CONTENTS
Abbreviations vi
General Analytical Techniques and Principles 1 - 32
Guidelines for Good Laboratory Practices 33 - 52
Analytical Methods 53 - 328
Other Analytical Techniques 329 - 342
Animal Experimentation 343 - 368
Statistical Methods 369 - 400
Appendix 401 - 424
SECTION A
General Analytical Techniques and Principles
Contents
1. Colorimetry and spectrophotometry 3
2. Fluorimetry 4
3. Flame photometry, atomic absorption and emission spectrophotometry 5
4. Chromatography
a. Paper and thin layer chromatography 6
b. Column chromatography 8
c. Gel filtration chromatography 8
d. Ion exchange chromatography and automatic acid analysis 9
e. Affinity chromatography 9
f. Gas liquid chromatography 9
g. High pressure liquid chromatography (HPLC) 10
h. Gas chromatograph Mass spectrometer (GC-MS) 10
5. Electrophoresis
a. Paper electrophoresis 11
b. High voltage electrophoresis 12
c. Gel electrophoresis 12
d. Western blot
12
6. Radioisotope methodology
a. Interaction of radiation with matter 13
b. Physical properties of a radio nuclide 14
c. Units of radioactivity 14
d. Radiation detection and measurement 15
e. Safe handling of radioisotopes
17
7. Immunoassays
a. Radioimmunoassay 18
b. Enzyme linked immunoassay 18
c. Radial immunodiffusion technique
18
8. Microbiological assays 18
9. Centrifugation 21
10. Dialysis and ultrafiltration 22
11. Lyophilisation and flash evaporation 23
12. pH 24
13. Balances 26
14. Collection of biological samples 26
15. Metabolic balance studies 28
16. Pharmacokinetics 29
SECTION B
Guidelines for Good Laboratory Practices
Contents
a. Personal 35
b. Facilities 35
c. Equipment 36
d. Standard operating procedures 37
a. Calculation of standard deviation and the coefficient
and the coefficient of variation
b. Control charts for acceptable limits 43
c. Calculation of cumulative sums 44
d. Conventional control chart 45
e. Cusum graph
45
a. First aid kit 48
b. Universal antidote powder 49
c. General procedure 49
d. Artificial respiration 50
e. Corrosive acids 50
f. Caustic alkalies 50
g. Cyanides 51
SECTION C
Analytical Methods
I. Food Analysis 55
II. Proteins, Amino Acids and Metabolites 59
III. Carbohydrates 102
IV. Lipids 112
V. Nucleic Acids and Nucleotides 130
VI. Vitamins and Coenymes 139
VII. Minerals 175
VIII. Hormones 196
IX. Enzymes 226
X. Drugs 264
XI. Food Toxins 273
XII. Bacteriology 285
XIII. Cytogenetics 293
XIV. Histopathology 297
XV. Hematology 306
XVI. Immunology 313
XVII. Other Metabolites 319
SECTION D
Other Analytical Techniques
Contents
a. Benedict-Roth respiration apparatus 331
b. Conditioning of the subject 332
c. The test
332
a. Absolute ethyl alcohol 339
b. Absolute diethyl ether 339
c. Absolute methyl alcohol 340
d. n-Propyl alcohol 340
e. Iso-propyl alcohol 340
f. Acetone 340
g. Methyl ethyl ketone 340
h. Benzene 341
i. Petroleum ether 341
j. Ethyl acetate 341
k. Pyridine 341
l. Methylene chloride 341
m. Chloroform 341
SECTION E
Animal Experimentation
Contents
1. Regulations for animal experimentation 345
2. Selection of animal species 345
3. Factors affecting the experimentation 346
4. Nutritional requirements of animals and formulation of diets 346
a. Nutritional requirements 346
b. Classification of diets 346
c. Formulation of diets 347
d. Feeding methods 348
5. Anaesthesia 348
6. Techniques and sites of blood collection 350
a. Rat 351
b. Mouse 351
c. Hamsters 351
d. Guinea pig 352
e. Rabbit 353
f. Cat 354
g. Dog 354
h. Monkey 354
i. Chicken 354
7. Euthanesia 356
8. Common surgical techniques 356
a. Liver biopsy 356
b. Partial hepatectomy 357
c. Ovariectomy 357
d. Orchidectomy 357
Table A Housing and environmental requirements for 359
commonly used laboratory animals
Table B Parameters of reproduction in common laboratory animals ... 360
Table C Mean and ranges of hematological and electrolyte values ... 361
Table D Mean and ranges of blood chemistry reference values 362
Table E Recommended dietary allowances for different animal species 363
Table F Composition of stock diets 363
Table G1 Mineral mix (AIN-93G-MX) than supplies the 364
recommended concentrations of elements for the AIN-93G diet
Table G2 Vitamin mix (AIN-93-VX) that supplies the recommended ... 365
concentrations of vitamins for AIN-93G and AIN-93M diets
Table H Dosages of tranquilliser and anticholinergic drugs 366
Table I Dosages of analgesic, hypnotic/sedative and injectable 367
anaesthetic agents
SECTION F
Statistical Methods
Contents
1. Concepts in statistics 371
2. Normal distribution 372
3. Measures of central tendency 372
a. Arithmetic mean 372
b. Median 372
c. Mode 372
4. Measures of dispersion or variation 373
a. Range 373
b. Standard deviation 374
c. Standard error 375
d. Coefficient of variation 375
5. Tests of significance 375
a. t test 376
b. Normal curve test 378
c. F test for equalities of the variances 380
d. Modified t test 381
6. Analysis of variance 383
7. Chi-square test of association 386
8. Correlation coefficient 387
9. Experimental designs 389
10. Sample size 390
Table-A Distribution of t-Probability 394
Table-B Fisher and Yates variance ratio 5 Per cent 395
Table-C Fisher and Yates variance ratio 1 Per cent 396
Table-D Fisher and Yates variance ratio 0.1 Per cent 397
Table-E Distribution of probability Chi-square 399
Table-G Random numbers 400
SECTION G
Appendix
Contents
1. Boiling points 403
2. Details on common acids and alkalies as purchased 403
3. Equivalent weights of some common chemicals 403
4. Preparation of common buffers 404
a. Acetate buffer pH 3.6-5.6 404
b. Barbital buffer pH 6.8-9.2 404
c. Borate buffer pH 8.1-9.0 404
d. Borate buffer pH 9.3-10.7 405
e. Carbonate buffer pH 9.7-10.9 405
f. Citrate buffer pH 3.0-6.2 405
g. Citrate phosphate buffer pH 2.6-7.0 406
h. Glycine HCL buffer pH 2.2-3.6 406
i. Glycine NaOH buffer pH 8.6-10.6 406
j. Krebs Ringer phosphate and bicarbonate 406
solutions pH 7.4
k. Phosphate buffer (Pottassium salt) buffer 408
pH 5.8-8.0
l. Phosphate buffer (Sodium salt) pH 5.7-8.0 408
m. Sodium bicarbonate 5% CO2 buffer pH 6.0-8.0 409
n. Sodium carbonate biocarbonate buffer pH 9.2-10.8 409
o. Succinate buffer pH 3.8-6.0 409
p. Tris HCl buffer pH 7.2-9.0 410
5. Gel filtration media 410
6. Ion Exchange resins 411
a. Cation 411
b. Anion 412
c. Cellulose 412
d. Sephadex and bio-gel ion exchange resins 413
7. Ammonium sulphate fractionalism of proteins 415
8. Solutions used in forming density gradients 416
a. Sucrose 416
b. Caesium chloride 417
c. Ficoll .. 417
9. Nomogram for computing relative centrifugal force 418
10. Classification of Whatmans filter papers (numbers) 419
11. Radioactive isotopes used in biochemical investigation 419
12. Composition of liquid scintillation counting fluids 420
13. Relation between transmittance (T) and optical density (OD) 420
14. International system of units and conversion tables 421
15. Four-place logarithms 422
16. Periodic table 424