ⅤⅢ．Spectrophotometric Techniques 3
A．Apparatus 3
1．Microscope Colorimeter and Spectrophotometer 3
(a) Drop Techniques 4
Norberg Drop Technique 4
Glick-Ferguson Drop Technique 4
Rotman Drop Technique 5
Edstrom Drop Technique 5
Ornstein-Lehrer Shrinking-Drop Technique 11
(b) Capillary Cuvette Techniques 12
Holter-Lovtrup-Krugelis Arrangement 12
Malmstrom-Glick Modification 13
2．Adaptation of Commercial Spectrophotometers 17
(a) Lowry et al．Adaptation of the Beckman Spectrophotometer 18
(b) Glick，Grunbaum，and Greenberg Modification 20
(c) Other Modifications of the Beckman Spectrophotometer 25
(d) Adaptation of the Coleman Spectrophotometer 27
3．Transfer of Liquid to Cuvettes 27
B．Optical Extinction In Measurement 28
C．Methods 29
Sodium 29
Bott Method for Sodium 31
Stone and Goldzieher Method for Sodium 32
Potassium 34
Eckel Method for Potassium 36
Calcium 38
Weybrew，Matrone，and Baxley Modification of the Sendroy Method for Calcium 44
Gillis，Van Der Stock，and Hoste Loretin Method for Calcium 46
Natelson and Penniall Alizarin Method for Calcium 46
Chiamori and Henry Chloranilate Method for Calcium 47
Brush Erio SE Procedure for Calcium 49
Kingsley and Robnett Modification of the Baar Method for Calcium 50
Magnesium 51
Glick，Freier，and Ochs Titan Yellow Method for Magnesium 54
Levine and Cummings Eriochrome Black T Method for Magnesium 55
Iron 56
Ashing 60
Kingsley and Getchell Bathophenanthroline Method for Iron 60
Ramsay Dipyridyl Method for Iron 61
Wootton Adaptation of the Ultraviolet Absorption Method of Bastian，Weberling，and Palilla for Iron 62
Zinc 63
Malmstrom and Glick Modification of the Dithizone Method for Zinc 66
Fredricks，Tanaka，and Valentine Modification of the Hoch and Vallee Method for Extraction of Zinc from Blood Cells and Measurement by the Dithizone Method 68
Chloride 70
Stiff Modification of the Sendroy Method for Chloride 73
King and Bain Modification of the Haslewood-King Method for Chloride 76
Lowry，Roberts，Leiner，Wu，and Farr Rhodanine Method for Chloride 77
Phosphorus and Phosphate 79
Preparation of Sample 79
Molybdenum Blue Methods 80
Labile Phosphates 82
Phospholipid 84
Nucleic Acid 85
Yellow Phosphomolybdic and Phosphomolybdovanadic Acid Methods 85
Other Methods 87
Lowry，Roberts，Leiner，Wu，and Farr Molybdenum Blue Method for Inorganic and Labile-，Acid-Soluble-，Lipid-，Nucleic Acid-，and Residual-Phosphorus 88
Peel and Loughman Modification of the Lowry and Lopez Molybdenum Blue Method for Inorganic Phosphate in the Presence of Labile Phosphate Esters 92
Bonting-Greenberg-Glick Adaptation of the Taussky and Shorr Modification of the Summer Molybdenum Blue Method for Inorganic Phosphate and Labile Phosphate Esters 93
Hirata and Appleman Modification of the Phosphomolybdic Acid Method for Total Phosphorus 94
Mokrasch Phosphomolybdic Acid Method for Free Phosphate in the Presenee of Labile Phosphate Compounds 95
Michelsen Modification of the Phosphomolybdovanadic Acid Method for Phosphate 96
Phosphatases 96
Lowry，Roberts，Wu，Hixon，and Crawford Modification of the Bessey，Lowry，and Brock Direct Absorption Method for Alkaline and Acid Phosphatases 100
Vescia Direct Absorption Method for Acid Phosphatase 102
Seligman，Chauncey，Nachlas，Manheimer，and Ravin Diazo Dye Method for Acid and Alkaline Phosphatases 102
Allen and Slater Modification for Alkaline Phosphatase of the Gomori Diazo Dye Method for Esterase 104
Lowry，Roberts，Wu，Hixon，and Crawford Molybdenum Blue Method for Adenosinetriphosphatase 104
Phosphoglucomutase 105
Buell，Lowry，Roberts，Chang，and Kapphahn Molybdenum Blue Method for Phosphoglucomutase 106
Phosphoglucoisomerase 107
Buell，Lowry，Roberts，Chang，and Kapphahn Fructose Method for Phosphoglucoisomerase 108
Aldolase，Phosphofructokinase，and Hexokinase 109
Lowry，Roberts，Wu，Hixon，and Crawford Modification of the Sibley and Lehninger Method for Aldolase 112
Buell，Lowry，Roberts，Chang，and Kapphahn Aldolase Methods for Phosphofructokinase and Hexokinase 113
Sulfur Compounds and Sulfatases 115
Spencer Modifications of the Benzidine Method of Dodgson and Spencer for Sulfate 119
Spencer Modified Chloranilate Method for Sulfate 121
Spencer Modified Benzidine and Chloranilate Methods Adapted to Dodgson and Spencer Procedures for Sulfatases 122
Malmstrom and Glick Adaptation of the Direct Absorption Method of Huggins and Smith for Phenol Sulfatase 123
Sulfate and Glucuronate Conjugation 125
Glick and Stecklein Adaptation of the Bernstein and McGilvery Method for Enzymatic Conjugation of m-Aminophenol 125
Total Nitrogen and Ammonia 127
Digestion of Sample for Determination of Total Nitrogen 127
Brüel，Holter，Linderstrom-Lang and Rozīts Procedure 129
Doyle and Omoto Modification 131
Shaw and Beadle Modification 131
Boell and Shen Modification 132
Lubochinsky and Zalta Procedure 132
Exley Procedure 132
Grunbaum，Schaffer，and Kirk Procedure 134
Direct Estimation of Ammonia 136
Lubochinsky and Zalta Method 137
Brown，Duda，Korkes，and Handler Modification 138
Exley Adaptation of the Russell Method 139
Estimation of Ammonia after Separation 141
Urea 142
Engel and Engel Modification of the Xanthydrol Method 143
De Nieves and Pike Adaptation of the Archibald Method for Urea 145
Uric Acid，Creatine，Creatinine，and Allantoin 145
Protein and Proteases 146
Lowry，Rosebrough，Farr，and Randall Phenol-Reagent Method for Protein 153
Duspiva Adaptation of the Anson Phenol-Reagent Method for Protease 154
Nayyar and Glick Adaptation of the Greif Bromsulfalein Binding Method for Protein and Protease 155
Lovtrup Adaptation of the Pechmann Color-Liberation Method for Cathepsin and Trypsin 157
Martin，Golubow，and Axelrod Direct Absorption Method for Chymotrypsin 158
Bundy Direct Absorption Method for Chymotrypsin 159
Amino Acid and Peptide 160
Copper Methods 162
Ninhydrin Methods 162
Other Methods 163
Kay，Harris，and Entenman Method for Amino Acids Separated by Paper Chromatography 164
Woiwod Copper Method for Free Amino-Nitrogen 166
Spies Direct Copper Method for Amino Acid 168
Grunbaum and Glick Method f or Hydroxyproline 168
Prockop and Udenfriend Method for Hydroxyproline 170
Hutterer and Singer Method for Hydroxyproline 172
Peptidases 173
Dye Formation Methods 174
Direct Absorption Methods 176
Ravin and Seligman Dye Formation Method for Carboxypeptidase 177
Green，Tsou，Bressler，and Seligman Dye Formation Method for Leucine Aminopeptidase 178
Allen Direct Absorption Method for Dehydropeptidase Ⅰ 179
Mitz and Schlueter Direct Absorption Method for Acylase Ⅰ 180
Schmitt and Siebert Direct Absorption Method for Peptidase 181
Arginase 182
Malmgren and Sylvén Adaptation of the Xanthydrol Method for Urea to Determination of Arginase 182
Glucose 183
Greenberg and Glick Modification of the Glucose Oxidase Method for Glucose 185
Glycogen 186
Greenberg and Glick Modification of the Glucose Oxidase Method for Glycogen 188
Phosphorylases 189
Glycogen Phosphorylase 189
Purine Nucleoside Phosphorylase 190
Buell，Lowry，Roberts，Chang，and Kapphahn Modification of the Inorganic Phosphate Method for Glycogen Phosphorylase (Synthesis) 191
Greenberg and Glick Modification of Methods for Glycogen Phosphorylase (Degradation and Synthesis) 192
Robins，Smith，and McCaman Adaptation of the Kalckar Method for Purine Nucleoside Phosphorylase 194
Hexosamine 195
Parekh and Glick Use of the Exley Method for Total Hexosamine 197
Heparin 199
Parekh and Glick Modification of the Jacques，Monkhouse，and Stewart Metachromatic Dye Method for Heparin 200
Hyaluronidase 201
Glick，Ottoson，and Edmondson Adaptation of the Tolksdorf et al．Modification of the Turbidimetric Method for Hyaluronidase and Hyaluronidase Inhibitor 202
Ascorbic，Dehydroascorbic，and Diketogulonic Acids 204
Glick，Alpert，and Stecklein Adaptation of the Indophenol Dye Method for L-Ascorbic Acid and the Dinitrophenylhydrazine Coupling Method for Dehydro-L-Ascorbic plus Diketo-L_Gulonic Acids 209
β-Glucuronidase 211
Wattenberg and Glick Adaptation of the Phenolphthalein Glucuronide Method for β-Glucuronidase 213
β-D-Galactosidase and α- and β-D-Glucosidases 214
Cohen，Tsou，Rutenburg，and Seligman Method for β-D-Galactosidase，and Cohen，Rutenburg，Tsou，Woodbury，and Seligman Method for β-D-Glucosidase 216
Goldbarg，Tsou，Rutenburg，Rutenburg，and Seligman Method for α-D-Glucosidase 218
Pardee，Jacob，and Monod Modification of the Lederberg Method for β-D-Galactosidase 219
Nucleic Acids 219
Ceriotti，Keck，and Bonting-Jones Adaptations of the Indole Method of Dische for Deoxyribonucleic Acid 222
Scott，Fraccastoro，and Taft Ultraviolet Absorption Method for Ribonucleic and Deoxyribonucleic Acids 225
Ceriotti Modification of the Orcinol Method for Ribonucleic Acid 227
McIntire and Sproull Ultraviolet Absorption Method for Deoyribonucleic Acid 228
Edstrom and Edstrom-Zawiak Ultraviolet Absorption Method for Ribonucleic and Deoxyribonucleic Acids and Single Cells 229
Deoxyribonuclease and Ribonuclease 232
Kurnick and Kurnick-Sandeen Methyl Green Binding Methods for Deoxyribonucleases 234
Fiers and Moller Ultraviolet Absorption Method for Ribonuclease 235
Houck Turbidimetric Method for Ribonuclease 239
Extraction and Fractionation of Lipids 240
Schmidt-Nielsen Method for Extraction and Fractionation of Lipids 240
Schaffer，Grunbaum，and Kirk Scheme for Extraction and Fractionation of Lipids 242
Total Lipid 246
Fatty Acid 246
Phospholipids and Sphingolipids 247
Robins，Lowry，Eydt，and McCaman Adaptation of the Analytical Scheme of Schmidt et al．for Determination of Separate Classes of Phospholipids and Sphingolipids in the Same Sample 248
Cholesterol and Cholesterol Esters 253
Cavanaugh and Glick Adaptation of the Schoenheimer-Sperry Method for Cholesterol and Cholesterol Esters 254
Adaptation by Glick and Lopez to the Cavanaugh-Glick Procedure of Modifications by Brown et al．and Zak and Epstein for Cholesterol and Cholesterol Esters 256
Lipolytic Enzymes 258
Purr Chromogenic Substrate Method for Esterase 260
Sumner，Herr，and Yesair Phenol Method for Esterase 262
Seligman and Nachlas Azo Dye Method for Esterase and Lipase 263
Doyle，Rappaport，and Doyle Adaptation of the Gomori Azo Dye Method for Esterase 265
Cholinesterase 265
Lowry，Roberts，Wu，Hixon，and Crawford Phenol Red Method for Cholinesterase 268
Ravin，Tsou，and Seligman Azo Dye Method for Nonspecific Cholinesterase 270
Coenzyme A 271
Greenberg and Glick Procedure of the Adaptation by Malmstrom and Glick of the Sulfanilamide Acetylation Method (Kaplan-Lipmann) for Coenzyme A 273
Histamine 276
Lowry，Graham，Harris，Priebat，Marks，and Bregman Dinitrofluorobenzene Method for Histamine 277
Catalase 280
Peroxidase 281
Straus N，N-Dimethyl-p-Phenylenediamine Method for Peroxidase 282
Cytochrome Oxidase 283
Hess and Pope Direct Colorimetric Method for Cytochrome c Oxidase 285
Straus N，N-Dimethyl-p-Phenylenediamine Method for Cytochrome c Oxidase 286
Sulfhydryl Groups 287
Pyridine Nucleotides 288
Dehydrogenases 291
Methods Based on Measurement of Pyridine Nucleotides 291
Lactic，Malic，and Glutamic Dehydrogenases and a-Keto Acids 291
Glucose-6-Phosphate and 6-Phosphogluconate Dehydrogenases 291
Glutathione Reductase 292
Phosphoglycerokinase and Glyceraldehyde Phosphate Dehydrogenase 292
Robins，Roberts，Eydt，Lowry，and Smith Quinolylhydrazine Method for Malic，Lactic，and Glutamic Dehydrogenases and a-Keto Acids 293
Greenberg and Glick Adaptation of the Direct Absorption Method for Glucose-6-Phosphate and 6-Phosphogluconate Dehydrogenases 295
Methods Based on Measurement of Reduced Tetrazolium Salts 297
Succinic Dehydrogenase 298
Lactic Dehydrogenase 300
a-Glycerophosphate Dehydrogenase 300
Influence of CoQ10 on Dehydrogenases 300
Glick and Nayyar Soluble-Formazan Method for the Succinie Dehydrogenase System 301
Fried，Greenberg，and Antopol Soluble-Formazan Method for Enzymatic a-Glycerophosphate Oxidation 303
Nachlas，Margulies，Goldberg，and Seligman Colloidal-Formazan Method for Lactic Dehydrogenase 305
Nachlas，Margulies，and Seligman Colloidal-Formazan Method for Succinic Dehydrogenase 306
Methods Based on Measurement of Reduced Dichlorophenol-indophenol 307
DPN-Diaphorase and Lactic Dehydrogenase 307
Succinic Dehydrogenase 308
Lactic Dehydrogenase 308
Glucose-6-Phosphate Dehydrogenase 309
Schemes for Combined Analyses 310
Scheme of Paul for determination of Lipid-Phosphorus，Total Lipid，Carbohydrate，Total Nucleic Acid，Deoxyribonucleic Acid，and Protein-Nitrogen in the Same Tissue Sample (1 mg Dry Weight) 311
Ⅸ．Titrimetric Techniques 317
A．Microliter Burettes 317
1．Calibrated Capillary Tube Burettes 319
(a) Screw-Drive Burettes 319
Linderstrom-Lang and Holter Burettes 319
Kirk Burette 322
Burette Attachment to Natelson Microgasometer 322
(b) Gravitational Burettes 322
Conway Burette 324
Grunbaum and Kirk Burette 327
Pecár Burette 327
(c) Calibration 328
2．Micrometer Screw Burettes 329
Scholander Burette 330
Allen Burette 331
Commercial Forms 333
3．Gravimetric Burettes 333
Pecár Burette 334
4．Ocular Micrometer Burettes 334
Loscalzo and Benedetti-Pichler Burette 334
B．Titration Vessels 338
C．Potentiometric Burette Assemblies 341
Linderstrom-Lang，Palmer，and Holter Assembly 341
Other Assemblies for Chloride Titration 342
Sisco，Cunningham，and Kirk Assembly 342
Grunbaum，Schaffer，and Kirk Assembly 343
Hull and Kirk Versatile Differential Electrode Assembly 344
Versatile Commercial Assembly 345
D．Coulometric Assemblies 345
Schreiber and Cooke Assembly 347
Ramsay，Brown，and Croghan Assembly 348
E．Amperometric Assembly 352
F．Methods 352
Sodium 352
Lindner and Kirk Method for Sodium 354
Potassium 355
Norberg Method for Potassium 356
Schall Method for Potassium 358
Sodium and Potassium Combined 360
Linderstrom-Lang Method for Sodium and Potassium 361
Calcium 362
Lindner and Kirk Cerimetric Method for Calcium 366
Freier Modification of the Acidimetric Method for Calcium 368
Griswold and Pace Complexometric Method for Calcium 369
Mori Complexometric Method for Calcium 371
Magnesium 372
Griswold and Pace Complexometric Method for Magnesium 373
Iron 373
Chloride 374
Linderstrom-Lang，Palmer，and Holter Electrometric Method for Chloride 376
Phosphorus and Phosphatase 377
Total Nitrogen and Ammonia 378
Brüel，Holter，Linderstrom-Lang，and Rozīts Method for Ammonia from Acid Digests，with Subsequent Modifications 380
Grunbaum，Schaffer，and Kirk Method for Ammonia from Acid Digests 384
Urea 385
Kinsey and Robison Method for Urea 386
Urease 387
Amide，Peptide，and Nitrate Nitrogen 387
Borsook and Dubnoff Methods for Amide，Peptide，and Nitrate Nitrogen 388
Acid，Alkali，Amino，and Carboxyl Groups 390
McConnell Method for a-Amino Acids 391
Proteolytic Enzymes 392
Linderstrom-Lang and Holter Acidimetric Acetone Method for Proteolytic Enzymes 393
Linderstrom-Lang and Duspiva Alkalimetric Alcohol Method for Proteolytic Enzymes 394
Weil Formol Method for Trypsin 396
Arginase 397
LInderstrom-Lang，Weil，and Holter Methods for Arginase 397
(a) Urease Method 397
(b) Acetone-Alcohol Titration Method 398
Reducing Sugars 399
Holter and Doyle Modification of the Linderstrom-Lang and Holter Iodometric Method for Reducing Sugars 399
Stern and Kirk Modification of the Cerimetric Method for Reducing Sugars 400
Amylase 401
Glycogen 402
Heatley Method for Total Glycogen 402
Heatley and Lindahl Method for Desmo- and Lyoglycogen 404
Total Fatty Acid 404
Schmidt-Nielsen Indicator Titration Method for Total Fatty Acid 405
Grunbaum，Schaffer，and Kirk Potentiometric Titration Method for Total Fatty Acid 406
Iodine Number of Lipids 408
Schmidt-Nielsen Method for Iodine Number 409
Grunbaum，and Kirk Method for Iodine Number 410
ALiesterase，Lipase，and Cholinesterase 413
Glick Acidimetric Method for Aliesterase and Lipase 414
Glick Acidimetric Method for Cholinesterase 414
Catalase 415
Holter and Doyle Adaptation of the Iodometric Method for Catalase 416
Von Hahn Adaptation of the Perborate Method for Catalase 417
Sulfhydryl Groups 417
Ⅹ．Flame Photometric Techniques 421
A．Methods Requiring Atomization of Sample Solutions 422
B．Methods Requiring Dried Sample on a Platinum Wire 425
Ⅺ．Microbiological Techniques 429
A．Microscopic Microbiological Assay 431