CONTENTS

 

Abbreviations                                                                           …                           vi

 

SECTION-A

General Analytical Techniques and Principles                             …         1     -       32

 

SECTION-B

Guidelines for Good Laboratory Practices                                 …       33     -       52

 

SECTION-C

Analytical Methods                                                                   …       53     -     328

 

SECTION-D

Other Analytical Techniques                                                      …     329     -     342

 

SECTION-E

Animal Experimentation                                                            …     343     -     368

 

SECTION-F 

Statistical Methods                                                                   …     369     -     400

 

SECTION-G

Appendix                                                                                 …     401     -     424


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                                                                 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


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                                                                  SECTION B

                                           Guidelines for Good Laboratory Practices

                                                                    Contents

 

  1. Good laboratory practices                                                                          …             35

a.                   Personal                                                                                       …             35

b.                  Facilities                                                                                       …             35

c.                   Equipment                                                                                    …             36

d.                  Standard operating procedures                                                     …             37

 

  1. Quality control                                                                                           …             41

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

  1. Safety in the laboratory                                                                              …             46 

 

  1. First aid in accidents and poisoning                                                             …             48

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

 


                                                               
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                                                                    SECTION C

                                                                Analytical Methods

                                                                       Contents

 

              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


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                                                                SECTION D

                                                    Other Analytical Techniques 

                                                        Contents

 

  1. Basal metabolic rate                                                                                   …           331

a.                   Benedict-Roth respiration apparatus                                             …           331

b.                  Conditioning of the subject                                                           …           332

c.                   The test

                                                                                                               …           332

  1. A typical cell fractionation procedure                                                          …           334

 

  1. Density gradient centrifugation technique for synoptozomes                         …           335

 

  1. Polyacrylamide get electrophoresis                                                             …           338

 

  1. Purification of water                                                                                   …           338

 

  1. Purification of the common organic solvents                                                …           339

 

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

 

  1. Glassware washing                                                                                  …            342

 


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                                                                    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

 

 

 

 

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                                                            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


 

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                                                                            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 Whatman’s 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

 

 

 

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