Laboratory Manual for Practical Biochemistry

Contents
Section 1: Basic Laboratory Principles
1. Laboratory Equipments, Glassware, Laboratory Hazards and
General Laboratory Rules......................................................................................3
Laboratory Equipments 3
Laboratory Glassware 4
Laboratory Hazards 7
Laboratory Safety Procedure 7
Laboratory First Aid 10
2. Laboratory Units, Basic Calculations and Preparation of Solutions ...............11
Units 11
Preparation of Molar and Normal Solutions 13
Preparation of Percent Solutions 14
3. Acids, Bases, Alkalies, pH, Indicators and Buffers ...........................................15
Acids and Bases 15
Alkali 15
Indicators 15
Buffers 16
Section 2: Qualitative Experiments
4. Carbohydrates .................................................................................
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6. Proteins ..................................................................................................................30
Definition 30
Elemental Composition 30
Functions 30
Classification 30
7. Color Reactions of Proteins ..................................................................................32
Protein and its Color Reactions 32
8. Precipitation Reactions of Proteins .....................................................................38
Methods of Precipitation Reactions 38
Significance of the Precipitation Reactions 39
Scheme for Identification of Unknown Proteins 41
9. Reactions of Nonprotein Nitrogenous (NPN) Substances ..................................42
Nonprotein Nitrogenous Substances 42
10. Scheme for Identification of Physiologically Important Substances ................44
11. Urine Analysis (Normal Urine) ............................................................................45
Analysis of Normal Constituents in Urine 45
Determination of Specific Gravity 45
12. Analysis of Abnormal Constituents in Urine ......................................................50
Physical Characteristics 50
Chemical Constituents 50
13. Inborn Errors of Metabolism ...............................................................................54
Screening Tests for Inborn Errors of Amino Acid Metabolism 54
Screening Tests for Inborn Errors of Carbohydrate Metabolism 56
14. Analysis of Gastric Juice ......................................................................................58
Tests for Normal Constituents 58
Tests for Abnormal Constituents 59
15. Analysis of Saliva ..................................................................................................61
Collection of Saliva 61
16. Preparation of Hemin Crystals ............................................................................63
Preparation of Hemin Crystals 63
17. Sample Collection and Preservation ...................................................................64
Blood Sample Collection and Preservation 64
Urine Collection and Preservation 65
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Contents
Section 3: Quantitative Experiments
18. Estimation of Blood Sugar by Folin-Wu Method ...............................................69
Principle 69
Procedure 69
Calculation 70
Clinical Aignificance 71
Other Methods 71
19. Estimation of Blood Glucose by O-Toluidine Method .......................................72
Principle 72
Procedure 72
Calculation 73
20. Estimation of Blood Urea by Diacetyl Monoxime (DAM) Method ..................74
Principle 74
Procedure 74
Calculation 75
Clinical Significance 75
Other Methods 76
21. Estimation of Serum Total Protiens, Albumin and Albumin/Globulin Ratio
by Biuret Method ..................................................................................................77
Principle 77
Procedure 77
Calculation 78
Clinical Significance 78
22. Estimation of Serum Albumin by Dye-Binding Method ..................................80
Principle 80
Procedure 80
Calculation 80
Other Methods 81
23. Estimation of Serum Inorganic Phosphate by Fiske and
Subba Row Method ...............................................................................................82
Functions of Phosphorus 82
Principle 82
Procedure 83
Calculation 83
Clinical Significance 83
Other Method 84
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24. Estimation of Serum Cholesterol by Zak’s Method ...........................................85
Principle 85
Procedure 85
Calculation 86
Clinical Mignificance 86
Other Methods 86
25. Estimation of Serum Uric Acid by Caraway Method .......................................87
Principle 87
Procedure 87
Calculation 88
Clinical Significance 88
Other Methods 88
26. Estimation of Serum Bilirubin by Malloy and Evelyn Method ........................89
Principle 89
Procedure 89
Calculation 90
Clinical Significance 90
27. Estimation of Serum Creatinine by Jaffe’s Alkaline Picrate Method ..............92
Creatine 92
Principle 92
Calculation 93
Clinical Significance 93
28. Estimation of Vitamin C (Ascorbic Acid) in Plasma by
Sauberlich Method ................................................................................................94
Principle 94
Procedure 94
Calculation 94
Clinical Sgnificance 95
29. Estimation of Vitamin A in Serum by Bessey Method ......................................96
Principle 96
Procedure 96
Calculation 96
Clinical Significance 96
30. Estimation of Serum Transaminases by Reitman and Frankel Method ..........97
Principle 97
Procedure 97
Calculation 98
Clinical Significance 98
Other Methods 99
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31. Estimation of Serum Alkaline Phosphatase by
Kind and King Method .......................................................................................100
Principle 100
Clinical Significance 101
32. Estimation of Serum Calcium by O-Cresolphthalein
Complexone Method ...........................................................................................102
Principle 102
Calculation 103
Clinical Significance 103
Other Methods 103
33. Estimation of Urine Creatinine By Jaffe Alkaline Picrate Method ................104
Principle 104
Procedure 104
Calculation 104
Clinical Significance 105
34. Estimation of Urinary Proteins by Turbidimetric Method .............................106
Principle 106
Procedure 106
Calculation 106
Other Methods 107
Section 4: Clinical Biochemistry Techniques
and Recent Aspects of Clinical
Biochemistry
35. Colorimetry—General Principle and Procedure .............................................111
Principle 111
Procedure 113
Calculation 113
Applications of Colorimetric Assays 113
36. Potentiometry ......................................................................................................114
Indicator Electrodes 115
pH Meter 115
Ion-Selective Electrodes 117
37. Flame Photometer—General Principle and Procedure ...................................118
Flame Photometry 118
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38. Electrophoresis—General Principle and Procedure ........................................121
Electrophoresis 121
39. Chromatography—General Principle and Procedure.....................................124
Chromatography 124
40. Automation in Clinical Biochemistry ................................................................128
Types of Analyzers 128
41. Quality Control in Clinical Biochemistry .........................................................130
Investigation of Clinical Laboratory 130
42. Oral Glucose Tolerance Test...............................................................................134
Assessing the Status of Glucose Tolerance 134
43. Case Reports ........................................................................................................139
44. Normal Values of Biochemical Parameters ......................................................147
Section 5: Reagent Preparations
45. Reagent Preparations .........................................................................................153
Reagents for Qualitative Experiments 153
Reagents for Quantitative Experiments 155
Index 159