fluorescein in 50% ethanol.

Sodium Fluoride TS

Dry about 0.5 g of sodium fluoride (NaF) at 200° for 4 h. Weigh accurately 0.222 g of the dried sodium fluoride, and dissolve it in sufficient water to make exactly 100 ml. Transfer 10.0 ml of this solution into a 1,000-ml volumetric flask, dilute to volume with water, and mix. Each ml of this final solution corresponds to 0.01 mg of fluorine (F).

Sodium Hydrogen Sulfite TS

A 33.3% w/v solution of sodium hydrogen sulfite in water. Prepare freshly before use.

Sodium Hydroxide TS

Dissolve 4.3 g of sodium hydroxide in water to make 100 ml (approximately N).

Sodium Hydroxide TS (5%), Methanolic

Dissolve 5 g of sodium hydroxide in 5 ml of water, then add sufficient methanol to make 100 ml. Use supernatant.

Sodium Indigotindisulfonate TS

See indigo carmine TS.

Sodium Nitrite TS

A 10% w/v solution of sodium nitrite in water. Prepare freshly before use.

Sodium Nitroferricyanide TS

A 5% w/v solution of sodium nitroferricyanide [Na2Fe(NO)(CN)₅.2H₂O] in water. Prepare this solution fresh.

Sodium Nitroprusside TS

See sodium nitroferricyanide TS.

Sodium Phosphate TS

See sodium phosphate TS, dibasic.

Sodium Phosphate TS, Dibasic

A 12% w/v solution of clear crystals of dibasic sodium phosphate (Na₂HPO₄.7H₂O) in water.

Sodium Phosphate TS, Monobasic

A 62.4% w/v solution of monobasic sodium phosphate (NaH₂PO₄.2H₂O) in water (approximately 4 M).

Sodium Starch Glycolate TS (5%)

Moisten 5 g of sodium starch glycolate with a few drops of ethanol, add 100 ml of water and boil for 2-3 min, and cool.

Sodium Starch Glycolate TS (1%)

Dilute 10 ml of sodium starch glycolate TS (5%) to 50 ml with distilled water. Prepare freshly before use.

Sodium Sulfide TS

A 10% w/v solution of sodium sulfide (Na₂S.9H₂O) in water. Prepare this solution fresh.

Sodium Sulfide TS (PbT)

Dissolve 10 g of sodium sulfide (PbT) in sufficient water to make 100 ml and filter.

Sodium Thiosulfate TS

Use 0.1 N sodium thiosulfate.

Stannous Chloride TS

Dissolve 3.2 g of stannous chloride (SnCl₂.2H₂O) in 40 ml of 0.3 N hydrochloric acid. Transfer the solution to a 100-ml volumetric flask and dilute to the mark with 0.3 N hydrochloric acid. Prepare fresh daily. The stannous chloride solution should be titrated with sulfuric periodic acid TS before use and adjusted so that 10.0 ml of the stannous chloride reagent will titrate 10.2 ml of the periodic acid reagent. For the titration, 5 ml of concentrated hydrochloric acid are added to 10 ml of stannous chloride plus 1 ml of starch indicator (a blue colour indicates the endpoint).

Starch TS

Triturate 1 g of arrowroot starch with 10 ml of cold water, and pour slowly, with constant stirring, into 200 ml of boiling water. Boil the mixture until a thin, translucent fluid is obtained. (Longer boiling than necessary renders the solution less sensitive.) Allow to settle, and use only the clear, supernatant liquid. Prepare this solution fresh.

Starch Iodide Paste TS

Heat 100 ml of water in a 250-ml beaker to boiling, add a solution of 0.75 g of potassium iodide (KI) in 5 ml of water, then add 2 g of zinc chloride (ZnCl2) dissolved in 10 ml of water, and, while the solution is boiling, add with stirring a smooth suspension of 5 g of potato starch in 30 ml of cold water. Continue to boil for 2 min, then cool. Store in a well-closed container in a cool place. This mixture must show a definite blue streak when a glass rod dipped in a mixture of 1 ml of 0.1 M sodium nitrite, 500 ml of water, and 10 ml of hydrochloric acid, is streaked on a smear of the paste.

Starch Mucilage TS

See Starch TS.

Sulfanilic Acid TS

A 0.8% w/v solution of sulfanilic acid (p-NH₂C₆H₄SO₃H.H₂O) in acetic acid. Store in a tight container.

Sulfanilic Acid/a-Naphthylamine TS

Dissolve 0.5 g of sulfanilic acid in 150 ml of acetic acid. Dissolve 0.1 g of a-naphthylamine in 0.26 g of hydrochloric acid and 150 ml of acetic acid, and mix. When a pink colour is produced upon standing, add zinc dust to decolourize.

Sulfuric Acid TS

Add a quantity of sulfuric acid of known concentration to sufficient water to adjust the final concentration to between 94.5 and 95.5% of H2SO4.

Sulfuric Acid TS, Dilute

A solution containing 10% w/v of H2SO4. Prepare by cautiously adding 57 ml of sulfuric acid (95-98%) or sulfuric acid TS to about 100 ml of water, then cool to room temperature, and dilute with water to 1,000 ml (approximately 2 N).

Sulfuric Acid/Periodic Acid TS

Dissolve 3.42 g of periodic acid (H5IO6) in 100 ml of 0.25 M sulfuric acid. Transfer the solution to a 500-ml volumetric flask and dilute to the mark with 0.25 M sulfuric acid (approximately 0.03 M sulfuric acid/periodic acid).

Tannic Acid TS

Dissolve 1 g of tannic acid (tannin) in 1 ml of ethanol, and add water to make 10 ml. Prepare this solution fresh.

Tannic Acid/Glacial Acetic Acid TS

Dissolve 10 mg of tannic acid in 80 ml of glacial acetic acid while shaking, and add 32 ml of phosphoric acid. Prepare freshly before use.

Tartrate Solution TS, Alkaline

Dissolve 34.6 g of potassium sodium tartrate (Rochelle salt) and 10 g of sodium hydroxide in water, dilute to 100 ml, let stand two days, and filter through glass wool.

Thymol Blue TS

Dissolve 0.1 g of thymol blue in 100 ml of ethanol, and filter if necessary. For pH determinations, dissolve 0.1 g in 4.3 ml of 0.05 N sodium hydroxide, and dilute with carbon dioxide-free water to 200 ml.

Thiourea TS

A 10% w/v solution of thiourea in water.

Thymolphthalein TS

Dissolve 0.1 g of thymolphthalein in 100 ml of ethanol, and filter if necessary.

Tin (II) Sulfate TS

Add 10 g of tin (II) sulfate to 100 ml of 1% sulfuric acid. Agitate continuously for several hours decanting the solution from the insoluble fraction at frequent intervals.

Triketohydrindene Hydrate TS

See Ninhydrin TS.

Trinitrophenol TS

(Picric acid TS). Dissolve the equivalent of 1 g of anhydrous trinitrophenol in 100 ml of hot water. Cool the solution, and filter if necessary.

Uranyl Acetate TS

Dissolve 1 g of uranyl acetate in 20 ml of water by shaking well and filter.

Uranyl Zinc Acetate TS

Dissolve 10 g of uranyl acetate [(CH₃COO)₂UO₂-2H₂O)] by heating with 50 ml of water and 5 ml of acetic acid (a solution containing approximately 30% w/v of CH₃COOH, in water, approximately 5 N). Dissolve 3 g of zinc acetate [(CH₃COO)₂Zn] in 30 ml of water and 3 ml of 30% w/v acetic acid (approximately 5 N). Mix the two solutions, allow to cool to room temperature, and remove by filtration any solid material which separates.

Vanadic Acid/Molybdic Acid TS

Dissolve 1.12 g of ammonium metavanadate in about 300 ml of warm water, add 250 ml of nitric acid. Combine the cooled solution with another solution of 27 g of ammonium molybdate in about 400 ml of warm water, then add sufficient amount of water to make 1,000 ml. Use after 3 to 4 days of preservation in a dark-coloured bottle.

Xylenol Orange TS

A 0.1% w/v of xylenol orange in ethanol.

Zinc Amalgam TS

Add about 10 g of granulated zinc to sufficient mercury, about 20 ml, to produce a liquid amalgam on cooling, and heat at 150°, with stirring, until the zinc is dissolved. Zinc amalgam may be used repeatedly until the content of zinc is reduced to 0.2% w/w, as determined by the following process. Fill a pycnometer with mercury at 25° ±1°, and weigh. Repeat the operation, using the amalgam. Calculate the proportion of zinc from the formula:

Percentage w/w of zinc = (13.534 - A) / 0.000875

where A = (wt of amalgam - 13.534) / wt of mercury

Zinc Sulfate TS

A 10% w/w solution of zinc sulfate (ZnSO₄.7H₂O) in water.

Zinc Sulfate TS, Standard

Dissolve 0.440 g of zinc sulfate in sufficient water to produce 1,000 ml, and dilute 50 ml of the solution to 1,000 ml with water. Each ml of the solution contains 5 μg of zinc.

Zirconium/Alizarin TS

Dissolve 0.80 g of zirconium nitrate [Zr(NO₃)₂.5H₂O] in water, add a few drops of 4 N nitric acid, and make up to 100 ml with water. Dissolve 0.10 g of alizarin sulfonate monohydrate in 20 ml of water, and make up to 100 ml with ethanol. Mix 1 ml of the first solution with 1 ml of the second solution and add 18 ml of water. This solution must be clear and the dilution should be freshly prepared.

Zwikker's TS

Mix 1 ml of pyridine with 4 ml of a 10% aqueous solution of copper sulfate and 5 ml of water.

VOLUMETRIC SOLUTIONS

Normal Solutions

A normal solution contains 1 g equivalent weight of the solute per litre of solution. The normalities of solutions used in volumetric determinations are designated as 1 N; 0.1 N; 0.05 N; etc.

Molar Solutions

A molar solution contains 1 g molecular weight of the solute per litre of solution. The molarities of such solutions are designated as 1 M; 0.1 M; 0.05 M; etc.

Preparation and Methods of Standardization

The details for the preparation and standardization of solutions used in several normalities are usually given only for those most frequently required. Solutions of other normalities are prepared and standardized in the same general manner as described. Solutions of lower normalities may be prepared accurately by making an exact dilution of a stronger solution, but solutions prepared in this way should be restandardized before use.

Dilute solutions that are not stable, such as 0.01 N potassium permanganate and sodium thiosulfate, are preferably prepared by diluting exactly the higher normality with thoroughly boiled and cooled water on the same day they are to be used.

All volumetric solutions should be prepared, standardized, and used at the standard temperature of 20°, if practicable. When a titration must be carried out at a markedly different temperature, the volumetric solution should be standardized at that same temperature, or a suitable temperature correction should be made. Since the strength of a standard solution may change upon standing, the normality or molarity factor should be redetermined frequently.

Although the directions provide only one method of standardization, other methods of equal or greater accuracy may be used. For substances available as certified primary standards, or of comparable quality, the final standard solution may be prepared by weighing accurately a suitable quantity of the substance and dissolving it to produce a specific volume solution of known concentration. Hydrochloric and sulfuric acids may be standardized against a certified primary standard.

In volumetric assays described, the number of mg of the test substance equivalent to 1 ml of the primary volumetric solution is given. In general, these equivalents may be derived by simple calculation.

0.1 N Ammonia, (3.505 g of NH4OH per litre)

Add sufficient water to about 35 ml of ammonia TS to 1,000 ml. Standardize the solution with 0.1 N hydrochloric acid, using bromophenol blue TS as the indicator.

0.1 N Ammonium Thiocyanate, (7.612 g of NH4SCN per litre)

Dissolve about 8 g of ammonium thiocyanate, NH₄SCN, in 1,000 ml of water, and standardize by titrating the solution against 0.1 N silver nitrate as follows: transfer about 30 ml of 0.1 N silver nitrate, accurately measured, into a glass-stoppered flask. Dilute with 50 ml of water, then add 2 ml ferric ammonium sulfate TS and 2 ml of nitric acid, and titrate with the ammonium thiocyanate solution to the first appearance of a red-brown colour. Calculate the normality, and, if desired, adjust the solution to exactly 0.1 N. If desired, 0.1 N ammonium thiocyanate may be replaced by 0.1 N potassium thiocyanate where the former is directed in various tests and assays.

0.01 M Barium Chloride

Dissolve 2.44 g of barium chloride in sufficient water, freshly boiled and cooled, to make 1,000 ml.

0.1 N Bromine, (7.990 g of Br per litre)

Dissolve 3 g of potassium bromate, KBrO₃, and 15 g of potassium bromide, KBr, in sufficient water to make 1,000 ml, and standardize the solution as follows: transfer about 25 ml of the solution, accurately measured, into a 500-ml iodine flask, and dilute with 120 ml of water. Add 5 ml of hydrochloric acid, stopper the flask, and shake it gently. Then add 5 ml of potassium iodide TS, re-stopper, shake the mixture, allow it to stand for 5 min, and titrate the liberated iodine with 0.1 N sodium thiosulfate, adding starch TS near the end of the titration. Calculate the normality. Store this solution in dark, glass-stoppered bottles.

0.1 N Ceric Sulfate, (33.22 g of Ce(SO₄)₂ per litre)

Transfer 59 g of ceric ammonium nitrate, Ce(N0₃)₄.2NH₄NO.2H₂0, to a beaker, add 31 ml of sulfuric acid, mix, and cautiously add water, in 20 ml portions, until solution is complete. Cover the beaker, let stand overnight, filter through a sintered-glass crucible of fine porosity, add water to make 1,000 ml, and mix.

Standardize the solution as follows: weigh accurately 200 mg of primary standard arsenic trioxide, As₂O₃, previously dried at 100° for 1 h, and transfer to a 500-ml Erlenmeyer flask. Wash down the inner walls of the flask with 25 ml of sodium hydroxide solution (2 in 5), swirl to dissolve the sample, and when solution is complete add 100 ml of water, and mix. Add 10 ml of dilute sulfuric acid (1 in 3) and 2 drops each of orthophenanthroline TS and a solution of osmium tetroxide in 0.1 N sulfuric acid (1 in 400), and slowly titrate with the ceric sulfate solution until the pink colour is changed to a very pale blue. Calculate the normality. Each 4.964 mg of As₂O₃ is equivalent to 1 ml of 0.1 N ceric sulfate.

0.01 N Ceric Sulfate, for Tocopherol Assay (3.322 g of Ce(SO₄)₂ per litre)

Dissolve 4.2 g of ceric sulfate, Ce(SO₄)₂.4H₂O, or 5.5 g of the acid sulfate Ce(HSO4)4, in about 500 ml of water containing 28 ml of sulfuric acid, and dilute to 1,000 ml. Allow the solution to stand overnight, and filter.

Standardize this solution daily as follows: weigh accurately about 275 mg of hydroquinone, C6H6O2, dissolve it in sufficient 0.5 N ethanolic sulfuric acid to make 500 ml, and mix. To 25 ml of this solution add 75 ml of 0.5 N sulfuric acid, 20 ml of water, and 2 drops of diphenylamine TS. Titrate with the ceric sulfate solution at a rate of about 25 drops per 10 sec until the red point is reached which persists for 10 sec. Perform a blank determination using 100 ml of 0.5 N ethanolic sulfuric acid, 20 ml of water, and 2 drops of diphenylamine TS, and make any necessary correction. Calculate the normality of the ceric sulfate solution by the formula 0.05W/55.057V, in which W is the weight, in mg, of the hydroquinone sample taken, and V is the volume, in ml, of the ceric sulfate solution consumed in the titration.

0.05 M Disodium Ethylenediaminetetraacetate (EDTA), (16.811 g of C₁₀H₁₄O₈N₂Na₂ per litre)

Dissolve 18.7 g of disodium ethylenediaminetetraacetate in sufficient water, freshly boiled and cooled, to make 1,000 ml.

Standardize the solution as follows: weigh accurately about 0.2 g of chelometric standard calcium carbonate, CaCO3, transfer to a 400-ml beaker, add 10 ml of water, and swirl to form a slurry. Cover the beaker with a watch glass, and introduce 2 ml of dilute hydrochloric acid TS from a pipet inserted between the lip of the beaker and the edge of the watch glass. Swirl the contents of the beaker to dissolve the calcium carbonate. Wash down the sides of the beaker, the outer surface of the pipet, and the watch glass, and dilute to about 100 ml with water. While stirring preferably with a magnetic stirrer, add about 30 ml of the disodium EDTA solution from a 50-ml buret, then add 15 ml of sodium hydroxide TS and 300 mg of hydroxynaphthol blue indicator, and continue the titration to a blue end-point. Calculate the molarity by the formula W/100.09V, in which W is the weight, in mg, of CaCO3 in the sample of calcium carbonate taken, and V is the volume, in ml, of disodium EDTA solution consumed. Each 5.004 mg of CaCO₃ is equivalent to 1 ml 0.05 M disodium EDTA.

0.1 N Ferrous Ammonium Sulfate, (28.405 g of FeSO₄(NH₄)₂SO₄ per litre)

Dissolve 40 g of ferrous ammonium sulfate hexahydrate in a 100 ml portion of a mixture of 100 ml of sulfuric acid and 100 ml of water previously cooled, add water to 1,000 ml, and standardize as follows:

Titrate 25 ml of this solution with 0.1 N ceric sulfate, using 2 drops of orthophenanthroline TS as the indicator, until a red colour of the solution changes to pale blue. Calculate the normality from the volume of 0.1 N ceric sulfate consumed.

10 N Hydrochloric Acid

Prepare and standardize, as directed under 1 N hydrochloric acid, using 950 ml of hydrochloric acid.

6 N Hydrochloric Acid

Prepare and standardize, as directed under 1 N hydrochloric acid, using 570 ml of hydrochloric acid.

2 N Hydrochloric Acid

Prepare and standardize, as directed under 1 N hydrochloric acid, using 190 ml of hydrochloric acid.

1 N Hydrochloric Acid, (36.461 g of HC1 per litre)

Dilute 95 ml of hydrochloric acid with water to 1,000 ml. Standardize by one of the following methods:

Method I: Dissolve about 1.5 g sodium carbonate (standard reagent) previously dried at about 270° for 1 h and accurately weighed, in 100 ml of water, and titrate with hydrochloric acid, using 2 drops of bromophenol blue TS as the indicator. Near the endpoint, boil to expel carbon dioxide, cool and continue to titrate. Calculate the normality.

Method II: Add 130 ml of water and 5 drops of nitric acid to 20 ml of 1 N hydrochloric acid. While stirring constantly, add about 40 ml of silver nitrate solution (1 in 10) or even more if necessary, until the precipitation is completed. Boil the mixture gently for 5 min, allow to stand in the dark until the precipitate settles. Transfer the precipitate completely into a tared Gooch crucible, dry to constant weight at 110°, and wash with water, slightly acidified with nitric acid, until the washings give no reaction for silver. Dry to constant weight at about 110°. From the weight of silver chloride obtained, calculate the normality of hydrochloric acid.

0.5 N Hydrochloric Acid

Using 47.5 ml of hydrochloric acid, prepare and standardize, as directed under 1 N hydrochloric acid.

0.1 N Hydrochloric Acid

Prepare this solution by diluting 1 N hydrochloric acid with water to 10 volumes, or using 9.5 ml of hydrochloric acid, prepare as directed under 1 N hydrochloric acid. Standardize as directed under 1 N hydrochloric acid.

0.02 N Hydrochloric Acid

Dilute 0.1 N hydrochloric acid with water to 5 volumes, and standardize as directed under 1 N hydrochloric acid.

0.01 N Hydrochloric Acid

Dilute 0.1 N hydrochloric acid with water to 10 volumes, and standardize as directed under 1 N hydrochloric acid.

0.002 N Hydrochloric Acid

Dilute 0.1 N hydrochloric acid with water to 50 volumes.

0.001 N Hydrochloric Acid

Dilute 0.1 N hydrochloric acid with water to 100 volumes.

0.5 N Hydroxylamine Hydrochloride, (34.745 g of NH₇OHHC1 per litre)

Dissolve 35 g of hydroxylamine hydrochloride in 150 ml of water, and dilute to 1,000 ml with anhydrous methanol. To 500 ml of this solution add 15 ml of a 0.04% solution of bromophenol blue in ethanol, and titrate with 0.5 N triethanolamine until the solution appears greenish blue by transmitted light. Prepare this solution fresh before use.

0.1 N Iodine, (12.690 g of iodine per litre)

Dissolve 14 g of iodine in a solution of 36 g of potassium iodide dissolved in 100 ml of water. Add 3 drops of hydrochloric acid and water to 1,000 ml, and standardize as follows.

Weight accurately about 0.15 g of arsenic trioxide previously pulverized and dried to constant weight at 100°, and dissolve in 20 ml of 1 N sodium hydroxide by heating if necessary. Dilute with about 40 ml of water, add 2 drops of methyl orange TS, and add dilute hydrochloric acid until the yellow colour changes to pale pink. Add 2 g of sodium bicarbonate, dilute with 50 ml of water, and add 3 ml of starch TS. Titrate with 0.1 N iodine until a sustaining blue colour is produced. Store in a glass stoppered bottle and restandardize frequently. Calculate the normality. Each 4.946 mg of As₂O₃ is equivalent to 1 ml of 0.1 N iodine.

0.1 N Lithium Methoxide, (3.797 g of CH₃OLi per litre)

Dissolve 600 mg of freshly cut lithium metal in a mixture of 150 ml of absolute methanol and 850 ml of benzene. Filter the resulting solution if it is cloudy, and standardize it as follows: dissolve about 80 mg of benzoic acid, accurately weighed, in 35 ml of dimethylformamide, add 5 drops of thymol blue TS, and titrate with the lithium methoxide solution to a dark blue endpoint. (Caution. Protect the solution from absorption of carbon dioxide and moisture by covering the titration vessel with aluminium foil while dissolving the benzoic acid sample and during the titration.) Each ml of 0.1 N lithium methoxide is equivalent to 12.21 mg of benzoic acid.

0.1 N Magnesium Chloride, (4.761 g of MgC12 per litre)

Dissolve 10.5 g of magnesium chloride in freshly boiled and cooled water to make 1,000 ml.

0.1 M Mercuric Nitrate, (32.46 g of Hg(NO₃)₂ per litre)

Dissolve about 35 g of mercuric nitrate, Hg(NO₃)₂.H₂O, in a mixture of 5 ml of nitric acid and 500 ml of water, and dilute with water to 1,000 ml. Standardize the solution as follows: transfer an accurately measured volume of about 20 ml of the solution into an Erlenmeyer flask, and add 2 ml of ferric ammonium sulfate TS. Cool to below 20°, and titrate with 0.1 N ammonium thiocyanate to the first appearance of a permanent brownish colour. Calculate the molarity.

0.1 N Oxalic Acid, (4.502 g of H2C2O4 per litre)

Dissolve 6.45 g of oxalic acid, H₂C₂O₄.2H₂O, in sufficient water to make 1,000 ml. Standardize by titration against freshly standardized 0.1 N potassium permanganate as directed under Potassium Permanganate 0.1 N. Store this solution in glass-stoppered bottles, protected from light.

0.1 N Perchloric Acid, (10.046 g of HClO₄ per litre)

Transfer about 8.5 ml of 70% perchloric acid into a 1,000-ml flask, add 950 ml of glacial acetic acid, and shake well. Add 15 ml of acetic anhydride gradually by dividing into 1 ml portions, and then dilute with acetic acid to 1,000 ml. Allow the solution to stand overnight.

Standardize as follows: add 50 ml of glacial acetic acid to 0.4 g of potassium biphthalate, previously dried at 120° for 1 h and accurately weighed, and heat to dissolve on a water bath. Titrate with 0.1 N perchloric acid to the end point, at which the colour changes from violet to blue, using 1 ml of 0.05% acetic anhydride solution of crystal violet as the indicator, and calculate the normality by the following formula:

Normality factor = (Weight of potassium biphthalate (g) x 1,000 x 10) / (The number of ml of 0.1 N perchloric acid consumed x 204.22)

0.1 N Potassium Acid Phthalate, (20.42 of KHC₆H₄(COO) per litre)

Dissolve 20.42 g of primary standard potassium biphthalate, KHC₆H₄(COO)₂, in glacial acetic acid in a 1,000-ml volumetric flask, warming on a steam bath if necessary to effect solution and protecting the solution from contamination by moisture. Cool to room temperature, dilute to volume with glacial acetic acid, and mix.

0.1 N Potassium Bromate, (2.784 g of KBrO₃ per litre)

Dissolve 2.8 g of potassium bromate in sufficient water to make 1,000 ml.

Standardize as follows: transfer 40 ml of the solution into a glass-stoppered flask, and add 3 g of potassium iodide and 3 ml of hydrochloric acid. Stopper tightly, and allow to stand for 5 min in the dark. Titrate the free iodine with 0.1 N sodium thiosulfate, using starch TS as the indicator. Perform a blank test in the same manner as the sample.

0.1 N Potassium Dichromate, (4.903 g of K₂Cr₂O7 per litre)

Dissolve 4.904 g of potassium dichromate previously pulverized and dried to constant weight at 120°, in sufficient water to make 1,000 ml.

0.5 N Potassium Hydroxide, Ethanolic

Dissolve about 35 g of potassium hydroxide, KOH, in 20 ml of water, and sufficient aldehyde-free alcohol to make 1,000 ml. Allow the solution to stand in a tightly stoppered bottle for 24 h. Then quickly decant the clear supernatant liquid into a suitable, tight container, and standardize as follows: transfer quantitatively 25 ml of 0.5 N hydrochloric acid into a flask, dilute with 50 ml of water, add 2 drops of phenolphthalein TS, and titrate with the ethanolic potassium hydroxide solution until a permanent, pale pink colour is produced. Calculate the normality. Store this solution in a tightly stoppered bottle protected from light.

1 N Potassium Hydroxide, (56.109 g of KOH per litre)

Using about 70 g of potassium hydroxide, prepare and standardize as directed under 1 N sodium hydroxide. Each 204.2 mg of KHC₆H₄(COO)₂ is equivalent to 1 ml of 1 N potassium hydroxide.

0.5 N Potassium Hydroxide

Dilute 1 N potassium hydroxide with water freshly boiled and cooled to 5 volumes, or using about 35 g of potassium hydroxide, prepare as directed under 1 N potassium hydroxide.

0.1 N Potassium Hydroxide

Dilute 1 N potassium hydroxide with water, freshly boiled and cooled, to 10 volumes, or using about 7 g of potassium hydroxide, prepare as directed under 1 N potassium hydroxide. Standardize as directed under 1 N potassium hydroxide.

0.05 M Potassium Iodate, (10.70 g of KIO₃ per litre)

Dissolve 10.700 g of potassium iodate of primary standard quality, KIO3, previously dried at 110° to constant weight, in sufficient water to make 1,000 ml.

0.1 N Potassium Permanganate, (3.161 g of KMnO₄ per litre)

Dissolve about 3.3 g of potassium permanganate in 1,000 ml of water, and boil for about 15 min. Allow to stand in a tightly closed flask for at least 2 days, and filter through a fine porosity sintered glass crucible. Store in a glass-stoppered, light-resistant bottle, and restandardize before use.

Standardize as follows: dissolve 0.2 g of sodium oxalate previously dried at 110° to constant weight and accurately weighed, in about 250 ml of water. Add 7 ml of sulfuric acid, heat to about 70° and titrate with 0.1 N potassium permanganate while hot. Each 6.700 mg of Na₂C₂O₄ is equivalent to 1 ml of 0.1 N potassium permanganate.

0.01 M Potassium Sulfate, (1.743 g of K₂SO₄ per litre)

Dissolve 1.743 g of potassium sulfate, previously dried at 110° for 4 h, in sufficient water, freshly boiled and cooled, to make 1,000 ml.

0.1 N Silver Nitrate, (16.99 g of AgNO₃ per litre)

Dissolve about 17.5 g of silver nitrate, AgNO3, in 1,000 ml of water, and standardize the solution as follows: dilute about 40 ml, accurately measured, of the silver nitrate solution with about 100 ml of water, heat the solution, and add slowly, with continuous stirring, dilute hydrochloric acid TS until precipitation of the silver is complete. Boil the mixture cautiously for about 5 min, then allow it to stand in the dark until the precipitate has settled and the supernatant liquid has become clear. Transfer the precipitate completely to a tared filtering crucible, and wash it with small portions of water slightly acidified with nitric acid. Dry the precipitate at 110° to constant weight. Each 14.332 mg of silver chloride obtained is equivalent to 1 ml of 0.1 N silver nitrate. Protect the silver chloride from light as much as possible during the determination.

0.05 N Sodium Arsenite, (3.248 g of NaAsO₂ per litre)

Transfer 2.4725 g of arsenic trioxide, which has been pulverized and dried at 100° to constant weight, to a 1,000-ml volumetric flask, dissolve it in 20 ml of 1 N sodium hydroxide, and add 1 N sulfuric acid or 1 N hydrochloric acid until the solution is neutral or only slightly acid to litmus. Add 15 g of sodium bicarbonate, dilute to volume with water, and mix.

0.1 N Sodium Chloride, (5.844 g of NaCl per litre)

Dissolve 5.845 g of sodium chloride, previously dried at 110° for 2 h, in sufficient water to make 1,000 ml.

1 N Sodium Hydroxide, (39.997 g of NaOH per litre)

Dissolve 45 g of sodium hydroxide in about 950 ml of water, and add a saturated barium hydroxide solution, freshly prepared, until not further precipitate is formed. Shake the mixture thoroughly, and allow to stand overnight in a stoppered bottle. Decant the supernatant liquid or filter the solution, and standardize by one of the following methods. Store in a well-fitted, rubber-stoppered bottle, or in a bottle with a soda-lime tube, and restandardize frequently.

Method I: Dilute 25 ml of 1 N hydrochloric acid or 1 N sulfuric acid with 50 ml of water, freshly boiled and cooled, and titrate with 1 N sodium hydroxide, using 2 drops of phenolphthalein TS as the indicator.

Method II: Dissolve about 5 g of potassium biphthalate previously powdered, dried at 100° for 3 h and weighed accurately, in 75 ml of water, freshly boiled and cooled, and titrate with 1 N sodium hydroxide solution, using drops of phenolphthalein Ts as the indicator. Each 204.2 mg of potassium biphthalate is equivalent to 1 ml of 1 N sodium hydroxide.

0.5 N Sodium Hydroxide

Using about 22 g of sodium hydroxide, prepare, standardize and store, as directed under 1 N sodium hydroxide. Restandardize frequently.

0.2 N Sodium Hydroxide

Dilute 1 N sodium hydroxide with water, freshly boiled and cooled, to 5 volumes, or use about 9 g of sodium hydroxide and prepare as directed under 1 N sodium hydroxide. Standardize and store, as directed under 1 N sodium hydroxide. Restandardize frequently.

0.1 N Sodium Hydroxide

Dilute 1 N sodium hydroxide with water, freshly boiled and cooled, to 10 volumes, or use about 4.5 g of sodium hydroxide and prepare as directed under 1 N sodium hydroxide. Standardize and store, as directed under 1 N sodium hydroxide. Restandardize frequently.

0.05 N Sodium Hydroxide

Dilute 1 N sodium hydroxide with water, freshly boiled and cooled, to 20 volumes. Standardize and store, as directed under 1 N sodium hydroxide. Restandardize frequently.

0.02 N Sodium Hydroxide

Dilute 0.1 N sodium hydroxide with water, freshly boiled and cooled, to 5 volumes. Standardize and store, as directed under 1 N sodium hydroxide. Restandardize frequently.

0.01 N Sodium Hydroxide

Dilute 0.1 N sodium hydroxide with water, freshly boiled and cooled, to 10 volumes. Standardize and store, as directed under 1 N sodium hydroxide. Restandardize frequently.

0.1 N Sodium Methoxide, in Pyridine, (5.40 g of CH₃ONa per litre)

Weigh 14 g of freshly cut sodium metal, and cut into small cubes. Place about 0.5 ml of anhydrous methanol in a round-bottom 250-ml flask equipped with a ground-glass joint, add 1 cube of the sodium metal, and when the reaction subsides, add the remaining sodium metal to the flask. Connect a water-cooled condenser to the flask, and slowly add 100 ml of anhydrous methanol, in small portions, through the top of the condenser. Regulate the addition of the methanol so that the vapours are condensed and do not escape through the top of the condenser. After addition of the methanol is complete, connect a drying tube to the top of the condenser, and allow the solution to cool. Transfer 17.5 ml of this solution (approximately 6 N) into a 1,000-ml volumetric flask containing 70 ml of anhydrous methanol, and dilute to volume with freshly distilled pyridine. Store preferably in the reservoir of an automatic buret suitably protected from carbon dioxide and moisture. Standardize the solution as follows: weigh accurately about 400 mg of benzoic acid, transfer it into a 250 ml wide-mouth Erlenmeyer flask, and dissolve it in 50 ml of freshly distilled pyridine. Add a few drops of thymolphthalein TS, and titrate immediately with the sodium methoxide solution to a blue endpoint. During the titration, direct a gentle stream of nitrogen into the flask through a short piece of 6-mm glass tubing fastened near the tip of the buret. Perform a blank determination, correct for the volume of sodium methoxide solution consumed by the blank, and calculate the normality. Each 12.21 mg of benzoic acid is equivalent to 1 ml of 0.1 N sodium methoxide in pyridine.

0.1 M Sodium Nitrite, (7.900 g of NaNO₂ per litre)

Dissolve 7.5 g of sodium nitrite, NaNO2, in sufficient water to make 1,000 ml, and standardize the solution as follows: Weigh accurately about 500 mg of U.S.P. Sulfanilamide Reference Standard, previously dried at 105° for 3 h, and transfer to a beaker or a casserole. Add 50 ml of water and 5 ml of hydrochloric acid, and stir well until dissolved. Cool to 15°, and add about 25 g of crushed ice, then titrate slowly with the sodium nitrite solution, stirring vigorously until a blue colour is produced immediately when a glass rod dipped in the titrated solution is streaked on a smear of starch iodide paste TS. When the titration is complete, the endpoint should be reproducible after the mixture has been standing for 1 min. Calculate the molarity. Each 17.22 mg of sulfanilamide is equivalent to 1 ml of 0.1 M sodium nitrite.

0.1 N Sodium Thiosulfate, (15.82 g of Na₂S₂O₃ per litre)

Dissolve about 26 g of sodium thiosulfate, Na₂S₂O₃'5H₂O, and 200 mg of sodium carbonate, Na₂CO₃, in 1,000 ml of recently boiled and cooled water. Standardize the solution as follows: weigh accurately about 210 mg of primary standard potassium dichromate, previously pulverized and dried at 120° for 4 h, and dissolve in 100 ml of water in a 500 ml glass-stoppered flask. Swirl to dissolve the sample, remove the stopper and quickly add 3 g of potassium iodide, KI, and 5 ml of hydrochloric acid. Stopper the flask, swirl to mix, and let stand in the dark for 10 min. Rinse the stopper and inner walls of the flask with water, and titrate the liberated iodine with the sodium thiosulfate solution until the solution is only faint yellow in colour. Add starch TS, and continue the titration to the discharge of the blue colour. Calculate the normality.

0.01 N Sodium Thiosulfate

Dilute 0.1 N sodium thiosulfate with water, freshly boiled and cooled, to 10 volumes. Standardize as directed under 0.1 N sodium thiosulfate before use.

4 N Sulfuric Acid

This solution contains 196.155 g of H2SO4 per 1,000 ml.

Using 120 ml of sulfuric acid, prepare and standardize, as directed under 1 N sulfuric acid.

1 N Sulfuric Acid, (49.039 g of H₂SO₄ per litre)

While stirring, slowly add 30 ml of sulfuric acid to about 1,000 ml of water, allow to cool to 20°, and standardize with sodium carbonate (standard reagent) as directed under 1 N hydrochloric acid. Each 52.99 mg of Na₂CO₃ is equivalent to 1 ml of 1 N sulfuric acid.

0.5 N Sulfuric Acid

Using 15 ml of sulfuric acid, prepare and standardize, as directed under 1 N sulfuric acid.

0.2 N Sulfuric Acid

Using 6 ml of sulfuric acid, prepare and standardize, as directed under 1 N sulfuric acid.

0.1 N Sulfuric Acid

Dilute 1 N sulfuric acid with water to 10 volumes, or using 3 ml of sulfuric acid, prepare as directed under 1 N sulfuric acid. Standardize as directed under 1 N sulfuric acid.

0.02 N Sulfuric Acid

Dilute 0.1 N sulfuric acid with water to 5 volumes, and standardize as directed under 1 N sulfuric acid.

0.01 N Sulfuric Acid

Dilute 0.1 N sulfuric acid with water to 10 volumes, and standardize as directed under 1 N sulfuric acid.

0.5 N Sulfuric Acid, Ethanolic

Add cautiously, with stirring, 13.9 ml of sulfuric acid to a sufficient quantity of absolute ethanol to make 1,000 ml. Alternatively, this solution may be prepared by diluting 100 ml of 5 N sulfuric acid with absolute ethanol to make 1,000 ml.

0.1 M Thorium Nitrate, (48.01 g of Th(NO₃ )₄ per litre)

Weigh accurately 55.21 g of thorium nitrate Th(NO₃)₄.4H₂O, dissolve it in water, dilute to 1,000 ml, and mix. Standardize the solution as follows: transfer 50 ml into a 500-ml volumetric flask, dilute to volume with water, and mix. Transfer 50 ml of the diluted solution into a 400-ml beaker, add 150 ml of water and 5 ml of hydrochloric acid, and heat to boiling. While stirring, add 25 ml of a saturated solution of oxalic acid, then digest the mixture for 1 h just below the boiling point and allow to stand overnight. Decant through Whatman No. 42, or equivalent, filter paper, and transfer the precipitate to the filter using about 100 ml of a wash solution consisting of 70 ml of the saturated oxalic acid solution, 430 ml of water, and 5 ml of hydrochloric acid. Transfer the precipitate and filter paper to a tared tall-form porcelain crucible, dry, char the paper, and ignite at 950° for 1.5 h or to constant weight. Cool in a desiccator, weigh, and calculate the molarity of the solution by the formula 200W/264.04, in which W is the weight, in g, of thorium oxide obtained.

0.1 N Titanous Chloride, (15.426 g of TiC1 per litre)

Mix 200 ml of 15% titanous chloride and 150 ml of hydrochloric acid, and dilute with freshly boiled and cooled water to 2,000 ml. Transfer into a light-shaded bottle equipped with a buret, replace the air in the bottle with hydrogen, and allow to stand for 2 days before use. Standardize as follows: place 3 g of ferrous ammonium sulfate in a wide-mouthed 500 ml flask, dissolve in 50 ml of freshly boiled and cooled water in an atmosphere of carbon dioxide and add 25 ml dilute sulfuric acid (27 in 100). Pass carbon dioxide through the solution, then quickly add 40 ml of 0.1 N potassium permanganate and add 0.1 N titanous chloride until the endpoint is nearly reached. Immediately add 5 g of ammonium thiocyanate, and titrate the solution with 0.1 N titanous chloride to the end point, when the colour of the solution disappears. Perform a blank test in the same manner as the sample.

Normality factor = Volume of 0,1 N potassium permanganate added (ml) / The number of ml of 0.1 N titanous chloride consumed

0.5 N Triethanolamine, (74 g of N(CH₂CH₂OH)₃ per litre)

Transfer 65 ml (74 g) of 98% triethanolamine into a 1,000 ml volumetric flask, dilute to volume with water, stopper the flask, and mix thoroughly.

0.01 M Zinc Acetate, (1.835 g of Zn(CH₃COO)₂ per litre)

Dissolve 2 g of zinc acetate in sufficient water to make 1,000 ml. Standardize as follows: to 25 ml of 0.01 M zinc acetate, add 2 ml of ammonia/ammonium chloride buffer solution and sufficient water to about 100 ml. Titrate the solution with 0.01 M disodium ethylenediaminetetraacetate, using 3 drops of eriochrome black TS as the indicator.

0.025 M Zinc Chloride, (3.407 g of ZnC1₂ per litre)

Place about 1.6 g of zinc in a beaker, add 30 ml of dilute hydrochloric acid, cover with a watch glass, and allow to stand. Dissolve by heating gently on a water bath after the initial rapid release of hydrogen gas. Wash the watch glass and the inside wall of the beaker with water, evaporate to almost dryness on a water bath, cool, and add water to 1,000 ml. Standardize as directed for 0.05 M zinc sulfate. Calculate the molarity.

0.05 M Zinc Sulfate, (8.072 g of ZnSO₄ per litre)

Dissolve about 15 g of zinc sulfate, ZnSO₄.7H₂O in sufficient water to make 1,000 ml, and standardize the solution as follows: dilute about 35 ml 0.05 M zinc sulfate accurately measured, with 75 ml of water, add 5 ml of ammonia/ammonium chloride buffer TS and 0.1 ml of eriochrome black TS, and titrate with 0.05 M disodium ethylenediaminetetraacetate until the solution is deep blue in colour. Calculate the molarity.